THE INFLUENCE Of CERYAIN BACTERIAL ANflGENS
ON THE MORPHOGENESIS OF MOUSE THYMUS
AND PERIPHERAL LYMPHOID TISSUE

Thesis fat the Demo. of M. S.
MKMGAN STATE UNIVERSITY
iShirky Mu Sudden
1966

 

[1 Univvcrs'zy {I

 

ABSTRACT

THE INFLUENCE OF CERTAIN BACTERIAL ANTIGENS ON
THE MORPHOGENESIS OF MOUSE THYMUS AND
PERIPHERAL LYMPHOID TISSUE

by Shirley Mae Selden

10 10

Bacterins containing 10 or 2 x 10 organisms per milliliter were
prepared from Staphylococcus m, Iégtobacjillus case}, and Lsctobacillus
acidgphilus. Newborn mice were repeatedly given intraperitoneal injections
of specific bacterins and littermate controls were given 0.85% NaCl solu-
tion. In one part of the study dams were given bacterin injections. Some
newborn mice in following litters were given repeated injections of the
same bacterin, saline solution, or sterile skflm milk. The mice were ob-
served for growth retardation, and the lymphoid tissues from test animals
and controls were studied and compared.

Five runts were produced and a slight to moderate growth retardation
occurred frequently. The normal development of the peripheral lymphoid
tissues did not appear to be altered by the bacterin injections. lesions
produced in this experiment included thickened edematous splenic and renal
capsules containing aggregations of lymphocytes and plasma cells. Foci of
lymphocytes and/or plasma cells, increased amounuaof connective tissue,

and straphy of acinar cells were present in the pancreas of many of the

mice that were given bacterin injections.

THE INFLUENCE W CERTAIN BACTERIAL ANTIGENS ON
THE MDRPHOGENESIS 0F MOUSE THYNUS AND

PERIPHERAL LYMPHOID TISSUE

By
Shirley Mae Selden

“A THESIS

Submitted to
Michigan State university
in partial fulfillment of the requirements
for the degree of

MASTER (F SCIENCE

Department of Pathology

1966

ACKNWLEDGEMENTS

The writer wishes to express sincere appreciation to Dr. D. A. Schmidt,

major professor, for guidance, encouragement, and “special assistance
during the performance of the experiment and the preparation of this
thesis. It is a pleasure to thank Dr. G. L. Healer, Dr. S. D. Sleight,
and Dr. C. C. Horrill for their constructive criticisms and guidance in
writing this, thesis.

The writer is grateful for the assistance of Mrs. M. J. long, Mrs.
B. Bradley, and Mr. R. A. Brooks, for helpful suggestions made by Dr.
W. E. Giddens and Dr. H. A. Richardson, and for the processing of the
large number of tissues by Mrs. H. Sunderlin and the other histology
technicians.

The assistance of Mrs. H. Bennett, Dr. 0. Kaufmann, Dr. G. Jaggard,
and Dr. C. K. Whitehair in obtaining materials and equipment and the
advice of Dr. J. Gill on the statistical analysis are appreciated.

The writer is grateful for the financial assistance of a Michigan
State University Graduate Office Scholarship and expresses appreciation

to those who made it available.

11

TABLE as CMEM‘S
Page
mm!” 1m. 0 O O O O O O O O O O O O O O I O 0 O O O O O 0 O O O O 1

mm a LWUREe O O O O O O O O O O O O O O O O O O O O O C O O 3

e
e
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Development of the thymus.
Involutionofthethymus................... 6
Development of the peripheral lymphoid system. . . . . . . . . 7
Function of the central lymphoid system. . . . . . . . . . . . 7
Models used to study autoimmune processes. . . . . . . . . . . 9
MERIAISANDHETHmS........................14
Mice.............................14
Preparationofinoculmss...................15
Mouse inoculations and observations. . . . . . . . . . . . . . l6
Hematologictechnique.....................l9
Histologic technique . . . . . . . . . . . . . . . . . . . . . l9
RESUIIS...............................22
Growthretardation......................22
Bematologicfindings.....................27
Gross and histologic findings.......... ....... 27
Thymus.........................27
Spleen.........................30
Lymphnodes......................39

Intestine . . . . . . . . . . . . . . . . . . . . . . . 39
Lung..........................42

mart O O O O O O O O O O O O O O O O O O O O O O O O O 42

liver . .

Pancreas.

Adrenal .

Kidney. .

Nitrogen content
DISCUSSIm. . . . . . .
SUMMARY AND CONCLUSIONS
APPENDIX. . . . . . . .
REFERENCES. . . . . . .

mA. 0 O O O O O O O O

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of inoculums.

O O O O 0

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42

45
45
47

55
56
58

62

Table

10

LIST OF TABIES

Inoculation data for mice in Group I. . . . . . . . . . . .
mxul‘tim data for nice in Group II e e e e e s e e .e e e

Sui-nary of histologic findings and observations on growth
rateinmiceafcroupIeeeeeeeeeeeeeeeeee

Smary of histologic findings and observations on growth
r‘teinmic90fcrowlleeeeeeeeeeeeeeeeee

Hematologic findings in mice of Group I . . . . . . . . . .
Hematologic findings in mice of GroupII. . . . . . . . . .

Mean values obtained by point sampling counts of splenic
corpusclesinmiceofGroupI. . . . ... . .. . . .. .

Mean values obtained by point sampling counts of splenic
corpusclesinmiceofGroupII. . . . . . . . . . . . . . .

Results of Dunnett's t-test comparing mean nunbers of
lymphocytes in splenic corpuscles in mice of Group I. . . .

Results of F test comparing mean numbers of lymphocytes in
splenic corpuscles in mice of Group II. . . . . . . . . . .

Pass
. 17

Figure

10

ll

12

LIST OF FIGURES

Grid superimposed over splenic corpuscles illustrating
method used for point sampling counts. . . . . . . . . . .

A lZ-day-old runt that was given 2. .cidgp‘hilus bacterin

injections and littermate control. . . . . . . . . . . . .

A mouse that was given L. acidgghilus bacteria injections

andlittermatecontrol..................

Thymus from 3k-week-old mouse that was given L. case;
bacterin injections, but died of an unrelated infection.

Thymus from runt seen in Figure 2 that was killed at
age of 3 weeks while growth retardation was the only
sign Of 111n°". 0 O 0 O O O O O 0 O O O O O O O O O
Iwrint of thymus from normal mouse on day of birth.

Imprint of spleen from normal mouse on day of birth.

Spleen from 9-week-old mouse that was given L. acidophilus‘
b‘Cterin inj.ct1m0 O O O O O O O O O O O O O O O O O O O O

Imprint of spleen from 9~week-old control mouse that was

the

O

0

given 8‘ 11m salut 1m inject 1m 0 O 0 O O O Q O O O O O O

Imprint of spleen from 9dweek-old mouse that was given

1... acidophilus bacterin injections . . . . . . . . . . . .

Aggregation of mononuclear cells in the lamina propria of

the intestine of a normal mouse on the day of birth. . . .

Pancreas from 9-week-old mouse that was given I. acido-

ghilul bCCCQrin 10.1.Ct1m e e e e e e e e e e e e o e e e

Pancreas from Midweek-old mouse that was given 1:. acido-

Bhilu baCterin ;nJQCt10n. e e e e e e e e e e e e e e e o

Acinar cells of pancreas with prominent intranuclear

inelmim-11kabdie.eeeeeeeeeeeeeeeeeee

vi

Page

21

26

26

31

31

32
32

34

40

40

41

46

INTRODUCTION

In recent years investigators have introduced the concept of 2 lymphoid
systems (Dalmasso gt. 5L, 1963; Sutherland g; a!” 1965). These are the
central and the peripheral lymphoid tissues. The thymus is the prototype
of the central lymphoid tissue, to*which category the bursa of Fabricius
of the chicken and the appendix of the rabbit also belong. The spleen and
lymph nodes are the characteristic peripheral lymphoid tissues. They are
dependent on central lymphoid tissue for the development of normal immuno-
logic capacity in late embryonic and early postnatal life, but are more
autonomous in the mature organism. Research (Cooper gg_gl,, 1966) also
indicates that the thymus and bursa of Fabricius in the chicken are respon-
sible for the development of 2 different morphologic and functional cell
lines in the peripheral lymphoid system.

The immunologic mechanisms involving the thymus and peripheral lymphoid
tissues are of particular interest because of the part they may play in
autoimmunity and connective tissue diseases. Systemic lupus erythematosus
is the human disease considered most typically an autoimmune disease,
(Dameshek _e_t; 2.1;" 1961; Waksman, 1962). The idea has been proposed that
lactobacilli and a cross-reacting antibody may be involved in the cause of
systemic lupus erythemstosus (Stevens, 1964).

