“HEI‘S

 
 

. LIBRARY ,
.' Michigan Stan:
‘ ’Uninnity

l l! mu; will 1m ll nan Jill lsllfl Iflllzl a

This is to certify that the

thesis entitled

EFFECTS OF ESSENTIAL FATTY ACID DEFICIENCY
AND VARIOUS LEVELS OF DIETARY POLYUNSATURATED FATTY ACIDS
0N HUMORAL IMMUNITY IN MICE

presented by

James w. De Nille

has been accepted towards fulfillment
of the requirements for

M. S. degree in Nutrition

 

 

K/[C/ éfléz'c'at (97.1

Major professor

 

Date JulLTQ, 1979

 

0-7 639

OVERDUE FINES ARE 25¢ PER DAY
PER ITEM

Return to book drOp to remove
this checkout from your record.

 

 

 

EFFECTS OF ESSENTIAL FATTY ACID DEFICIENCY
AND VARIOUS LEVELS OF DIETARY POLYUNSATURATED FATTY ACIDS
ON HUMORAL IMMUNITY IN MICE

By
James w. De Nille

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1979

ABSTRACT
EFFECT OF ESSENTIAL FATTY ACID DEFICIENCY

AND VARIOUS LEVELS OF DIETARY POLYUNSATURATED FATTY ACIDS
ON HUMORAL IMMUNITY IN MICE

By
James w. De Wille

Six experiments were conducted to determine the
influence of an essential fatty acid deficient (EFAD) diet
and dietary polyunsaturated fatty acids on humoral
immunity in mice. The results indicated that:

l) Diets deficient in essential fatty acids (0% corn
oil) significantly reduced humoral immunity. This reduc-
tion occurred after feeding the EFAD diet for 28 days; and
preceded effects on growth or appearance.

2) Reduced immunity was demonstrated against T-cell
dependent and T-cell independent antigens; and in both
primary and secondary responses of mice fed the EFAD diet.

3) After 56 days of feeding the EFAD diet mice
switched to the control diet (l3% corn oil) for 7 days
demonstrated full recovery of the humoral response.

4) Diets containing various levels of polyunsaturated
fatty acids (from 2 to 70% of energy from corn oil) did
not adversely affect the humoral response.

These results indicate the importance of essential
fatty acids in maintaining the functional integrity of the

humoral response.

TABLE OF CONTENTS

LIST OF TABLES.

LIST OF FIGURES

LIST OF SYMBOLS AND ABBREVIATIONS
INTRODUCTION.

REVIEW OF LITERATURE.

Dietary Fat and the Immune System.

Membrane Lipids and Lymphocyte Activation:

MATERIALS AND METHODS
RESULTS

DISCUSSION AND CONCLUSIONS.
RECOMMENDATIONS

REFERENCES.

ii

Page

handicap»:

29
33
35

LIST OF TABLES

Table
1 Composition of the diets.

2 Body and organ weights of mice fed diets
containing various levels of corn oil for 35
or 70 days (Experiment l) . . . . .

3 Primary antibody response to sheep red blood
cells of mice fed diets containing various
levels of corn oil for 35 or 70 days
(Experiment 2). . . . . . .

4 Primary antibody response to sheep red blood
cells of mice fed diets containing various
levels of corn oil for 42 day (Experiment
3 . . . . . . . . . . . . . . . . . . . .

5 Secondary antibody response to sheep red blood
cells of mice fed an essential fatty acid
deficient (0% corn oil) or control (13% corn
oil) diet for 75 days (Experiment 5).

6 Primary antibody response to lipopolysaccharide-
sheep red blood cells of mice fed an essential
fatty acid deficient (0% corn oil) or control
(l3% corn oil) diet for 42 or 56 days
(Experiment 6). . . . . . . . . .

iii

Page
10

15

I6

2]

26

28

LIST OF FIGURES

Figure Page

1 Primary antibody response of mice fed an
essential fatty acid deficient (0% corn oil)
diet expressed as percent of control (13%
corn oil) response. . . . . . . . . . . . l9

2 Primary antibody response 2, 3, 4, 5. and 6 days

after intraperitoneal immunization with lxlO
sheep red blood cells . . . . . . . . . 24

iv

EFAD
PUFA
CMI
IgG
IgM
SRBC
MEM

LIST OF SYMBOLS AND ABBREVIATIONS

essential fatty acid deficient
polyunsaturated fatty acids
cell mediated immunity
immunoglobulin G
immunoglobulin M

sheep red blood cells

minimum essential medium

INTRODUCTION

It is well established that nutritional status influ-
ences the functional capacity of biological systems. In
the case of the immune system, the complexity of this
influence and its effect on health are just beginning to
emerge. Increased susceptibility to certain infectious
diseases has been a common observation in malnourished
children (l). More detailed studies indicate that protein-
calorie malnutrition as well as vitamin and mineral defi-
ciencies, alter host resistance by decreasing immune
competence (2,3). The association between nutrient intake
and disease susceptibility has also been investigated with
respect to dietary excesses (4,5).

In Western countries, the type and level of fat in the
diet has been positively correlated with the increased
incidence of certain tumors (4,6,7). In studies with
rodents, elevated levels of dietary polyunsaturated fatty
acids (PUFA) have been reported to enhance the development
of chemically induced tumors (8-ll). From this information
an association between dietary PUFA and enhanced suscepti-
bility to malignancy has been proposed (4).

Mertin has reported that PUFA inhibit lymphocyte

transformation in vitro (l2), and impair graft and tumor

rejection in vivo (9,13-15). Based on these observations

he has proposed that PUFA suppress cell-mediated immunity
(9). These views, however, have been challenged (l6-l9),
and currently the extent to which dietary PUFA influence the
immune system is open to question.

When PUFA are removed from the diet, biochemical evi-
dence of essential fatty acid deficiency (EFAD) rapidly
appears (20). This has been observed in patients receiving
fat-free solutions during parenteral nutrition (21,22). At
the same time, delayed wound healing and an increased inci-
dence of infection are common observations in these patients
(22).

Because of the lack of studies on the effect of EFAD
on the immune response, and the ambiguity of available data
on the effects of elevated levels of PUFA on the immune
system, the experiments described in this thesis were under-
taken. These experiments have focused on the effects of
varying the level of PUFAs in the diet (from 0-70% of energy

from corn oil) on the humural immune response of mice.

REVIEW OF LITERATURE

Dietary Fat and the Immune System

PUFA and Immunity. 'The investigation of PUFA and
immunity has focused largely on the effects of PUFA, admini-
stered by subcutaneous injection, on cell-mediated immunity
(CMI) (9,l2—15). Using this design Mertin reported that
PUFA inhibit allograft and tumor rejection capabilities
in mice; and concluded that PUFA may influence disease or
tumor susceptibility by inhibiting CMI (7,9,l2). Several
other investigators have also used subcutaneous injections
of PUFA to study their effects on CMI. In general, most
investigators agree that injections of PUFA are highly
toxic to animals (16-18), but there is not uniform agreement
with Mertin's conclusion that this treatment has immuno-
inhibitory effects (l6-l8). In other studies Mertin
assessed the effects of oral administration of PUFA on CMI
(9). These studies, while tending to support the hypothesis
that PUFA inhibits CMI, provided no evidence on the
mechanism(s) responsible for the observed results. Addi-
tional studies are clearly needed before the influence of

PUFA on immunity can be unequivocally established.