Growth retardationwwith reported peripheral lymphoid depletion has
been produced in mice by injections of sterile bacterins. Ekatedt and
Nishimmra (l964)_used bacterins prepared with a variant of the Smith strain

of Staphylococcus aureus and a strain (D 24) of Group A, Type 30 Streptococcus.

2

In the work to be reported here newborn mice were repeatedly injected
with bacterins prepared from Staphylococcus 52532;, Lactobacillus ppppi,‘and
lactpppcillus pcidgphilus. The mice were observed for growth retardation,
and lymphoid tissues from test animals and littermate controls that were
given saline solution injections were studied and compared at various ages.
Lung, heart, liver, pancreas, adrenal, and kidney sections were also
examined for changes which might indicate a reaction of the immunologic
system. In additional studies dams were injected with a specific bacterin.
Some newborn mice in following litters were injected with the same bacterin,
saline solution, or sterile skim milk. Tissues from mice that were not
given any injections were also examined.

The object of this study was to produce runting and lesions similar
to those found in autoimmune processes. The lactobacilli were used because
of the possible connection between lactobacilli and systemic lupus erythema-
tosus in young women. The Staphylococcus mwas used in an attempt to

repeat the runting reported by Ekatedt and Nishimura (1964).

REVIEW W LITERATURE

Early anatomists studied the structure of the thymus. In 1779,
William Hewson told of the lymphocyte distributing function of the thymus
and gave a preview of the concept of 2 different levels of functioning
lymphocytes (Dameshek, 1963). Beard (1899) described the transfonmation
of epithelial cells in the thymus into lymphocytes in the smooth skate
@115 M) and stated that this was the first source of lymphocytes
'within the body. Good 25,31. (1964) attributed the background for much
of the recent research on this subject to the writings of J. August
Hammer.

Despite the fact that much early work was done on the thymus, students
‘were taught for many years that it is a lymphoid organ with unknown function.
In recent years, such has beenlearned about the role the thymus plays in
immunobiology. Current research is concerned mainly with functions in
relation to lymphopoiesis, leukemogenesis, and imunologic mechanisms
(Smith, 1964). As the role of the central and peripheral lymphoid systems
becomes better understood, a close and careful look at the cells that com-
pose these tissues during their development and life history and the effects

of different conditions on these cells becomes essential.

Develppppnt of the thpppp

There are species differences in the development and structure of the
thymus and peripheral lymphoid tissues. ‘Mice are less mature immunologically

at birth than are rabbits and dogs and are more ideal for the study of the

4
development of the thymus and other lymphoid tissues (Good pp_pl,, 1962).
Huch of the description here will be confined to the mouse.

In mammals, the thymus develOps from the 3rd and 4th pharyngeal
pouches in early embryonic life (Good 23.51,, 1962). Embryonic thymus of
the mouse becomes grossly visible at dissection 11 days after fertilization.
At this time it is composed of vesicular epithelial cells surrounded by a
capsule of mesenchyme (Auerbach, 1964). Twelve days after fertilization
the lobes are separated from each other by a considerable distance and are
predominantly epithelial (Auerbach, 1960). The mesenchymal layer is only
1 to 3 cells thick, and the total population of the thymus is about 4000
cells. Hesenchyme and epithelium are distinct prior to the 13th day;
after this they become difficult to separate (Auerbach, 1964). The rudi-
ment at this time does not appear lymphoidal and has not reached its
definitive position (Auerbach, 1961).

During the next 4 days the lobes grow, become lymphoidal in nature,
and migrate posteromedially (Auerbach, 1960, 1961). The total lymphoid
cell population increases rapidly between the 16th and 18th days of embryonic
life. This is followed by a period of stability and then a period of less
rapid increase. A shift of cell types from large to small lymphocytes is
seen in size-distribution studies of the thymus in the 3~day period before
birth (Auerbach, 1964). Histologically the thymus of the newborn mouse
shows development of the cortex and medulla, and is composed of small
lymphocytes (thymocytes) and epithelial stromal cells (Good 35.31,, 1962).

The experimental approach to the analysis of mammalian lymphoid de-
velopment has become practical in recent years because of the development
of precise procedures for cell separation and the improvement of techniques

for transplantation and tissue culture (Auerbach, 1964). Some of the

5
recent work lends support to ideas that were put forth many years ago. In
an experimental study of the origin of cell types in the thymus, Auerbach
(1960) demonstrated that the epithelial cells of the thymus of the mouse
embryo differentiate into lymphocytes. He has also demonstrated that this
lymphopoiesis is dependent upon an interacting mesenchymal factor which
will function across a 20-micron millipore filter barrier. These experi-
mnts were carried out using techniques of transplantation to the anterior
chamber of isologous adult mouse eyes, chick chorioallantoic membrane,
and tissue culture. By using a combination of mouse and chick. thymic cells,
which can be readily distinguished from each other, Auerbach (1964) has
shown that mesenchyme provides an initial inductive stimulus to lymphocyte
formation and the stromal elements of the deve10ping structure, whereas
the epithelium is the source of the lymphoid cells of the developing thymus.

In the mouse the thymus is regularly the only organ with true lympho-
cytes at the time of birth (Good 5; .a_l_., 1962). The growth of the thymus
is most rapid during the first 14 postnatal days on the basis of relative
weight, and during the first 35 days on the basis of absolute weight (Smith,
1964).

There are some species differences in the thymus. The thymus of the mouse
is not divided into lobules as in most other species, but fibrous septa
occur in thymuses grafted subcutaneously in mice. Thymic lobules in vari-
ous species appear uniform in size, especially with regard to the width of
the lymphoid cortex (Netcalf, 1964). In the guinea pig the thymus is lo-
cated in the neck. In man it is predominantly intrathoracic but may extend
upward into the lower parts of the neck. In the opossum, portions are
located in both sites, and in hoofed animals there are separate cervical

and thoracic thymuses (Arnason gt; __., 1962; Metcalf, 1964). In the mouse,

6
Hassall's corpuscles are usually comprised of only 3 or 4 cells and a cen-
tral cavity is not conspicuous (Siegler, 1964). There are several opinions
about the origin of Hassall's corpuscles '(Hamar, 1921; Smith, 1964). The

weight of the human thymus increases from birth to puberty (Bamar’, 1921).

Involution of the thmus

At puberty the mass of the thymic parenchyma begins a gradual decrease.
This process has been termed age involution or physiologic involution
(Banner, 1921; Fisher, 1964). Age involution in the mouse begins at
approximately 6 weeks (htcalf, 1964). A variety of infections, malnu-
trition, tramnatic experiences, Roentgen-ray treatment, or pregnancy will
cause another form of thymic involution referred to as accidental involu-
tion. Adrenocortical hormones cause a marked involution (Hauler, 1921;
Fisher, 1964). Banner (1921) states that the thymus is never found in the
normal condition in subjects who have died of disease.

Age involution involves a reduction in the parenchyma of the organ and
a replacement of interstitial connective tissue by adipose cells. Mitotic
division of lymphocytes continues and the parenchyma remains as a system
of cords gradually growing smaller with age. The occurrence of mitoses,
the presence of Hassall's corpuscles and the power to react by accidental
involution remain (Hemar, 1921). The ethymus probably never completely
disappears. ‘ ‘

Throughout life the size and activity of the thymus are influenced by
complex interacting endocrine factors. Testosterone and estrogen are
thymolytic and thyroidectomy hastens involution. Hyperthyroidism is asso-
ciated with enlargement of the thymus. After adrenalectomy and in Addison's

disease the thymus becomes hyperplastic. In castrated animals the thymus

remains large and involution is not marked (Arnason g; 21..., 1962).

Develgmnt of the Eripheral lymphoid system

The spleen in the newborn mouse lacks development of the red and white
pulp and lymphocytes and plasma cells are virtually lacking (Good at. $1.,
1962). From birth until 3 weeks, it is cmposed mainly of myelopoietic,
erythropoietic, and reticular cells (Parrott .e_t_ 31., 1966). No lymphoid
tissue is found in the intestine at birth. The peripheral lymphoid struc-
ture of mice develops gradually. Its development in the spleen, intestine
and lymph nodes begins during the first week of life and continues for the
next month (Good at 91..., 1962).

Although the thymus of the newborn mouse has the normal adult type of
architecture, some extrinsic antigenic stimulus appears to be necessary for
the normal deve10pment of other lymphoid organs. In germfree animals the

lymph nodes remain rudimentary (Good 9_t. §_]_._., 1962).

Function of the central 1mhoid system

Several hypotheses have been made regarding the function of the thymus.
It is the producer of a large number of lymphocytes (Miller, 1961; Good 91;
915, 1962; Metcalf, 1964; Sainte-lhrie and Leblond, 1964).