The effects of PUFA on lymphocyte function in vitro
have also been studied. Mertin and Hughes reported that
both saturated and unsaturated fatty acids dissolved in
ethanol and added to lymphocyte cultures inhibit PHA induced
lymphocyte transformation (12). Other studies, however,
have indicated that culture conditions (19) and the mode
that fatty acids are added to the cultures (23) have a large
effect on whether or not PUFA inhibit lymphocyte transfor-
mation. This suggests that a number of factors may deter-
mine this effect, and raise doubts that PUFA specifically
inhibit lymphocyte function.

Malignancy. Studies associating dietary fat with
enhanced tumor susceptibility first appeared in the litera-
ture almost fifty years ago (24). Since that time the
influence of dietary fat on tumorgenesis has been extensi-
vely studied (25,26). A number of studies with rodents have
associated dietary PUFA with enhanced development of
chemically-induced tumors (27-30); but not all authors are
in agreement (31). The importance of these findings to
human malignancy has yet to be established (32). Epidemio-
logical studies have been unable to associate PUFA intake
with tumor incidence (33,34); and studies of PUFA and
human tumor incidence have not been well controlled (35,
36). Thus, the association between dietary PUFA and cancer
has been actively pursued for quite some time; but no clear-

cut relationship has yet been discovered.

Multiple Sclerosis. Because there is evidence that
auto-immunity may play a role in multiple sclerosis, and
because low serum levels of PUFA have been found in some of
these patients (37), an association between multiple
sclerosis and dietary PUFA was proposed (37). While results
of some human studies suggest beneficial effects of supple-
menting the diet of multiple sclerosis patients with PUFA
(38), no conclusive evidence that dietary PUFA effects the
overall course of the disease has been obtained (39).
Injecting myelin basic protein (with adjuvant) into suscep-
tible animals induces a condition called experimental
allergic encephalomyelitis(40). Reports that diets high
in PUFA slow the development of this disease have been
interpreted to mean that dietary PUFA inhibit immune func-
tions (33); however, direct experimental evidence for this
interpretation is lacking. Whether or not this experimental
model truly is representative of multiple sclerosis is open
to question, and the secondary role of dietary PUFA in this
condition is still unresolved.

Graft Rejection. Rejection of foreign tissues and
grafts is mediated by CMI. The suggestion that PUFAs may
inhibit CMI led to several studies involving kidney trans-
plant patients. While some pilot studies reported an
increase in graft survival with PUFA treatment (14), longer
term studies showed no significant effect (41).

Cardiovascular Disease. Reports of increased levels

of anti-milk antibodies in heart patients (42) and lipid

accumulation in auto-immune induced atheromas (43) has led
to the suggestion of a link between dietary fat, immune
functions, and cardiovascular disease.

Resistance to Infection. The influence of dietary fat
has been studied with respect to another aspect of immunity,
resistance to infection. Dogs fed high-fat diets were more
susceptible to canine hepatitis virus than dogs fed a
control diet (44); and chronically over-nourished dogs were
more susceptible to canine virus challenge than control
dogs (45). On the other hand, when dogs were fed an essen-
tial fatty acid deficient (EFAD) diet, they demonstrated
increased incidences of upper respiratory, skin, and ear
infections (46). Since EFAD has recently been described in
patients receiving intravenous hyperalimentation (47-49),
and infection is a common problem in these individuals (47),
the possibility that EFAD may play a role in enhancing
susceptibility to infection in these patients seems plau-

sible.

Membrane Lipids and Lymphocyte Activation
It is important to recognize that the fatty acid
composition of the diet is reflected to a significant extent
in membranes of many organs (50,51). This is of particular
concern in the case of the lymphocyte whose interaction
with antigen and foreign cells takes place initially at the

membrane surface. An alteration in fatty acid composition

of lymphocyte membranes could affect a number of events
crucial to lymphocyte activation and response. The initial
event in lymphocyte activation is binding of the antigen

or mitogen to the lymphocyte surface (52). Following this,
membrane phospholipid metabolism is stimulated (53), and
the redistribution of membrane receptors into a polar cap
(capping) has been observed (54). Each of these events
could be affected by changes in membrane fatty acid compo-
sition.

Binding. Binding of antigen or mitogen to the lympho-
cyte surface is necessary to initiate activation. We are
unaware of any studies describing the effect of dietary
modification of membrane lipids on subsequent lymphocyte-
ligand interactions. There is, however, evidence showing
that hormone binding to membrane surfaces is altered when
membrane lipids are disrupted with digitonin or phospho-
lipase A treatment (55). Alterations in lymphocyte membrane
fatty acids could affect binding characteristics to membrane
receptors and thereby influence the ability of the lympho-
cyte to respond to ligands.

Phospholipids Metabolism. Lymphocyte membrane phos-
pholipid metabolism is stimulated during lymphocyte activa-
tion (56). Marked increases in membrane phospholipid fatty
acid turnover, with increased incorporation of unsaturated
fatty acids into phospholipids has been reported (56-61).

This is supported by studies showing increased activity of

membrane associated lysolecithin acyl-transferases (58),
which catalyze the transfer of specific fatty acids into
phospholipids. This enzyme has a low Km for arachidonic
acid, consistent with the large increase in arachidonate
concentration in lymphocyte membrane phospholipids observed
following mitogen stimulation (53,58). Evidence is accumu-
lating to indicate that changes in membrane phospholipids
following ligand binding are among the most important events
in triggering lymphocyte activation (60). It would seem
reasonable, therefore, to postulate that modifications in
consumption of polyunsaturated fatty acids might alter cel-
lular phospholipid metabolism and that this could be an
important factor in modulating lymphocyte response.

Capping. Another early event associated with lympho-
cyte activation is the lateral diffusion of lymphocyte
receptors through the lipid bilayer to form polar caps (54).
This process, which has been extensively studied (54), is
greatly influenced by membrane fluidity properties (62).

The increased incorporation of unsaturated fatty acids into
lymphocyte membranes following stimulation increases membrane
fluidity (53) which facilitates movement of membrane recep-
tors into caps (53). Fluidity is determined in part by the
membrane PUFA/saturated fatty acid ratio (63). Since this
ratio reflects the dietary intake of these fatty acids (63)
it is entirely possible that dietary fat may alter lympho-
cyte membrane processes (such as capping) that are dependent

on membrane fluidity properties.

MATERIALS AND METHODS

Animals and Diets. Male A/J mice 21 to 28 days old
were housed in plastic solid-bottom cages, five mice per
cage, in a temperature (24 1 1°), light (12 hrs light per
day), and humidity-controlled room. Diets and water were
provided ad libitum. Formulation of the diets was on an
equal energy basis with casein providing 24% of the dietary
energy. Corn oil was used as a source of essential fatty
acids and was assumed to contain 59.5% linoleic acid and
0.8% linolenic acid as previously determined (64). Corn oil
was substituted for glucose on an equal energy basis. Com-
position of the diets fed is presented in Table 1.

Experiment 1. To determine the effects of various
levels of dietary PUFA on the humoral antibody response four
groups of mice (10 mice per group) were fed diets containing
0%, l3%, 50%, or 70% of energy from corn oil for 35 or 70
days. The antibody response was determined by the Jerne
plaque assay. Body weights, spleen weights, thymus weights,
and energy intake were assessed.