It is likely that thymectomy in mice and rats interferes with the de-
velopment of the peripheral lymphoid tissues beyond the stage of maturation
reached at the time the surgical procedure is carried out (Dalmasso .e_t_ 3),...
1963). The bursa of Fabricius in the chicken also plays a key role in the
development of the imunologic system (Good 3; $1., 1962). The function
of the thymus in initiating imunogenesis is not necessarily restricted to
early life. The thymus appears to be essential for complete recovery of
immme mechanisms following sublethal irradiation in the adult mouse

(Miller, 1962).

8 .

Burnet (1962), in an extension of the clonal theory of imunity, pro-
posed that the thymus be regarded as the chief "first level" immunologic
organ, and that the spleen, lymph nodes, bone marrow and local lymphoid cell
aggregations be regarded as "second level" immunologic organs. He hypothe-
sized that the thymus, and other central lymphoid tissues, were the sites
of origin of cells. concerned with the recognition of foreign chemical pat-
terns, and that the thymus releases "uncommitted" lymphoid cells, which
are distributed to second-level lymphoid organs. In the second-level
sites, on apprOpriate stimulation, these cells give rise to descendants
which are imunologically "omitted".

Several investigators give support to the idea that lymphocytes from
the thymus are distributed to peripheral lymphoid tissues (Good .e_t_ $1.,
1962; Arnason g 31., 1962; Parrott, 1966). Autoradiographic studies were
made by injecting tritiated adenosine labeled cells from the thymus and
spleen into neonatally thymectomized mice. Specific thymus-dependent
areas in the spleen and lymph nodes were demonstrated. These areas in
the spleen were located within the follicles inediately surrotmding the
central arterioles. The thymusadependent areas were depleted of small
lymphocytes, and large, pale-stained, nucleated reticular cells were more
prominent. In the lymph nodes of these mice some lymphoid follicles de-
velOped in the outer cortex, but the mid and deep cortex, except for a
narrow band adjoining the medulla, was depleted, of lymphocytes. This work
was interpreted to indicate that the thymus contributes cells directly to
a "mobilizable pool" of lymphocytes, and that another system primarily
responsible for the production of the plasma cell series probably exists

(Parrott g_t_ 51., 1966).

9

In the chicken the thymus-dependent system consists mainly of small
lymphocytes and is responsible for delayed hypersensitivity and homogreft
rejection. The bursa-dependent system is composed of plasma cells and the
larger lymphocytes seen in germinal centers. Functionally it is the ill-uno-
globulin producing system. The thymus-dependent system appears to be the
same in mammals and birds. Tonsils and other lymphoepithelial tissue of
the gastrointestinal tract in manuals may serve the same functions as the
bursa of Fabricius (Cooper 35 3.1., 1966).

Other workers, using lymphocytes labeled with tritiated thymidine and
autoradiographic analysis of their migration in mice, concluded. that the
majority of thymic lymphocytes do not leave the thymus (Matsuyama g; 5.1.,
1966). The functions of the production of a hormone or hormoneswhich
stimulate lymphopoiesis (Metcalf, 1956; Auerbach, 1964), and normal func-
tion of lymphoid tissue (Good 2; $1., 1962) are frequently attributed to
the thymus. Osoba (1965a, 1965b) has shown that neonatal thymic tissue
enclosed in a millipore diffusion chamber, and humoral substances which
have crossed the placental membranes, are capable of restoring the isnuno-
logic responses of a significant number of neonatally thymectomised mice
to normal. Claims of anti-cancer properties, growth-promoting, and growth-
retarding effects, not widely substantiated, have also been made (Osoba,

1965b) .

Models used to study autoimmune processes
Animal diseases which produce conditions with similarities. to human

 

eutoismune diseases include Aleutian disease of mink (Leader, 1964) and
the Combs-positive, hemolyt ic anemia of New Zealand mice (Burnet and

Helms , 1964) .

10

A deficiency of small lymphocytes throughout the body has been the
most frequently reported finding in laboratory models used to produce con-
ditions with similarities to autoimmune processes (Siskind and Thomas,
1959;‘Waksman 55.31., 1962; Ekstedt and Nishimura, 1964; Miller, 1965;
Stastny g£_gl,, 1965). Other findings were not always in agreement.

"Runt disease" results from the injection of immunologically competent
cells into newborn animals. Host'workers have considered it to be an-
immunologic reaction of the grafted cells against a tolerant host (Siskind
and Thomas, 1959; MacKay and Burnet, 1963). "Runt disease’may also be due
to an increased adrenocortical hormone level resulting from a graft-host
interaction (Ekstedt and Nishimura, 1964). Brooke (1964) reported iso-
lating Salmonella typhimurium var. cgpenhagen from the spleens of runts.
Active and passive immunization with this strain of g, typhimmrium did
protect against runting and salmonellosis, but treatment of the infected
spleen cells with sound waves, heat, and x-irradiation did not reduce the
incidence of runting.

The clinical signs reported for "runt disease" have included illness
after an initial period, failure to grow at a normal rate, diarrhea, and
death by 4‘weeks of age. Coombs-positive anemia, splenomegaly, hepato-
megaly with focal coagulative necrosis, and atrophic changes of other
lymphoid tissues were sometimes noted in the runted mdce (Siskind and
Thomas, 1959). Other workers have reported leukopenie, abnormal plasma
electrOphoretic patterns, thrombocytOpenia, negative lupus erythematosus
cell preparations and alopecia. The mice reportedly became hunched,
developed ruffled fur, and were hypothermic before they died (Oliner
t $1., 1961). Runted mice werealso tolerant of skin grafts from the

original cell donor's strain (Siskind and Thomas, 1959).

aim

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and:

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"Homologous disease" is produced in tolerant adult recipients by the
administration of homologous lymphoid cells. The reported changes in adult
rats involved the lymphoid and hematopoietic tissues, skin, joints, and
heart. The lymph nodes were characterized by fewer and smaller follicles
and some replacement with plasmacytoid cells and histiocytes. There was
also an increased prominence of sinusoidal architecture. The thymus medulla
sometimes had an increase in vacuolated, eosinOphilic, reticular cells,
and the cortex was involuted in rats dying between 24 and 27 days after
3 injections of homologous lymphoid cells. Loss of lymphocytes from the
follicles and a relative increase in reticular cells was noted in the spleen.
There was an associated increase in plasmacytoid cells and hemosiderin.de-
posits, but a loss of megakaryocytes. Fibrosis occurred in more advanced
cases. The skin lesions of "homologous disease"grossly and histologically
resembled autografts undergoing rejection in these rats. A migratory and
transient arthritis with a mononuclear reaction occurred. Histologic
changes in the heart involved the valves, myocardium, and epicerdima. '
These lesions were composed of focal edema, mononuclear inflametion, and
serous atrophy of fat. Vasculitis and arteritis with fibrinoid change
were seen occasionally (Stastny g; 31., 1963, 1965).

"Wasting disease" develOps in some neonatally thymectomized mice,
rats, and hamsters and is similar to "runt disease". This wasting syndrome
does not occur in germfree animals and appears to depend upon the presence
of a microbial flora (Cooper 5; a}... 1966). Serum antibody response to
some antigens is depressed following neonatal thymectomy and there is
permanent impairment of the ability to reject skin from donors of foreign
strains. lymphoid cells from neonatally thymectomized mice appear to be

less able to induce a graftoversus-host reaction in appropriate recipients

12
than similar cells from normal donors (Miller, 1965; Parrott and East,
1964; Sherman and Dameshek, 1964). The principal histologic finding in
the lymphoid tissues of neonatally thymectomized animals is a depletion of
small lymphocytes from their usual position in the spleen and lymph nodes
(Waksman g; 9.1., 1962). The age at which thymectomy must be performed in
order to cause this wasting syndrome varies with different species. Thy-
mectomized or bursectomized chickens in which the peripheral lymphoid tissue
has been destroyed by Roentgen-ray treatment provide other useful models
(Copper §_t_ 31., 1966).

Ekstedt and Nishimura (1964) observed runting in neonatal mice while
studying the immunologic reactions to species of staphylococci and strep-
tococci. Repeated intraperitoneal injections of sterile suspensions of the
Smith strain of g, 935223 and a strain (D 24) of Group A, Type 30 Strepto-
gggggg_were made. They reported that, in representative members of the
treated groups, there was loss of delineation of cortex and medulla in the
thymus and fewer lymphoid follicles in the spleen. The follicles present
were poorly developed. Few cells resembling mature lymphocytes were
present in the spleen. They were not able to identify lymph nodes, but
found Peyer's patches in some of the runted mice. No abnormality in
serum electrOphoretic patterns was seen. Circulating antibody could not
be detected in runted animals during the period of treatment with bacterins.
Precipitating antibody could be detected in surviving animals 2 weeks
after termination of the injections. Following termination of treatment
the runted animals gained weight and appeared healthy. Germfree mice were
‘more resistant to this runting than conventional animals. Runting was pro-
duced in neonatal germfree mice treated with a §, 333523 bacterin tO'which a

staphylococcal antiserum prepared in rabbits was added. These studies

13
suggested that small amounts of antibody might be required to complex‘with
the injected antigen to initiate the reaction.