Experiment 2. In this experiment two aspects of the
relationship between EFAD and the humoral response were
examined: 1) the length of time required to observe losses

in humoral immunity after switching to the EFAD diet, and

._.o_ .mmopzp—mu new

 

 

 

nu "Ame mocmcmwmc mmmv o.¢ .st Poem:_s mm.o .muwcopgo m=_po;u ”Axe mocmcmwmc mmmv ¢.o
1. .xms =_Emu_> mm.o .m:_:o_spme mo.om .=_mmmo "Apmmma m mm com o :PV em:_mu:oo x_E Pommmp
__o :coo
o“ om m_ m m N 0 seem xmcmcw &
um om m ¢.m o.~ m.o 0 Amy —Po :Loo
e om um um Pm mm mm Amy mmoozpo
mm mm mm mm mm mm mm Amy _xwe _mmmm
N o m e m N P
um_o “cowomcmcm

 

mom_e as» co

co_uwmoasou ._ mpamh

11

2) the length of time required to reverse these losses by
feeding the control diet. Mice were fed the EFAD diet (0%
corn oil) or the control diet (13% corn oil) for up to 70
days. One group of mice was switched from the EFAD to the
control diet on day 56 of the experiment. Plaque assays
were performed at the times designated in Figure 1. Spleen
and body weights were also recorded.

Experiment 3. To determine if reduced amounts of
dietary polyunsaturated fatty acids would affect the humoral
response, mice were divided into five groups (10 mice per
group) and fed diets containing 0, 2, 5, 9, or 13% of energy
from corn oil for 42 days. Plaque assays were performed and
spleen and body weights were recorded.

Experiment 4. To examine the sequence of development of
the primary antibody response in mice fed the EFAD diet (0%
corn oil) or control diet (13% corn oil), plaque assays were
performed 2, 3, 4, 5, and 6 days after mice were immunized
(intraperitoneal injection) with 1x108 sheep red blood cells
(SRBCs)5. Spleen and body weights were also assessed.

Experiment 5. Experiments 1 to 4 focused on the
effects of EFAD on the primary antibody response. To evalu-
ate the effects of EFAD on the secondary response, mice fed
the EFAD diet (0% corn oil) or the control diet (13% corn
oil) were immunized with 1x108$RBCs on day 42 and again on
day 70. The secondary response, which is exclusively immu-

noglobulin G (IgG), was measured on day 75 by plaque assay;

12

spleen and body weights were also recorded.

Experiment 6. Optimal antibody response to SRBCs

 

requires the interaction of T helper cells and B-cells (65).
To determine the influence of EFAD on B-cell response inde-
pendent of significant T-cell influence, mice fed the EFAD
diet (0% corn oil) or the control diet (13% corn oil) were
immunized with 10 ug of E. coli lipopolysaccharide (LPS).
Plaque assays were performed by a modification of the pro-
cedure of Jacobs and Morrison (66).

Determination of Antibody Mediated Response (Jerne

Plaque Assay). Antibody response was assessed by a modifi-

 

cation of the Jerne plaque assay. Briefly, mice were
injected intraperitoneally with 1x108 SRBCs and killed by
cervical dislocation at the times indicated in each experi-
ment. A spleen cell suspension was prepared by gently
teasing the spleen cells from the spleen capsule into
sterile Hank's Balanced Salt Solution. Spleen cells were
then washed, resuspended in minimal essential medium (MEM),
and plated with 2x108 SRBCs and MEM containing 0.6% agarose.
Plates were incubated at 370 in a humidified chamber.

Both direct immunoglobulin M (IgM) and indirect (IgG)
plates were prepared in duplicate. Following an initial 90
minute incubation, the direct (IgM) plates were flooded with
guinea pig complement. After reincubation for 30 minutes,
plaques became visible. Each plaque represented lysis of
SRBCs by antibody released by a single plasma cell in the

presence of complement. The indirect plates were similarly

13

treated, however, to visualize IgG producing cells, rabbit
anti-mouse IgG was added to the plates 30 minutes prior
to the addition of guidea pig complement. Background
plaques were negligible. Indirect plaques were corrected
for the small numbers of IgM plaques (9%) that developed.
Results were expressed as plaque-forming cells per spleen.
Determination of Lipopolysaccharide-SRBC Primary
Response. A modification of the method of Jacobs and
Morrison (66) was used. Mice were immunized with 10 ug of
E. coli lipopolysaccharide (LPS) in 0.1 ml of a sterile
solution containing 0.9% NaCl and 100 mM phosphate (pH 7.4).
LPS coated SRBCs were prepared by boiling LPS for 60
minutes in 0.25 N NaOH. neutralizing the LPS containing
solution with HCl, and then adding 4.0 mg of LPS to each
milliliter of packed, washed SRBCs. Following a 30 minute
incubation at 37°, the LPS-SRBCs were washed four times
with sterile phosphate buffered saline and the plaque assay
performed.

Data Analysis. All data were treated statistically by

 

either the student's "t" test. or one way analysis of
variance with treatment differences determined by Tukey's

test;

RESULTS

Experiment I. To determine the effect of various

 

levels of dietary polyunsaturated fatty acids on humoral
immunity, mice were fed diets containing 0% (EFAD), 13%
control), 50%. or 70% of energy from corn oil for 35 or 70
days. Body weights, energy intake, spleen weights, and
thymus weights did not differ among the treatment groups
after 35 days (Table 2). After 70 days. body weights, and
spleen weights of mice fed the 0% corn oil diet (EFAD)
were significantly lower than all other treatment groups
(Table 2), but thymus weights did not differ. There was no
evidence of dermal lesions at any time during the study.
Although no differences in any of the physical para-
meters measured were apparent after 35 days. differences
in the direct (IgM) response were evident by this time
(Table 3). Mice fed the EFAD diet (0% corn oil) responded
only 66% as well as controls (13% corn oil): but there was
no difference in the direct response between mice fed the
control diet and mice fed the elevated levels of corn oil
(Table 3). The indirect (196) response was not altered
after 35 days (Table 3). Extending the feeding period to
70 days resulted in a greater loss in antibody response in

the mice fed the EFAD diet. At 70 days. the direct (IgM)

l4

15

wmmcm>m

.mmmo emu mo_e m>_dm

.m «.mp ma; S;m_mz scan Pm_u_=¢

uuwmco xuzum am one we when m H mm mew: mow:
-wewcmwm ace mqucomgmazm ucmcm$$_c ;u_z chF msmm asp :P mcmnE=z

.Amo.o v av ucmcmmeU >Fucmo
.mowe o— cow :mmz_

 

mw.o new mom emu amN Away 93 massec
m~.e comp am__ nm_P amm Amsv p: emm_am
om.P “we awe awe mmm NAsme\aaa8\_auxv mxape_ seemed
ou.o cam. _m Uo. mm m. mm ¢.e~ Amv a: seam Passe
n m

mama ON

me.P amm mom mam new Amev a: massgc
mm.m one, ape, cep_ amo_ Away 33 cmm_am
NN._ awe awe awe cam Nhsau\ammU\Peuxv oxaucu smtmcm
mm.o mN.¢~ am.¢~ mm.e~ am.¢~ Ame 3: seem .aepe
mama mm

mm cu om mp o cmumamcmm

 

P_o :cou soc; mmcmcm acouwwa &

.P “cmewcmaxm .wmzmv on so mm cow

Pwo :cou we m—m>m_ m:o_cm> m:_cwmacoo mumwu now move we macm. m3 cameo can zoom .N w—nmh

16

ecu Ame save

more .ueeswgeexe awe om esp :H .Amm xeev
mppee eeepe ems emecm
mm ex» ea .pemce »

.Aou xeev swamp whee m em:_Eceaee emceemec

m—Pee beeps ems eeegm op x F new: »_Pee=euwceeecucw ee~_czsew ace;
meuep whee m eecwsceuee emceemec use Aom aeev

op x p ;u_3 xppmmceuwceeecucw ee~wceeew use: mews .pceewceexm awe
wage as one we mzee m H mm ecez mew: .Amo.o v av ucegmmwwe apucee

upwpcmwm use mue_gemcee=m acegmwwpe saw: ecpp eEem ecu cw memeszz .ee_e op gem :eezP

 

 

mom.~ em~¢.oe noom.ee emom.m¢ emmm.mm Auma peecwecm
omm.m eP—o.me eo-.om noou.~e momm.~N A: HV uemc_o
mxeo ox
mpm.~ emoe.me empm.om enmm.mm epmn.mm Awmmv ueecwecm
mpm.m nocv.n¢ amme.~e comp.mm mom¢.wm AzmHv ueeg_a
seepem\m__ee mechec meeepe mxmo mm

mm 05 om mp o

 

pwo cceu sec» magma“ Aceam_o a

 

.F
m:_:Peucee mauve new

acmewceexu . mXee on so mm gee Pwe :cee ee mpe>ep meewge>
move we mppeepeeepe we; emenm eu emceemmc xuea_u:e ALeEPL¢ .m epnmh

17

response was reduced to 58% of the control response; and
the indirect (190) response was reduced to 73% of the
control values. As in the 35 day study, the antibody
response of the mice fed the elevated levels of corn oil
did not differ from values obtained in mice fed the control
diet. This suggests that: l) humoral immunity is signifi-
cantly reduced by feeding diets deficient in essential
fatty acids, but 2) humoral immunity is not adversely
affected by feeding elevated levels of PUFAs.

Experiment 2. Results from Experiment 1 indicated
that mice fed an EFAD diet for 35 days exhibit normal growth
and appearance; but have an impaired humoral immune capa-
city. To determine the length of time required for an EFAD
diet to significantly impair the primary antibody response,
mice were fed the diet containing 0% corn oil (EFAD) for up
to 70 days. The capacity of these mice to respond to SRBCs
was compared to that of mice fed the control diet (13% corn
oil). After 21 days both the direct (IgM) and indirect
(196) responses of mice fed the EFAD diet were reduced to
approximately 80% of the control values (Figure l). Signi-
ficant reductions were apparent at 28 days when the direct
response was reduced to 64% of control and the indirect
response to 71% of the controls. From 28 to 42 days both
responses decreased slightly; with no further reductions
apparent beyond 42 days. The significance of this plateau

is not known at this time.

Figure l.

18

Primary antibody response of mice fed an essential
fatty acid deficient diet (0% corn oil). Response
expressed as percent of control (13% corn oil)
response. Each point represents the mean of 10
mice. Standard errors ranged from 3 to 13% of
mean values. Mean of control direct (IgM)
response = 42,761 plaque forming cells per

spleen; mean of control indirect (IgG) response =
44,422 plaque forming cells per spleen. A sig-
nificantly different from control (P < 0.05).

B = significantly different from refed (P < 0.05).

19

Figure l

 

 

 

 

 

“zumgmmxmnjmo 025.com 954.5
a: o : Puma—o

_
_ T
_ ....._ 1
D
_ e. 1
R
_ .. 1
N"
_ m -
D
_ m n. 1
F. c.
E R L
_ u
_ m 1
_ _ _ g
.l. ._.,. m a o
nu
mw Jomhzoo “.0 ex.

28354249566370

14 2|

.7

 

EFAD

 

 

 

 

.6528 Le .x.
.zuwdmajue ezimec meefia
8 3 . Bumfiz.

25 1" "I"- REFED CONTROL DIET
1

28354249566370

TIME (DAYS)

I4 2|

.7

20

To determine if the deleterious effects of the EFAD
diet could be reversed. mice fed the EFAD diet were switched
to the control diet after 56 days. After 7 days of feeding
the control diet, both the direct (IgM) and indirect (IgG)
responses were restored to control levels (Figure 1). This
recovery was also apparent after 14 days, when the responses
of the refed mice equalled or exceeded control levels
(Figure 1). This suggests that losses in humoral immunity
induced by the EFAD diet can be rapidly reversed by feeding
a diet containing essential fatty acids.

Experiment 3. It was apparent from experiments 1 and

 

2 that a clear difference in antibody response could be
demonstrated between mice fed a 0% (EFAD) or 13% (control)
corn oil diet. To more closely determine the influence of
the level of corn oil in the diet on humoral immunity, mice
were fed diets containing 0, 2. 5, 9, or 13% of energy from
corn oil for 42 days. The 2% corn oil diet was chosen to
approximate the essential fatty acid requirement of the
mouse for growth, (about 1.2% of energy from linoleic acid)
(70). Final body weights did not differ among the experi-
mental groups; however. the direct (IgM) response of the
mice fed the diet containing 0% corn oil (EFAD) was reduced
to 54% of controls (13% corn oil), and the indirect (190)
response was reduced to 65% of controls (Table 4).
Responses of mice fed the diets containing 2 to 13% corn

oil did not differ. These results indicate thato 1) normal

21

.ANe xeev gene, mace m eecwsceuee emceemec use Awm xeev wwwee eeewa we; amenm mow x w
saw: appeeceuwgeaecacw eerczsew mew: mew: .Amo.o v a .ms N H mm .m> m A msv mezecm

wwe :cee walN ecu can» cezep awgceewwwcmwm ecmz “ewe Fwe :Lee No eew mews we mucmwez
ceewem wmcww .Am m.o A m.MN u ceeev meeecm aceEaeecu ocean Lewwwe we: ewe mwzmwez

anon Pecww .uemce aeeum am one we mxee m A wN ego: mews Pw< .Amo.o v av pcmcmwwwe
xwuceewwwcmwm ewe muewcemceeem ucmcewwwe saw: ecww eEem as» cw mcmnsaz .eews ow cow :eez

 

 

—
«Nm.N ewem.em nomm.mm noom.Nm noeN.m¢ emow.mm Aomwv ueecwecw
omm.N nome.¢m nome.~¢ novo.me Doom.me eONm.mN Asmmv geecwo
ceewem\mpwme mechew escape
mm mp m m N o

 

Pwo :Leu sec; xmcecm xceuewo N

.m acmswcmexm .meee Ne Lew wwe :cee we mpe>ew mzewce>

mcwcweucee mpewe new mews we wwwee eeewn we; eemcm e» emceemec xeeewuce xgeewce .e ewnew

22

functional capacity of the humoral immune system, as well

as normal growth and appearance, is maintained by feeding
diets containing 2% corn oil (approximately 1.2% of energy
from linoleic acid); and 2) significant losses in humoral
immunity can be demonstrated in mice fed EFAD diets before a
depression in growth is apparent.