Stevens (1964) suggested that systemic lupus erythematosus and other
autoimmune conditions might be caused by cross-reactions between antibodies
to bacterial polysaccharides and similar chemical groupings of polysaccharides
in body membranes. The antibodies found in systemic lupus erythematosus
reacted with DNA of mammalian, fish, and bacterial origin. This disease
is usually confined t0‘women of child-bearing age, at which time large
numbers of lactobacilli are present in the normal flora of the vagina.

Most experimental models which have been considered similar to systemic
lupus erythematosus do not show the elevation in gamma-globulin found in
this condition, and it has been suggested that they are mediated by lymphoid

cells rather than humoral antibody.

1‘5
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MATERIAIS AND METHODS

£133

Ten pairs of Swiss Webster mice were obtained from Spartan Research
Animals, Incorporated, Haslett, Michigan. The mice were housed as monoga-
mous pairs. The female was allowed to give birth in the presence of the
male, and sometimes mating occurred at postpartum estrus. They were
given water ad libitum and fed a standard mouse and rat ration.1

Newborn mice were cared for by the dam. Mice in excess of 9 to 11
per litter were killed to provide conditions favorable to uniform growth
rates. The mice were sexed and moved to separate cages when they were 3
weeks old.

A total of 161 young mice was used in this study. Bacterin injections
were given to 75, saline solution injections were given to 47, and milk
injections were given to 3. The normal development of the lymphoid tis-
sues at various ages was studied in 36 mice that were not given injections.

Individual mice in each litter were identified by the removal of
different toes. During the early part of the experiment an attempt was
made to identify the mice with various stains, but this method did not
prove satisfactory. The dye could not be seen clearly on the hairless

mice, and the mother often removed the stain.

 

1Purina Laboratory Chow, Ralston Purina Co., St. Louis, Mo.

14

Press!
*

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15
Preparation of inoculums
A hemolytic strain of Staphylococcus m was obtained fras‘Mrs.
Marian Bennett, Veterinary Clinic, Michigan State University. A culture of
lactobacillus gags; was obtained from Dr. Oliver Kaufmann, Department of
Microbiology and Public Health, Michigan State University, and one of 223-

bacillus gcidgphilus from the Dairy laboratory, Philadelphia, Pennsylvania.

The latter was a consercial preparation supplied. in vials.1 One vial was
added to 1 quart of autoclaved skim milk and incubated at 37 C for '24 hours.

2

Subcultures were then made in brain-heart infusion broth. The lactobacilli

cultures were maintained on 312 assay medium2 and the .§_. 93% culture on
nutrient agar.3 These were maintained at 4 C and subcultured at biweekly
intervals.

Bacteria used in preparing bacterins were propagated in brain-heart
infusion broth at 37 C for 18 to 24 hours. The cultures were centrifuged
and the organisms washed once in 0.851 NaCl solution. The washed bacteria
were autoclaved at 115 C, 15 pounds pressure, for 15 to 20 minutes.

The bacteria were counted by the Wright method (Carpenter, 1965), and
the stock suspensions diluted with sterile 0.851 NaCl solution to contain
1010 organism per milliliter. Bacterins subsequently prepared were stan-
dardized by visual cmparison of the turbidity with the previously standardised
suspensions. The bacterins were prepared in sterile vacuum tubesdprepared

for blood collection.‘ In the latter part of the experiment the concentration

 

lFlav-o-lec Flakes, Dairy Laboratories, Philadelphia, Pa.
2Difco Laboratories, Detroit 1, Mich.
3Eugonagar, Baltimore Biological Laboratory, Baltimore, Md.

l'Vacutainer, Becton, Dickinson and Co., Rutherford, N.J.

16

of the L. acidgphilus bacterin was increased to 2 x 1010 organisms per milli-
liter by the removal, after sedimentation, of 1/2 of the total volume.
Each tube of bacterin was stored at 4 C and tested for sterility at the
time it was used.

Milk was reconstituted from a dehydrated preparation1 according to the
instructions on the package. The milk was distributed in small tubes and
autoclaved at 115 C, 15 pounds pressure, for 15 to 20 minutes. Each tube

of milk was stored at 4 C and tested for sterility at the time it was used.
Mouse inoculations and observations

Grm . Mice were injected intraperitoneally with a specific bacterin
or 0.85% NaCl solution as soon after birth as possible and thereafter on
alternate days. The number of mice in each litter and the amount and type
of injection given are listed (TABLE 1). All mice given injections were
weighed every other day for the first 3 weeks and at less frequent inter-
vals after this. The mice were observed daily for normal activity and signs

of growth retardation.

Grog I . Three dams were each given 2 intraperitoneal injections, 4
or 5 days apart, of 0.5 m1. of bacterin. One was given the §_. m bac-
terin, the 2nd the L. gaggi- bacterin, and the 3rd the _I_.. acidophilus bac-
terin. Subsequent litters born to these mice and the injections they
received are indicated (TABLE 2). Observations made of these injected

mice were the same as in the previous group.

 

ICarnation Instant Nonfat Dry mm, Carnation Co., Los Angeles,

Calif.

17

TABLE 1. Inoculation data for mice in Group I.

_‘ - -"

Number of mice and inoculum

 

 

 

101° 2 x 1010
Number organisms organisms
Litter of fiml. '** lml.
number Mflce* bacterin bacterin
If” If" II.“ It" 3 0c: 2
e: re re 33 g
s a r: a 5‘: a ..
a a e 3“ ...» s
H
i
1 7 3 2 --~ --- 2 ~--
4 7 1 3 --- --- 3 ---
5 9 4 3 --- --- 2 ---
6 5 2 2 --- --- 1 ---
8 6 2 2 --- ~-- 2 ---
9 7 --- 3 --- --- --- a#
10W 6 2 2 --- --- 2 «-
2-1 4 --- --- 2 --- 2 ---
2-3 10 --- --- --- 4 4 2
2-5 7 --- --- -°- 3 4 ---
2-7 9 --- --- --- 5 4 ---
2-9 3 -~- --- --- 5 3 ---
2-10 7 ~-- --- --- 3 4 ---
351 4 ~-- ~-- --- 2 2 ---
Total 96 14 17 _ 2 22 35 6

AAA—L x ' I A ._.|_l _._‘ __ ' .1 .— 4 _- . 4 _4 ... —_L'

 

*Does not include mice that died during first 3 days or those removed
to limit size of litter.
**Intraperitonea1 injections of 0.1 ml. amounts'were given on alternate
d‘y‘. ‘ . - m '
***Mflce were given 0.05 ml. until 1-2 weeks old and 0.1 m1. thereafter.
'#Injections‘were given on the 1st day, but the stain was removed by
the dam and identification was not recognizable.
# All mice in this litter were ill with an unidentified infection.

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18

 

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Hemat olggic technique

Packed cell volumes, total leukocyte counts and differential leukocyte
counts were performed on tail blood from a group of test mice and controls.
Absolute lymphocyte values were determined using the total leukocyte count
and the relative number of lymphocytes. Bone marrow imprints were stained

with Wright's stain followed by Giemsa stain.

Histolggic technique
Mice were killed with diethyl ether at variousages. The thymus,

spleen, cervical and axillary lymph nodes, Peyer's patches, lung, heart,
liver, pancreas, adrenals, and kidneys were fixed in 10% buffered formalin
and saved for histologic examination.

Tissues were embedded in paraffin. The processing time for dehydra-
tion, clearing, and infiltration was shortened to 20 minutes in each solu-
tion in an attempt to prevent excessive hardening of the tissues. Sections
were cut at 5 to 6 microns and stained with hematoxylin and eosin.

Imprints of thymus, spleen and axillary lymph nodes were made. These
isprints were stained with a modified Shorr's stain and with Wright's
stain followed by Giemsa stain. Imprints were air dried before staining
by the Wright-Giemsa technique. Imprints for the Shorr's stain were
fixed while wet and stored. in an alcohol-ether solution until stained.
The modification of Shorr's stain used in this experiment may be found in
the appendix.

The spleen, Peyer's patches, and lymph nodes were examined for deple-
tion of small lymphocytes and the presence or absence of germinal centers.