Experiment 4. When A/J mice are fed a nutritionally
adequate diet, optimal primary response to SRBCs occurs five
days after immunization. Following this protocol, the first
three experiments reported here indicated that mice fed an
EFAD diet for 28 days or more produce significantly fewer
plaque forming cells than control fed mice. It would,
therefore, be important to assess the development of the
antibody response after immunization for differences in the
onset of response, sequence of appearance of IgM and IgG,
and peak response. To accomplish this, mice fed an EFAD
(0% corn oil) or control (13% corn oil) diet were assayed
2, 3, 4, 5, and 6 days following immunization with SRBCs.
The direct (IgM) response of the mice fed the EFAD diet was
significantly reduced from the earliest detectable day (day
3) through to day 6 (Figure 2). Compared to values for mice
fed the control diet, mice fed the EFAD diet produced 52% of
control values on day 3, 79% on day 4, 54% on day 5, and 61%
on day 6. Also, peak (IgM) response of the EFAD mice
occurred on day 4, compared to day 5 for controls. The

significance of the altered peak response is not known at

Figure 2.

23

Primary antibody response 2, 3, 4, 5, and 6 gays
after intraperitoneal immunization with 1x10

sheep red blood cells. Mice were fed an essential
fatty acid deficient (0% corn oil) or control

(13% corn oil) diet for 56 days. Each * indicates
significant differences (P < 0.05) between the two
treatment groups. Each point represents the mean
(i SEM) response of 8 mice.

24

Figure 2

 

 

 

 

 

 

‘
coco.
.‘
coco
- “—
O
O
O
O
O
O
C
..
Ilia.»
.'..
.."..'..
...'.
'....
lb
AU
3“
Mm nu
o m l
Pb .r—
u
I
u
_ — _ _
nv Av nu Av nv
A? 10 a‘ II

zwudmm \ no. a 3.50 92.28.... 30¢:
a 2 9— . Puma-a

 

 

6

5

4

3

2

 

 

1.

nu

n"

T. nu

N"

o n 1
n» .e

I

I

I

u

_ _ _ _ _

nu nu nu nu nu nu
a. 4. a. .4 .1

zumdmx me. .. mime eziceh. ”.53....
3 a: 535:.

DAYS POST lMMUNIZATlON WITH SHEEP RED BLOOD CELLS

25

this time.

Low levels of IgG production were also detectable by
day 3. At this point the indirect (196) response of the
EFAD mice was only 54% of control values. By day 4, how-
ever, the indirect response of the two groups did not differ.
Peak indirect (196) responses of both EFAD and controls were
observed on day 5, with the EFAD peak response at 64% of
controls. At day 6, EFAD response was 68% of controls.

It has been known for some time that the switch from
IgM to I90 production requires T—cell helper function (65).
Results from this experiment suggest that consumption of
the EFAD diet, which consistently reduced IgM production,
may be less effective in reducing IgG production, and raises
the possibility that EFAD may impair B-cell function more
than T-cell function.

Experiment 5. An essential component of humoral immu-
nity is the secondary or "memory" response. This response
occurs after a second encounter with the same antigen and
is characterized by a high rate of antibody production which
is exclusively 196. To determine the effect of EFAD on the
memory response, mice fed diets containing 0 or 13% corn oil
received immunizations with SRBCs on days 42 and 70. The
response of mice fed the EFAD diet was only 57% of the con-
trols (Table 5). This indicates that EFAD further compro-
mises humoral immunity by reducing the generation of memory

cells, and therefore reducing the memory response.

26

Table 5. Secondary antibody response to sheep red blood

cells of mice fed an essential fatty acid defi-
cient (0% corn oil) or control (13% corn oil)
diet for 75 days]. Experiment 5.

 

% Dietary Energy From Corn Oil

 

O 13 SE

 

Indirect (IgG) 98,000a 171.900

plaque forming cells/spleen

b 12,168

 

1

Mean for 10 mice. Numbers in the same line with different
superscripts are significantly different (P < 0.05). Mice
were 21 i 3 days of age at study onset. Response assessed
(day 75) following primary immunization (da§ 42) and
secondary immunization (day 70) with l x 10 sheep red
blood cells. Final body weights (23 t 0.7 vs. 27 i 0.5 g)
and spleen weights (116 i 8 vs. 138 t 5 mg) were signifi-
cantly lower (P < 0.05) in the essential fatty acid
deficient group compared to controls.

27

Experiment 6. Optimal response to SRBCs requires
interaction between both major populations of lymphocytes;
11cells(thymus derived) and B-cells (bone marrow derived).
Certain antigens (T-cell independent antigens) have been
identified that do not require significant T-cell helper
influence to elicit antibody response. In these cases, the
response is largely B-cell dependent, and the class of
antibody produced is exclusively IgM (66). In experiments
1 to 4 impairment of the direct (IgM) response of EFAD
mice appeared to be greater than the indirect (IgG) impair-
ment. This suggests that the EFAD may result in a greater
loss in numbers or in functional capacity in B-cells than
in T-cells. To assess B-cell function without significant
T~cell helper influence, mice fed EFAD and control diets
were immunized with E. coli lipopolysaccharide (LPS), a
primarily T-cell independent antigen. In a modification of
the plaque assay, LPS was absorbed to the SRBC surface and
the plaque assay performed as usual. Under these conditions,
the primary response of the mice fed the EFAD diet was
reduced to 62% of controls after 42 days, and 58% of con-
trols after 56 days (Table 6).

These results are consistent with our previous assess-
ments of the direct (IgM) response and support the hypothesis
that EFAD significantly impairs B-cell function. The extent
to which EFAD effects T-cell function is not known at this

time.

28

Table 6. Primary antibody response to lipopolysaccharide-
sheep red blood cells of mice fed an essential
fatty acid deficient (0% corn oil) or control
(13% corn oil) for 42 or 56 days . Experiment 6.

% Dietary Energy From Corn Oil

 

 

 

o 13 SE
plaque forming cells/spleen

42 Days .

Direct (IgM) 38,081a 61,450b 4,642

56 Days

Direct (IgM) 29,000a 49,675b 3,768

1

Mean for 10 mice. Numbers in the same line with dif-
ferent superscripts are significantly different (P <
0.05). Mice were 21 i 3 days old at study onset.
Response assessed four days after intraperitoneal
immunization with 10 ug of lipopolysaccharide. In the
42 day experiment final body weights (22.1 t 0.5 9) did
not differ, however, spleen weights of the essential
fatty acid deficient mice were significantly lower

(P < 0.05) than controls (93 i 2 vs 111 i 6 mg). In the
56 day experiment, final body weights were significantly
lower in the essential fatty acid deficient group

(22 i 0.6 vs 26 i 0.6 g) (P < 0.05) and final spleen
weights were also significantly lower (89 i 7 vs.

113 i 3 mg) (P < 0.05).