The point sampling method was adapted to count the number of small

20
lymphocytes and larger cells in the area imediately surrounding the cen-
tral arterioles in the splenic corpuscles (Er'ankfl, 1955; Weibel, 1963).
The cells were counted in those splenic corpuscles in which the central
arteriole seen in the section was near the center of the follicle. A grid
with 64 intersections was‘ placed in the eyepiece of the microscope. The
central arteriole was centered and the number of intersection points
falling on various cell types was recorded (Figure l). The oil imersion
objective was used. Intersection points were counted as falling on small
lymphocytes, reticular cells, granulocytes, and interstitial spaces.
Large lymphocytes, endothelial cells, and early granulocytes were not

differentiated and were counted as reticular cells in this examination.

.m _-
“jafl‘.”

G5: égfl

 

Figure 1. Grid superimposed over splenic corpuscles
illustrating method used for point sampling counts. "Test
mouse that was given _L_. casei bacterin injections (left)
and control that was given saline solution injections
(right). Hematoxylin and eosin. 7501!.

S S R R S R R L S R L R S L L L
L L S R S R R L S L S L R R R S
R R R R S L L R R S R S R R L S
L R S S S R R R L R L R R S L S
R S S R R R R R L L R R R R L S
R R S R S R L L L L L R S S L R
S R R S S S S R L L L L S R R L
R S R S R S L L S L L L L L L S

Intersection points counted as falling on lymphocytes
(L), reticular cells (R), interstitial spaces (S) or
granulocytes (G).

d ....3. :5... a. .51.} 4,344.32:
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RES UIIS

Growth retardation
The number of mice in which there was no growth retardation, slight

to moderate growth retardation, and marked retardation are listed (TABLES

3 and 4). Those having slight to moderate growth retardation generally
gained steadily each day but less than their littermate controls. Mice
with marked growth retardation gained at a much slower rate and will be

referred to as runts.

A runt that was given the L. acidophilus bacterin and a littermate . 4
control given saline solution injections are shown (Figure 2). This photo-
graph was taken on the 12th day of age. At this time the runt weighed _
2.49 Ga. and the littermate control 7.50 Gm. The runt appeared very active
and the dam was observed nursing this mouse several times. It did not
assume the hunched position described by other workers (Oliner .e_t_ 5.,
1961) as one of the characteristics of "runt disease". This mouse and
the littermate control were killed on the 21st day of age. The runt gained
weight steadily for the first 17 days but lost 0.38 Gm. in the 4 days be-
fore it was killed.

Another runt that died at the age of 10 days is sham (Figure 3). _
This runt had also been given the L. acidgphilus bacteria and the litter-
mate control pictured with it, saline solution. This runt had made no
significant weight gain since birth and remained hairless.

Five runts‘were produced in this experiment. Three runts occurred in

Group I; l was given the L. casei bacterin and 2 the L. acidophilus bacterin.

Two runts occurred in Group II; 1 was given the L. casei bacterin and the

fi

22

23

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Number of Mice

 

TABLE 4. Smry of histologic findings and observations on growth rate in mice of Group II.

 

24

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 2. A 12-day-old runt that was given
.L. acidoghilus bacterin injections (bottom) and
littermate control. Notice lack of hair develop-
ment and difference in size.

IWWWWW'
Figure 3. A mouse that was given'g. gcido-
ghilus bacterin injections (top) and littermate

control. The former died at the age of 10 days,
remained hairless, with no significant weight gain.

 

27

other the g. M bacterin. Mice with growth retardation during the first
4 weeks later gained at a rapid rate and became normal in appearance, al-
though the bacterin injections were continued.

During the experiment several mice became ill between the ages of 2 and
4 weeks. The test mice were affected more often than the controls, but both
mice injected with saline and those without injections became sick and some
died. The sick mice frequently assumed a hunched position and lost weight.
Some had signs of diarrhea. Bacteriologic cultures and gross and micro-
scopic examinations of tissues did not reveal the cause of the illness.
Because there was weight loss and periods of no weight gain with this ill-
ness, mice that were affected are included with those showing slight to

moderate growth retardation (TABLES 3 and 4).

Hemat olgic f indingg

The mean packed cell volumes, total leukocyte counts and absolute lympho-

cyte counts, and the ranges obtained for each, in mice that were given spe-
cific injections are listed (TABLES 5 and 6). The number of mice on which
the blood tests were performed and their ages are also given in the tables.
LymphOpenia was not found, and anemia was indicated in only 2 mice. No

abnormalities were seen on examination of the bone marrow imprints.

gross and histolggic findings

The number of mice with different histologic findings for each type of
injection are also listed (TABLES 3 and 4). The histologic findings in

each organ will be discussed separately.

Thmus. No significant gross findings were present in the thymuses
of mice killed if growth retardation was the only sign of ill health. In

mice that died as the result of an unidentified infection, “the thymuses

were usually smaller than normal. The normal delineation between cortex

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and medulla, present at the time of birth and thereafter, was not seen in
9 test mice and in l saline control (Figure 4). All the mice with this
loss of normal architecture had signs of illness several days before death
or before being killed. The thymus of the runt (Figure 2) killed when
growth retardation was the only visible effect of the bacterin injections
retained the normal delineation between cortex and medulla (Figure 5), as
did all other mice with growth retardation_,that were killed before other
signs of illness were seen. Each normal appearing thymus was composed of
a cortex with tightly packed small lymphocytes and a medulla composed of
epithelial and stromal cells, few lymphocytes, and small Hassall's corpuscles.
Cilia-lined cysts, sometimes seen in the thymus of the mouse (Smith, 1964),
were present in 2 mice. A few adipose cells were sometimes seen in sec-
tions from mice 12 weeks old, but involution was not marked in any of the
thymuses examined.

Cellular detail was better in imprints than in sections. The cells
seen in the imprints were mainly small and medium size lymphocytes
(Figure 6). No differences between the thymic cells of control and test

mice were seen.

£21333. The spleen in the newborn mouse was composed mainly of erythro-
poietic and myelopoietic cells (Figure 7) with a connective tissue capsule.
Splenic corpuscles, composed of pale-staining reticulum-cells, were first
seen in a normal mouse at the age of 6 days. One section from a normal
lO-day-old mouse contained “small darkly-stained lymphocytes in the splenic
corpuscles._

Flat, grayish-white areas on the capsule of, the spleen were seen by

_ gross examination in 14 test animals. Thickening of the capsule and edema,

3l

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' .. .1;

 

Figure 4. Thymus from 3’g-week-old mouse
that was given 3. casei bacterin injections but
died of an unrelated infection. Loss of normal
delineation between cortex and medulla with con-
gestion. Hematoxylin and eosin. 188x.

 

Figure 5. Thymus from runt seen in Figure
2, that was killed at the age of 3 weeks while
growth retardation was the only sign of illness.
Normal delineation between cortex and medulla.
Hematoxylin and eosin. 188x.

32

u‘/ l

K.

‘\
V ‘7‘

 

Figure 6. Imprint of thymus from nonmel
mouse on day of birth. Predominant cells are
small and medium size lymphocytes. Wright-Giemsa.

750x.

' ml

 

".V

Figure 7. Imprint of spleen fran normal
mouse on day of birth. Predominant cells are
erythropoietic and myeIOpoietic. Wright-Giemsa.
750K.

33
noted microscOpically, were present in the spleens of 50 test animals
(35 out of 55 'in Group I and 15 out of 27 'in Group II). Manyof the
thickened capsules contained lymphocytes and plasma cells. Aggregations
of lymphocytes were sometimes found surrounding blood vessels (Figure 8).
Neutrophils, eosinOphils, and macrophages were also present in sane in-
stances. This lesion was more frequent in the mice injected with the
_I_._. acidgphilus bacterin than in those given other injections.

The mean nunbers of lymphocytes, interstitial spaces, and reticular
cells obtained from splenic corpuscles‘using the point sampling technique
in mice of Groupsl’I and II are listed (TABLES 7 and 8). The nunber of
lymphocytes counted was lower in the test mice than in saline controls,
and the number of reticular cells was higher.

Dunnett's t-test (Dunnett, .1955) was used to test the significance of
the mean numbers of lymphocytes in the mice that were given the various
bacterins in Group I as compared to controls that were given saline so-
lution. The results of this analysis are given (TABLE 9). The decreased
number of lymphocytes was significant at the 12 level in mice that were
given the L. acidgphilus bacterins.

An F test for non-orthogonal comparisons (0stle, 1963) was used to
test the significance of the mean numbers of lymphocytes obtained from
splenic corpuscles in Group II. The results of this analysis are listed
(TABLE 10). The decreased number of lymphocytes was significant at the
12. level in mice that were given the §_. m bacterin versus the saline
controls, when the dam was given previous injections of the§. m bac-
terin. The decreased number of lymphocytes was significant at the 52
level in mice that were given the L. acidophilus bacterin versus the saline

controls, when the dam was given previous injections of the g. ggidophilg

31$

 

Figure 8. Spleen from 9-week-old mouse
that was given 1,. acidgphilus bacterin injections.
Notice thickened edematous capsule containing
lymphocytes and plasma cells surrounding blood
vessel. Hematomylin and eosin. 188x.

35

TABLE 7. ‘Hean values obtained by point sampling counts of splenic
' corpuscles in mice of Group I.

 

 

Inoculum
0.852 1010 organisms 21:10lo organisms
NaCl lml. bjagterins (ml, bacterin
solution §_. aureus L. casei L. acidghilus L. acidgphilus
Nmnber of
mice 33 12. 17 2 21
Mean number
of lympho-
cytes 21.8 17.2 19.1 8.5 16.1
Mean number
of inter-
atitial
spaces 16.7 14.7 14.0 10.0 15.8
man number

of reticu-
lar cells 24.9 31.8 30.2 44.5 32.3

36

 

 

 

 

 

 

 

 

 

 

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37

TABLE 9. Results of Dtmnett's t-test comparing mean numbers of lympho-

cytes in splenic corpuscles in mice of Group I.

 

 

 

Inoculum , t
Saline versus 1010 organisms/m1.
_L_. acidgphilus bacterin 3.13*
Saline versus 2 x 1010 organisms/m1. _
L. acidOphilus bacterin 3.80*
Saline versus 1010 organisms/m1.
L. casei bacterin 1.69
Saline versus 1010 organisms/ml.
fi. aureus bacterin 2.16

 

*‘Significant at the 1% level.

:

QH-_ ...-M.

Tao-fi

cal. ~ CHI.

Tut.~ 7L.—

TIM-h

L.‘

38

TABLE 10. Results of F test comparing mean numbers of lymhocytes in
splenic corpuscles in mice of Group II.

 

44g

Bacterin dam received

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum test mice received F
S. aureus _S_. aureus 1
versus 8.52*
saline g. aureus
L. casei L. casei
versus 0.38
saline L. casei
L. acidghilus L. acidgphilus .
versus 6.20**
saline L. acidgphilus
§_. aureus L. casei L. acidgphilus -
versus 6.92**
§. aureus L. casei L. acidophilus
L. casei L. acidghilus
versus 0.94
L. casei L. acidghilus
L. acidgphilus L. acidgphilus L. gcidgphilg
versus + 1.42
saline milk L. acidgphilus
L. acidghilus L. gcidgphilus
versus 0.20
milk L. acidophilus

 

*Significant at the 11 level.
“Significant at the 57. level.

but!
of th

each

can
nice
area

m:

by I
It ‘
lyu
Pa:
in

an:

mi

tel

39

bacterin; and in mice that were given the §_. m bacterin versus the total
of those given the L. 933;; and L. acidgghilus bacterins, when the dams of
each group were given the corresponding bacterins.

Spleen imprints of control mice given saline solution contained areas
composed primarily of lymphocytes (Figure 9), whereas some iaprints from the
mice injected with the different bacterins contained few or none of these
areas. Erythropoietic and myelopoietic cells were the predominant types

present in the spleen imprints of these test mice (Figure 10).

Lmh nodes. Cervical and axillary lymph nodes could usually. be found
by gross examination in mice 3 weeks of age or older. The youngest age
at which a cervical lymph node was found was 6 days. Enlarged edematous
lymph nodes were occasionally present. The presence of germinal centers
was variable. Germinal centers were observed in 63% of the control mice
in Group I and in 581 of the controls in Group 11. They were seen in 351
and 50% of the test mice in Groups I and II, respectively. Plasma cells
were prominent in only a few, and areas with many fibroblasts were found in
some lymph nodes of both test and control animals. No abnormalities were

seen in lymph node imprints.

Intestine. An aggregation of large, pale-staining mononuclear cells
was present in the lamina propria of 1 mouse on the day of birth (Figure
11). This mouse had not been given any injections. Follicles containing
some reticulum cells and some lymphocytes were seen in the intestine of
a 3-day-old mouse. Lynphoid nodules were regularly observed in normal
mice 10 days of age or older. Peyer's‘ patches were usually visible at
gross examination of mice 3 weeks of age or older and were found in both

test and control animls. The numbers of test and control mice in which

 

Figure 9. Imprint of spleen from 9-week-old
control mouse that was given saline solution injections.

Area composed predaninant 1y of lymphocytes. Wright-
Giemsa. 750X.

. I!
r A =

 
          

s’ a
"_, .
nor .5? i "
Figure 10. Imprint of spleen from 9~week-old
mouse that was given L. acidgphilus bacterin injec-
tions. The predaninant cell types are erythrOpoietic

and myelopoietic with only occasional lymphocytes
present. Wright-Giemsa. 750X.

41

 

Figure 11. Aggregation of mononuclear
cells in the lamina propria of the intestine of
a normal mouse on the day of birth. Hematoxylin
and 0081B. 750xe

42
Peyer's patches and germinal centers were present are listed (TABLES 3
and 4).
Cross sections of parasites were found in the lumens of 271 of all

mice. These were interpreted to be gpicularis tetraptera.

_hfl. Congestion of varying degrees, usually slight, was seen in
large numbers of both test and control mice. The diethyl ether used for
killing the mice may have caused this congestion. Congestion (and atelec-
tasis was marked in mice that were sick and died during the experiment,
but it did not appear extensive enough to have been the cause of death.
Some of the lungs contained focal aggregations of neutrophils and others

lymphoid follicles. Slight emphysema was also present in a few instances.

Heart. In 1 test mouse and 2 controls a mild endocarditis and slight

mononuc lear inf i ltrat ion were not ed .

Li_v_e£. Grossly visible surface lesions were seen on the liver in
8 mice. Seven of these were test mice and 1 was a saline control. These
lesions appeared as raised white areas.

Histologic lesions were more frequent in test mice, but were also
found in control mice. In Group I, histologic lesions were present in 427.
of the test mice and 2020f the controls; and in Group II they were present
in 552 of the test mice and 61 of the controls. These lesions were of 2
types. One was a granulcnatous type reaction with macrophages and giant
cellswhich, in a few instances, surrounded calcified or amorphous material.
The other type of lesion found on the surface of the liver was composed of
an increased amount of connective tissue, lymphoid cells, plasma cells, a

few neutrOphils and eosinOphils.

43
Necrosis was not prominent in the liver. Pleomorphic nuclei were a
common finding, and binucleated hepatic cells were occasionally seen.
HematOpOietic cells were found in the livers of normal mice until the
age of 3 weeks. After this age their presence was variable. Hematopoietic
cells were seen in the livers of some test mice 12 weeks old. The incidence
of hematopoietic cells in the livers of control mice was lower than that in

the test mice (TABLES 3 and 4).

Pancreas. Several histologic changes were seen in the pancreas. In-
creased loose areolar connective tissue surrounding lobules of acinar cells
was present in 632 of the test mice in Group I and 857. of the test mice in
Group II. The incidence of this finding was also higher in those mice
given the L. acidghilus bacterins (951 in Group I and 1002 in Group II).
There was an apparent relationship between atrophy of the acinar cells and
increased connective tissue. AtrOphy of the acinar cells was prominent in
642 of the test animals in Group I which had increased connective tissue and
in 522 of those in Group II. Focal aggregations of lymphocytes and plasma
cells were a cannon finding (Figure 12). There were also areas in which
lymphocytes were diffusely scattered among the acinar cells. A few neutro-
phils and eosinOphils were seen in acne sections. In some areas the atro-
phied acinar cells appeared to be replaced by adipose tissue. In 1 mouse,
aggregations of lymphoid and plasma cells surrounded some of the islets;
however, this was an exception, and the islets generally did not appear to
be involved. Large cells with eccentric nuclei and globules of casino-
philic material were present, in 1 section. These were thought to be plasma

cells with Russell bodies (Figure 13).

 

Figure 12. Pancreas from 9-week-old mouse
that was given L. acidg hilus bacterin injections.
Notice focal collection of plasma cells and lympho-
cytes. Acinar cells appear atrophic (compare with
Figure 14). Hematoxylin and eosin. 750x.

     

Figure 13. Pancreas from 4’5-week-old mouse”
that was given L. gcidghilus bacterin injectiOns.
Notice lymphocytes and plasma cells. Large cells
with eccentric nuclei (arrow) contain globules

of eosinophilic staining material and are plasma
cells with Russell bodies. Hematoxylin and eosin.
750x.