DISCUSSION AND CONCLUSIONS

In recent years numerous investigations have been
directed towards understanding the relationship between
diet and immunity. Results from these studies have indi-
cated that protein-calorie malnutrition (1-3), as well as
various vitamin and mineral deficiencies (71-73) reduce
immune-competence. The results presented here indicate
that EFAD also causes a significant reduction in immunity.
Significant losses in humoral immunity were observed in EFAD
mice challenged with T-cell dependent and T-cell independent
antigens. These losses occurred before any changes in
growth or appearance, and indicate that decreased immune-
competence precedes the gross symptoms of EFAD.

Because PUFAs have been reported to inhibit various
aspects of immunity, we also investigated the effects of
various levels of dietary PUFAs on the humoral response. In
these experiments, corn oil was used as the dietary source
of PUFAs, and the amount of corn oil varied from 2 to 70%
of dietary energy. The 2% corn oil diet was chosen to
approximate the EFA requirement of the mouse for growth
(approximately 1.2% of energy from linoleic acid) (70).
Under the conditions of these experiments, diets containing

as much as 50 to 70% of energy from corn oil had no effect.

29

30

on the antibody mediated response. It is concluded from
these experiments that EFAD significantly reduces humoral
immunity in the mouse; while elevated levels of dietary
PUFAs have no effect. Several explanations can be offered
for these findings.

In mice fed the EFAD diet, losses in humoral immunity
were generally accompanied by a decrease in spleen weight,
and a proportional decrease in splenic lymphocyte numbers
(averaging approximately 106 lymphocytes per mg spleen
weight in both EFAD and control mice). When results were
expressed on a per million spleen lymphocyte basis, rather
than on a total organ basis, the EFAD response remained
significantly lower than controls. Thus, in addition to the
reduced numbers of lymphocytes populating the spleen, there
was a significant reduction in the ability of the residual
lymphocytes to respond to antigen. Interaction between
antigen and specific lymphocyte membrane receptors initiates
lymphocyte stimulation and response. Since essential fatty
acids are important components of membranes, the effects of
EFAD on immunity could result from alterations in membrane
structure induced by EFAD.

It has been established that the lipid composition of
the diet is reflected in tissues and membranes (50, 74);
and that the physical properties of the membrane lipids
affect cell function (50, 63). Feeding an EFAD diet has
been shown to decrease lymphocyte linoleic and arachidonic

acid content (37), and to decrease membrane PUFA

31

concentration (50). This decrease in membrane PUFAs could
result in a decrease in membrane fluidity (63), which has
recently been shown to be important in "capping" (62), and
other membrane events associated with lymphocyte stimula-
tion (75). Thus, changes in lymphocyte membrane fatty acid
composition, resulting in a decrease in membrane fluidity,
could impair lymphocyte activation and result in fewer
numbers of cells capable of responding to antigen. In
addition, recent work by Ferber and Resch (53) showed that
marked changes in lymphocyte membrane fatty acid turnover,
with increased incorporation of unsaturated fatty acids
(specifically linoleic and arachidonic acid) occur upon
activation of the lymphocyte (53). It would seem reasonable,
therefore, that the reduced availability of linoleic and
arachidonic acids, as occurs with EFAD, could reduce
lymphocyte activation and response.

We are not aware of any previous studies on the effects
of dietary PUFAs, or EFAD, on humoral immunity. However,
the relationship between PUFAs and cell-mediated immunity
has been studied (9, 33). Results presented here indicate
that elevated levels of dietary PUFAs do not adversely
affect humoral immunity; and that EFAD significantly reduces
humoral immunity. This is not in agreement with the
results of Mertin, who reported that PUFAs inhibit cell-
mediated immunity, and that "PUFA deficiency" potentiates

cell-mediated immunity (9). It is important to recognize

32

that the experiments reported here assessed humoral immunity,
as opposed to Mertin, who assessed cell-mediated immunity
(9). In addition, our studies focused on the effects of
PUFAs as supplied in the diet; whereas in the majority of
Mertin's studies, PUFAs were administered by subcutaneous
injection or stomach tube (9, 13-15). The extent to which
these differing routes of administration may stress the
animals, and in this way affect results, is not known.

In summary, little is currently known about the rela-
tionship between nutritional status and immune functions.
This is particularly true for essential fatty acids.

Results presented here indicate that EFAD profoundly reduces
humoral immunity. This supports the hypothesis that
essential fatty acids play a crucial role in maintaining

the functional integrity of the humoral immune system.
Although the mechanisms for this effect are not yet known,
essential fatty acids are important components of membranes
and lipoproteins, and are precursors of prostaglandins.
Because each of these could affect lymphocyte activation
(62, 75, 76), essential fatty acids are in a unique position

to influence immunity.

RECOMMENDATIONS

The overall purpose of these experiments was to assess
the influence of an essential fatty acid deficient (EFAD)
diet, and various levels of dietary polyunsaturated fatty
acids on humoral immunity. Based on the results, it is
apparent that EFAD can significantly reduce the humoral re-
sponse, but diets containing elevated levels of polyunsatu-
rated fatty acids have no effect. This indicates that EFAD
impairs B-cell function, but does not directly address the
effects of EFAD, or dietary PUFAs, on T-cell function.
Thus, future experiments should be designed to assess the
effects of EFAD, and various levels of dietary PUFAs, on
in vivo cell-mediated immunity (CMI). In addition, in vitro
experiments should be carried out to more closely determine
the role of essential fatty acids in lymphocyte function.
To accomplish this, the role of essential fatty acids in the
sequence of events leading to lymphocyte activation should
be studied. These events include: ligand binding to the
lymphocyte surface, membrane phospholipid fatty acid meta-
bolism, receptor "patching" and "capping", and RNA and DNA
synthesis during blastogenesis. To more closely monitor
the role of essential fatty acids in lymphocyte activation,

studies should be carried out using a variety of culture

33

34

conditions including standard medium supplementation with
fetal bovine serum (FBS), supplementation with sera from
mice fed the various experimental diets, serum-free cul-
tures, and serum-free cultures with selected fatty acids.

Information obtained from the studies described in
this thesis, as well as those proposed for future consi-
deration, should help us understand how essential fatty
acids are involved in modulation of the immune system and
secondly, should provide a model system whereby influences
of other fatty acids on the immune system can also be
studied.

A better understanding of the role of dietary fat in
human health will make a significant contribution to future
decisions involving recommendations for the type and amount
of fat in the American diet (77). Also, because these
studies will focus on the effects of dietary fat at the
cellular level, additional insights will be gained regarding
the role of diet in membrane-associated events leading to

biological activity in the cell.

REFERENCES

10.

11.

REFERENCES

Scrimshaw, N.S., Taylor, C.E., & Gordon, J.E. (1968)
Interactions of Nutrition and Infection, WHO Monograph
No. 57.

Chandra, R.K. & Newberne, P.M. (1977) Nutrition,
Immunity and Infection, Plenum Press, New York.

Suskind, R.M. (1977) Malnutrition and the Immune
Response, Raven Press, New York.

Hopkins, G.J. & West, C.E. (1976) Possible roles of
dietary fat in carcinogenesis. Life Sci. 19, 1103-
1116.

Newberne, P.M. (1966) Overnutrition on resistance of
dogs to distemper virus. Fed. Proc. 25, 1701-1710.

Gori, 6.8. (1977) Diet and cancer. J. Am. Diet.
Assoc. 71, 375-379.

Pearce, M.L. & Dayton, S. (1971) Incidence of cancer
im men on a diet high in polyunsaturated fat. Lancet 1,
464-467.

Gammal, E.B., Carroll, K.K., & Plunkett, E.R. (1967)
Effects of dietary fat on mammary carcinogenesis by

7,12-dimethylbenz (a)-anthracene in rats. Canc. Res.
27, 1737-1742.

Mertin, J. & Hunt, R. (1976) Influence of polyun-
saturated fatty acids on survival of skin allografts

and tumor incidence in mice. Proc. Natl. Acad. Sci.
73, 928-931.

Hopkins, G.J., & West, C.E. (1977) Effect of dietary
polyunsaturated fat on growth of a transplantable
adenocarcinoma in C3HAVYfB mice. J. Natl. Cancer
Inst. 58, 753-756.

Hopkins, G.J., Hard, 6.0., & West, C.E. (1978) Carci-
nogenesis induced by 7,12-Dimethylbenz(a)-anthracene

in C3HAVYfB mice: Influence of different dietary fats.
J. Natl. Canc. Inst. 60, 849-853.

35

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

36

Mertin, J. & Hughes, 0. (1975) Specific action of
polyunsaturated fatty acids on lymphocyte transfor-
mation induced by PHA and PPD. Int. Archs. Allergy
Appl. Immun. 48, 203-210.

Mertin, J. (1976) Effect of polyunsaturated fatty
acids on skin allograft survival and primary and

secondary cytotoxic response in mice. Transplantation
2] 3 1'4.

Mertin, J., Mead, C.J., Hunt, R. & Sheena, J. (1977)
Importance of the spleen for the immuno-inhibitory
action of linoleic acid in mice. Int. Arch. Appl.
Immun. 53, 569-573.

Meade, C.J. & Mertin, J. (1976) The mechanism of
immuno inhibition by arachidonic and linoleic acid:
effects on the lymphoid and reticuloendothelial
systems. Int. Arch. Appl. Immun. 51, 2-24.

Hughes, 0., Caspary, E.A., & Wisnieski, H.M. (1975)
Immuno-suppression by linoleic acid. Lancet 2, 501-
502.

Salamar, J.R. & Millar, D. (1975) Linoleic acid as an
immuno-suppressive agent. Lancet 1, 857.

Brock, J. & Field, E.J. (1975) Unsaturated fatty
acids and transplantation. Lancet 1, 1382-1383.

Tonkin, C.H. & Brostoff, J. (1978) Do fatty acids
exert a specific effect on human lymphocyte transfor-

mation in vitro? Int. Archs. Allerg. Appl. Immun. 58,
171-176.

Wene, J.D., Connor, W.E. & DenBesten, L. (1975) The
development of essential fatty acid deficiency in
healthy men fed fat-free diets intravenously and
orally. J. Clin. Invest. 56, 127-134.

Fleming, C.R., Smith, L.M., & Hodges, R.E. (1976)
Essential fatty acid deficiency in adults receiving
total parenteral nutrition. Am. J. Clin. Nutr. 29,
976-983.

Meng, H.C. & Wilmore, D.W: (1976) Fat Emulsions in
Parenteral Nutrition. AMA, Chicago, Illinois.

Weyman, C., Morgan, S.J., Belin, J., and Smith, A.0.
(1977) Phytohemagglutinin stimulation of human lympho-
cytes. Effect of fatty acids on uridine uptake and
phosphoglyceride fatty acid profile. Bioch. Biophys.
Acta 496:155-166.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

37

Watson, A.E. and Mellanby, E. (1930) Tar cancer in
mice. II-The condition of the skin when modified by
external treatment or diet, as a factor in influencing
the cancerous reaction. Brit. J. Exp. Path. 11:311-322.

Visek, W.J., Clinton, S.K. and Truex, C.R. (1978)
Nutrition and experimental carcinogenesis. Cornell
Vet. 68:3-29.

Hopkins, G.J. and West, C.E. (1976) Possible roles of
dietary fats in carcinogenesis. Life Sci- 19:1103-
1116.

Gammal, E.B., Carroll, K.K., and Plunkett, E.R. (1967)
Effects of dietary fat on mammary carcinogenesis by

7,12-dimethylbenz(a)-anthracene in rats. Cancer Res.
27:1737-1742.

Hopkins, G.J. and West, C.E. (1977) Effect of
dietary polyunsaturated fat on growth of a trans-
plantable adenocarcinoma in C3HAVYfB mice, J. Natl.
Cancer Inst. 58:753-756.

Hopkins, G.J., Hard, 0.0. and West, C.E. (1978) Car-
cinogenesis induced by 7,12-dimethylbenz(a)-anthracene
in C3H-4VYfB mice. Influence of different dietary fats.
J. Nat. Cancer Inst. 60:849-853.

Santiago-Delpin, E.A. and Szepsenwal, J. (1977)
Prolonged survival of skin and tumor allografts in
mice on high-fat diets. J. Nat. Cancer Inst. 59:
459-461.

Dayton, 5., Hashimoto, S., and Wollman, J. (1977)
Effect of high-oleic and high-linoelic safflower oils
on mammary tumors induced in rats by 7,12-dimethyl-
benz(a)-anthracene. J. Nutr. 107:1353-1360.

Sell, S. (1978) Tumor immunity: Relevance of animal
models to man. Human Path. 9:63-69.

Meade, C.J. and Mertin, J. (1967) Fatty acids and
immunity. Adv. Lipid Res. 9:127-165.

Miller, A.B. (1977) Role of nutrition in the etiology
of breast cancer. Cancer 39:2704-2708.

Heyden, S. (1974) Polyunsaturated fatty acids and
colon cancer. Nutr. Metab. 17:321-328.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

38

Pearce, M.L. and Dayton, 5. (1971) Incidence of
cancer in men on a diet high in polyunsaturated fat.
Lancet 1:464-467.

Tsang, W.M., Belin, J., Monro, J.A., Smith, A.D.,
Thompson, R.H.S. and Zilkha, K.J. (1976) Relation-
ship between plasma and lymphocyte linoleate in
multiple sclerosis. J. Neur. Neurosurg. Psych. 39:
767-771.

Millar, J.H.D., Zilkha, K.J., Langman, M.J.S., Wright,
H.P., Smith, A.D., Belin, J. and Thompson, R.H.S.
(1973) Double-blind trial of linoleate supplementa-
tion of the diet in multiple sclerosis. Br. Med. J.
1:765-768.

Bates, 0., Fawcett, P.R.W., Shaw, D.A. and Weightman,
D. (1977) Trial of polyunsaturated fatty acids on
nonrelapsing multiple sclerosis. Br. Med. J. 2:932-
933.

Selivonchick, D.P. and Johnston, P.V. (1975) Fat
deficiency in rats during development of the central
nervous system and susceptibility to experimental
allergic encephalomylitis. J. Nutr. 105:288-305.

McHugh, M.I., Wilkinson, R., Elliot, R.W., Field, E.S.,
Dewar, P., Hall, R.R., Taylor, R.M.R., and Uldall,
P.P. (1977) Immuno-suppression with polyunsaturated
ggtty acids in renal transplantation. Transpl. 24:

3- 67.

Davies, D.F. (1976) Immunological aspects of athero-
sclerosis. Proc. Nutr. Soc. 35:293-295.