4S

Sonm increased connective tissue was also present in the mice that
were given injections of sterile milk. The dam had been given injections
of the L. acidgphilus bacterin previous to the birth of this litter. In-
creased connective tissue was not noted in the pancreatic tissues of any
control mice given saline solution; however, a small amount was found in 2
mice, 7 and 12’: weeks of age, which were given 1 injection on the day of
birth and none thereafter. The identification of the lsttinjection is
unknown}

Adipose cells and a few lymphocytes were seen in the pancreatic tis-
sue of some of the control mice given saline solution. A lymphoid follicle
was found in the pancreas of a 5-day-old mouse that was given '1 injection1
on the day of birth and in a 395~week=old mouse that had not been given any
injections. The latter had appeared sick for several days.

Prcminent inclusion-like bodies were seen in the nuclei of acinar
cells of both test and control animals (Figure 14). These inclusion-like
bodies were noted in 472 of the test animals and 492 of the controls in
Group I and 401 and 532, respectively, in Group II. They were also found

in mice that were not given any injections.

gr nal. Sections of adrenal glands were not adequate to confirm
cortical hyperplasia. Accessory adrenals were found in 3 mice. Neutro-

phils were seen in the medulla of l adrenal gland.

Kidney.4 Slight congestion and proliferation of reticuloendothelial.
cells were present in both control and test mice. A thickening of the cap-

sule was present in this organ in some test mice. This was visible at

 

1The stain used to identify the mouse was removed by the dam.

46

 

Figure 14. Acinar cells of pancreas with
prominent intranuclear inclusion-like bodies
(arrow). Hematoxylin and eosin. 75ax.

 

47
gross examination in 9 of the test mice, and always appeared more prominent
on the left kidney than on the right. Microscopically lymphocytes, plasma
cells and occasionally neutrophils and macrophages were seen in the thickened

capsules. This lesion resembled those seen in the capsule of the spleen.

Nitrggeg content of inoculums

It was desirable to know if the milk used for injection contained more
or less nitrogen than the bacterins. Results of semi-micro Kjeldahl nitrogen
determinations1 indicated that the 2 x 1010 organisms per milliliter L.
gcidgphilus bacterin contained 0.04% N, the 1010 organisms per milliliter

L. casei and §_. aureus bacterins each contained 0.01% N, and the milk con-

tained 0.601 N.

 

fi

lThe semi-micro Kjeldahl nitrogen determinations were made by'Mrs.
Betty Bradley, Department of Animal Husbandry, Michigan State University.

DISCUSS ION

Marked growth retardation was seen in a few instances following re-
peated intraperitoneal injections of g. m, L. 9531, or L. acidgphilus
bacterin, and a slight to moderate growth retardation occurred frequently.
The results do not indicate that the incidence of growth retardation was
increased in those litters in which the dam was given previous injections
of the specific bacterins.

The results of this experiment do not agree with those previously re-
ported in which marked depletion of lymphoid tissues was the predominant
finding in runted mice (Ekstedt and Nishimura, 1964). The thymuses of
severely or moderately runted mice in the work reported here did not have
a loss of delineation between cortex and medulla unless the mice appeared
to be ill from some infectious process. Loss of delineation between cor-
tex and medulla, along with decreased size, was the main change reported
by Ekstedt and Nishimura (1964) in the thymuses of the rents they produced
with bacterin injections.

Other workers have used the number of splenic corpuscles or their
size as criteria for denoting lymphoid depletion in the spleen (Ekstedt
and Nishimura, 1964; Osoba, 1965). In the work reported here it was ob-
served that the number of splenic corpuscles seen in a longitudinal section
and their diameter depended on where the section was cut. In cross section
the mouse spleen is (triangular in shape. A section cut longitudinally
through one of the spices would have a single bead-like strand of splenic

corpuscles. A section cut longitudinally through the thickest part would

48

49
have from 2 to 4 rows of splenic corpuscles. The number and size of splenic
corpuscles seen in runted mice did not appear to be markedly decreased.

A more exact method for evaluation of lymphoid depletion was desired so
the point sampling method was used. The use of this method demonstrated that
the mean lymphocyte counts of splenic corpuscles for the test mice were lower
than for the control mice and, correspondingly, the umber of reticular cells
in the same area was higher in the test mice. The number .of interstitial
spaces was variable. The difference in the number of lymphocytes was sta-
tistically significant in Group I in mice that received the L. scidgphilus
bacterins but not in those that received the L. 3594.. or the g. m bac-
terins. In Group II the difference was most significant between the saline
controls and test mice that had received g. m bacterin following pre-
vious injection of the dam with §_. m bacterin. Thisfinding might
indicate that the dam produced antibodies to the _S_. m and these anti-
bodies were passed through the placenta, so that they later complexed with
the antigen in the neonatal mice and participated in initiating this reaction
in the spleen. Although these findings indicate that there could be a sig-
nificant decrease in the mmber of lymphocytes present in the splenic cor-
puscles, they are not conclusive. A relative difference in the distribution
of the cells in the areas counted could result frma a swelling of the white
pulp, with edema around the central arteriole. This is one of the changes
which occur in the spleen following the intravenous administration of large
amounts of antigenic materials (Congdon, 1964; _Cosgrove and Upton, 1965).
Examination of the lymph nodes and Peyer's patches indicated that the bac-
terin injections did not delay or prevent their normal development.

LymphOpenia wasnot noted, and evidence of anemia was present in only

2 instances. The leukocyte counts seemed to vary with age, and a more

50
complete study of normals would be necessary to evaluate this finding.
Peripheral blood obtained from the tail vein contains a higher number of
white blood cells (mean 21,510) than ventricle blood (man 3,717), and
there is also some variation among strains (Fekete, 1941).

It is possible that the amount of material remaining in the peritoneal
cavity of the mouse on initial injection might influence the final outcome
in this experiment. Difficulty with leakage of the injected material from
the site of injection was experienced with mice less than 1 week old. In
an attempt to prevent this a smaller volume of inoculum was used. The con-
centration of the L. acidgphilus bacterin was increased so that the number
of organisms previously used was contained in a smaller volume. With this
change in technique a severely runted mouse resulted (Figure 3) and led to
the use of the increased concentration of L. acidgphilus.

The use of milk injections in some controls was introduced to determine
if the growth retardation produced was the specific result of a reaction to
bacterial substance or if it would result with any injected protein. The
small number of mice injected with milk, and the fact that the dam had re-
ceived previous injections of L. acidghilus bacterin prevented any valid
conclusions being drawn from this part of the experiment.

The increased growth and edema of connective tissue and the large num-~
bars of plasma cells and lynphocytes present in sect ions of pancreas and on
the surface of the spleen and kidneys are of particular interest. The high
incidence of these lesions in the mice receiving the bacterin injections
suggests that they are a manifestation of some reaction to the injected
substance.

The exact nature of this reaction in the pancreas is, open to specula-

tion. The presence of lymphocytes and plasma cells is suggestive of a

51

delayed hypersensitivity reaction (Waksman, 1959). Several mechanisms
appear possible under the conditions of this experiment. The first is that
an immune reaction may occur against antigenic components of organisms when
they are introduced into a site where they normally do not occur, or against
organisms found in the normal flora under adverse conditions. If there is
a similarity between the injected material and some tissue substance, a
cross-reaction might occur'which could lead to this type of lesion in the'
pancreas. Assuming that an antibody formed against the injected bacterins
could cross-react with ce11.sembranes in the pancreas, a progressive inter-
action of antibody and tissue substance could occur. It is also possible
that a small amount of cross-reacting antibody might become attached to the
acinar cells and alter the cells enough that they could become antigenic.
Many bacterial cell‘walls, including lactobacilli and streptococci groups
APP, contain N-acetyl-glucosamine. Mammalian.membranes contain hyaluronic
acid, which is composed of N-acetyl-glucosamine and glucuronic acid. Thus,
it is possible that a shuilarity between the sequence of N-acetyl-glucosmmine
end-groups in mamalian membranes and bacterial cell walls is thevbasis for
an immune reaction resulting in a cross-reacting antibody (Stevens, 1964).

Other possible causes for the pancreatic lesions in the experiment
might be related to traumatic injury resulting from repeated injections,
or injection of the bacterin into the pancreas itself. Traumatic injury
might result in the release of tissue substances normally not in contact
with the ismune apparatus of the body. The released substances might
attract antibody-forming cells and the bacterin might serve as an adjuvant.
The possibility of the bacterin being injected directly into the pancreas
appears remote, but if this did occur, a local inflammatory reaction could

follow. Another possibility is that a localized peritonitis may have

52
developed as a result of the multiple injections; however, if this were true
these lesions would have occurred in the saline controls also. This was not
observed.