Minick, C.R., Murphy, G.E. and Campbell, W.G. (1966)
Experimental induction of athero-arteriosclerosis by
the synergy of allergic injury to arteries and a lipid-
rich diet. J. Exp. Med. 124:635-651.

Fiser, R.H., Rollins, J.B. and Beisel, W.R. (1972)
Decreased resistance against infectious canine hepa-
titis in dogs fed a high-fat ration. Am. J. Vet. Res.
33:713-719.

Newberne, P.M. (1966) Overnutrition on resistance of
dogs to distemper virus. Fed. Proc. 25:1701-1710.

Hansen, A.E., Beck, 0., and Weiss, H.F. (1948) Suscep-
tibility to infection manifested by dogs on a low-fat
diet. Fed. Proc. 7:289 (abs.).

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

39

Meng, H.C. and Wilmore, D.W. (1976) Fat Emulsions in
Parenteral Nutrition. AMA, Chicago, Illinois.

O'Neill, J.A., Caldwell, M.D. and Meng, H.C. (1977)
Essential fatty acid deficiency in surgical patients.
Ann. Surg. 185:535-542.

Fleming, C.R., Smith, L.M. and Hodges, R.E. (1976)
Essential fatty acid deficiency in adults receiving
total parenteral nutrition. Am. J. Clin. Nutr. 29:
976-983.

King, M.E., Stavens, B.W. and Spector, A.A. (1977)
Diet-induced changes in plasma membrane fatty acid
composition affect physical properties detected with a
spin-label probe. Biochem. 16:5280-5285.

Sun, G.Y. and Sun, A.Y. (1974) Synaptosomal plasma
membranes: acyl group composition of phosphoglycerides
and (Na+, K+)-ATPase activity during fatty acid defi-
ciency. J. Neurochem. 22:15-18.

Stobo, J.D. (1977) Use of lectins as probes of
lymphocyte structure and function. In: The Lympho-
cyte: Structure and Function-Part II, pp. 493-510,
(J.J. Marchalonis, ed.), Marcel Dekker, Inc., New York.

Ferber, E. and Resch, K. (1977) Structure and physio-
logic role of lipids in the lymphocyte membrane. In:
The Lymphocyte: Structure and Function - Part II,

pp. 593-620, (J.J. Marchalonis, ed.), Marcel Dekker,
Inc., New York.

Schreiner, D.F. and Unanue, E.R. (1976) Membrane and
cytoplasmic changes in B lymphocytes induced by
ligand-surface immunoglobulin interaction. Adv.
Immun. 24:37-165.

Pohl, S.L., Krans, M.J., Kozyreff, V., Birnbaumer, L.
and Rodbell, M. (1971) The glucagon-sensitive adenyl
cyclase system in plasma membranes of rat liver. IV
Evidence for a role of membrane lipids. J. Biol. Chem.
246:4447-4454.

Resch, K. (1976) Lymphocytes as a tool for the study
of cell activation. In: Receptors and Recognition 1:
61-117.

Resch, K. and Ferber, E. (1972) Phospholipid meta-
bolism of stimulated lymphocytes. Eur. J. Biochem. 27:
153-161.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

4O

Ferber, E. and Resch, K. (1973) Phospholipid meta-
bolism of stimulated lymphocytes: activation of
acyl-CoA: lysolecithin acyl-transferases in microsomal
membranes. Biochim. Biophys. Acta 296:335-349.

Ferber, E., DePasquale, G.E. and Resch, K. (1975)
Phospholipid metabolism of stimulated lymphocytes:
composition of phospholipid fatty acids. Biochim.
Biophys. Acta 398:364-376.

Resch, K., Buillon, 0., Gemsa, 0., and Averdunk, R.
(1977) Drugs which disrupt microtubules do not
inhibit the initiation of lymphocyte activation.
Nature 265:349-351.

Northoff, H., Dorken, B., and Resch, K. (1978) Ligand-
dependent modulation of membrane phospholipid metabolism
in ConA-stimulated lymphocytes. Exp. Cell Res. 113:
189-195.

Kosower, E.A., Kosower, N.S., Faltin, F., Diver, A.,
Saltoun, G., and Frensdorff, A. (1974) Membrane
mobility agents: A new class of biologically active
molecules. Biochim. Biophys. Acta 363:261-266.

Bloj, B., Morero, R.D., Farias, R.N. and Trucco, R.E.
(1973) Membrane lipid fatty acids and regulation of
membrane-bound enzymes. Allosteric behavior of
erythrocyte Mg++-ATPase, (Na+, K+)-ATPase and acetyl-
cholineasterase from rats fed different fat-supplemented
diets. Biochim. Biophys. Acta.3ll:67-79.

Reid, M.E., Bieri, J.G., Plack, P.A., & Andrews, E.L.
(1964) Nutritional studies with the guinea pig. X.
Determination of linoleic acid requirement. J. Nutr.
82, 401-408.

Katz, D.H. and Benacerraf, B. (1972) The regulatory
influence of activated T cells on B cell responses to
antigen. Adv. Immun. 15, 1-93.

Jacobs, D.M. and Morrison, D.C. (1975) Stimulation of
T-cell independent primary anti-hapten response in
vitro by Trinitrophenol-Lipopolysaccharide (TNP-LPS).
J. Immun. 144, 360-364.

Yeh, Y.Y. & Leveille, G.A. (1969) Effect of dietary
protein on hepatic lipogenesis in the growing chick.
J. Nutr. 98, 356-366.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

41

Leveille, G.A. & O'Hea, E.K. (1967) Influence of
periodicity of eating on energy metabolism in the rat.
J. Nutr. 93, 541-545.

Steel, R.E.D. and Torrie, J.H. (1960) Principles and
Procedures of Statistics, McGraw-Hill Book Company,
Inc., New York.

Anonymous. (1972) Nutrient requirements of the
laboratory mouse. National Academy of Sciences-
National Research Council, Washington, D.C.

Fraker, P.J., Haas, S.M. & Luecke, R.W. (1977) Effect
of zinc deficiency on the immune response of the young
adult A/J mouse. J. Nutr. 107, 1889-1895.

Kumar, M. and Axelrod, A.E. (1978) Cellular antibody
synthesis in thiamin, biotin, and folic acid deficient
rats. Proc. Soc. Exp. Biol. Med. 157, 421-423.

Willis-Carr, J.I. and St. Pierre, R.L. (1978) Effects
of vitamin 85 deficiency on thymic epithelial cells a T
lymphocyte differentiation. J. Immun. 120, 1153-1159.

Hopkins, G.J. and West, C.E. (1977) Diet-induced
changes in the fatty acid composition of mouse hepato-
cyte plasma membranes. Lipids 12, 327-334.

Zimecki, M. & Webb, D.R. (1977) The role of prosta-
glandins in the control of the immune response to an
autologous red blood cell antigen (Hb). Clin. Immun.
Immunopath. 8, 420-429.

Curtiss, L.K., DeHeer, D.H., & Edington, T.E. (1977)
In vivo suppression of the primary immune response by
a species of low density serum lipoprotein. J.
Immunology 118, 648-652.

Harper, A.E. (1977) Dietary goals - A skeptical view.
Am. J. Clin. Nutr. 31:310-321.

"I11111111117111111.11111“