The facts that the first injections were given at an age when the mouse
is presumed immunologically tolerant and that lactobacilli are notably poor
antigenic substances may detract from the idea that this is an immune reaction.
However, imune tolerance is a variable finding, and it may be that "cyto-
philic" antibodies are produced in response to lactobacilli, and suitable
procedures for identifying them.have not yet been introduced. The tissue
reactions reported in this experiment suggest that further work including
serum protein, imunologic, and histochemical studies might be of interest.

The mode in'which the pancreatic lesions might lead to growth retarda-
tion is also open to consideration. Atrophy of acinar cells in this experi-
ment might in some way interfere with the exocrine secretions of the pancreas
which include amylase, lipase, and trypsin. In the adult mouse this may not
cause any notable ill effects, but in a rapidly developing young mouse, it
may interfere with the processes of digestion or absorption to such an
extent as to cause growth retardation. Hellman and Jordan (1965) studied
the effects of pancreatic resection in mice at the age of 4 weeks. They
found no demonstrable change in growth rate, absorption of fat, nitrogen or
calcium. In the present‘work the growth retardation was produced at an
earlier age.

Although the islets of Langerhans did not appear to be affected, there
could be alterations involving insulin production which would also cause a
delayed development. Other mechanisms which could be considered include
the possible effects that the bacterin injections might have on humoral
substances produced by the thymus and the effects of stress on adreno-

cortical hormones and the pituitary.

53

Senile and lipomatous atrophy of the pancreas reportedly occur in mice
(Tuffery, 1963), and it is of interest to note human conditions which have
similarities to the lesions of this experiment. Polyarteritis nodcsa has
been reported to result in an infiltration of eosinophils, lymphocytes, and
plasma cells around the involved vessels in the pancreas (Anderson, 1964).
Arterioles and capillaries are involved in systemic lupus erythematosus,
and there may be secondary exudation of inflammatory cells, composed pri-
marily of lymphocytes and plasma cells. lesions identical to those in poly-
arteritis nodcsa have been found in serum sickness and hypersensitiveness
to sulfonamides (Boyd, 1961). No fibrinoid changes were noted in the sec-
tions of pancreas, spleen or kidney in this experiment, but the increased
connective tissue contained many small vessels, and foci of lymphoid cells
frequently appeared to be clustered around them.

In human pancreatitis, predisposing factors such as cholelithiasis and
alcoholism are not implicated in 25 to 302 of the cases. Histologic changes
include an intense lymphoid infiltration in the early phases followed by
progressive fibrosis and destruction of the acini (MacKay and Burnet, 1963).
Pancreas-specific isoantibodies have been demonstrated by gel diffusion and
tanned cell hemagglutination techniques, but autoantibodies have not been
demonstrated. Pancreas-specific antibodies have also been demonstrated in
cases of carcinoma and cystic fibrosis of the pancreas and in diabetes
mellitus (MacKay and Burnet, 1963; Humphrey and'White, 1964). Pancreas-
specific isoantibodies have been produced experimentally in rabbits and
rhesus monkeys, and the isoantigens in rabbit pancreas have been located
in the cytoplasm.of the apex of the acinar cells using fluorescent antibody

techniques (Hetzgar, 1964).

54

It was unfortunate that infection and illness occurred among the mice
used in this experiment. However, this did indicate 3 important facts:

(1) mice injected.with bacterins were more susceptible to infection than the
saline controls; (2) increased amount and edema of the connective tissue and
lymphocyte and plasma cell foci in the pancreas did not occur in control
once which had signs of illness; and (3) loss of delineation between the
cortex andrmedulla did not occur in the thymuses of severely runted mice
without other signs of illness but did occur in control mice that appeared
ill.

The effects that the Agpicularis tetraptera infestation may have had
in the experimental results cannot be measured. The significance of the
intranuclear inclusion-like bodies in the acinar cells of the pancreas is
not known. Intracytoplasmdc inclusion bodies are known to occur in the-
acinar cells of the pancreas and other epithelial cells in infectious ectro-
melia 00ingle, 1941). These coincidental’findings do point out the value
of using specific-pathogen-free or germfree animals in long-term investi-

gations and those involving reactions of the immune system.

SUMMARY AND CWCLIB IONS

Newborn mice were repeatedly given intraperitoneal injections of bac-
terins prepared from Staphylococcus 525223, Lactobacillus £333_, and Lagggr
bacillus gcidgphilus. In one part of the study dams were injected with a
specific bacterin. Some newborn mice in following litters were given re-
peated injections of the same bacterin, saline solution, or sterile skim
milk. Runting was seen in a few instances and a slight to moderate growth
retardation occurred frequently. The bacterin injections did not appear to
delay or prevent the normal development of the poripheral lymphoid tissues
and the thymus was not morphologically affected. An apparent quantitative
decrease in the number of lymphocytes surrounding the central arterioles of
splenic corpuscles was demonstrated. Lesions produced in this experiment
included thickened edematous connective tissue in renal and splenic cap-
sules, with some aggregations of lymphocytes and plasma cells. Large
numbers of plasma cells and lymphocytes, increased amounts of connective
tissue, and atrophy of acinar cells were present in sections of pancreas
of many test animals. The pancreatic changes may have resulted from.some
form of an immune reaction and may be the mechanism causing the growth

retardation.

55

Fixation:

APPENDIX

Modification of Shorr's Stain for Inprints

Imprints should be fixed while wet in a 50/50 alcohol-ether

solution; The imprints should be fixed for at least 15 minutes

but can'be stored in the fixing solution until a time con-

venient for sta ining.‘

Staining procedure:

Results:

1.

Pass imprints through absolute and 952 alcohols to
distilled water.

Stain in Harris's hematoxylin for 30 seconds.

Rinse in water to blue.

Place imprints in Shorr's staining solution for 1 minute.
Wash in 95% alcohol.

Rinse several times in absolute alcohol, clear 'in xylene,

and mount in Permount.

This is a good stain to demonstrate nuclear detail. Chromatin

masses stain blue, cytoplasm a light green, and erythrocytes

orange red .

56

57
Solutionsz‘ (Armed Forces Institute of Pathology, 1960)

Shorr ' s Staining Solution

AICOhOI, smOOOOOOO.......O00.0.00;...‘00000.001000000 m1.
-Biebr1Ch scanlet.00.0000......0......00.000000000000500 p.
orange GOOOOOOOOOOOOOO...............OOOOOOOO.OOOOOOZSO P.
F‘Bt:8reen’>FCFOOOOOOOOOO'O‘U‘OOOOOOOOOOOOOOO000.00.00.075 an.
thphOtmssti-c aCi-dOOOOOO....OOOOOOOOOOOOOOOOOOOOOSOO 8n.
Phosphomolybdic acid..........................,...0'.50_0 gm.
Glacial acetic acj-diO...OO.O.OIOOOOOOOOOOO:OOOOOO‘OIOOOO ‘1.

Harris 's Rematoxylin

Hmtoxylin cmtalseaseseessoeeeeaeeeeoeeeaeaoeeeoSeo ”a
AlCOhOl, 951......”.............o................50.0 n10
Mil-mar p0t”81m all-noseaeoeeaeeeae‘eeeeeeeeelooao 8n.
D13tilled WOtOruo.........u”.......o;........1000.0 m1.
tut-cutie uideeeeaaebeeeeaeeeaaeaeeeeaeeeeea'oaaoa‘eszas 8.0

 

REFERENCES

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VITA

The author was born April 12, 1931, in Grand Blanc, Michigan. Elemen-
tary education was obtained at the South Mundy School, and she graduated
from Linden High School, Linden, Michigan, in 1949. She was employed in a
Flint physician's office from 1950 to 1953, and in the laboratory of the
former Goodrich General Hospital, Goodrich, Michigan, from 1953 to 1956.
She received an A.S. degree from Flint Junior College, Flint, Michigan, in
1956, and a B.S. degree in Madical Technology from Wayne State University,
Detroit, Michigan, in 1958. Upon completion of 12 months of clinical
training at Detroit Receiving Hospital, Detroit, Michigan, the author wrote
and passed the national examination of the Registry of Medical Technologists,
American Society of Clinical Pathologists, in July of 1958. She was em-
ployed as a medical technologist at McLaren General Hospital, Flint, Michigan,
from July, 1958, until June, 1962, and as chief medical technologist at
Clinton mmorial Hospital, St. Johns, Michigan, from June, 1962, until
November, 1965. She began graduate study at Michigan State University on
a part-time basis in September, 1962, and as a full-time student in November,
1965. She is a member of the American Society of Mbdical Technologists, the
Michigan Society of Medical Technologists, and the Michigan Association of

Blood Banks .

62

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