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The spontaneously diabetic BB rat

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Title:
The spontaneously diabetic BB rat a model of autoimmunity and immunodeficiency
Creator:
Elder, Melissa Ellen, 1955-
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Language:
English
Physical Description:
ix, 183 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Autoantibodies ( jstor )
Diabetes ( jstor )
Diabetes complications ( jstor )
Diabetes mellitus ( jstor )
HLA antigens ( jstor )
Lymphocytes ( jstor )
Rats ( jstor )
Spleen cells ( jstor )
Type 1 diabetes mellitus ( jstor )
Autoimmune Diseases ( mesh )
Dissertations, Academic -- Pathology -- UF ( mesh )
Pathology thesis Ph.D ( mesh )
Rats, Inbred Strains ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 168-182.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Melissa Ellen Elder.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
028968634 ( ALEPH )
08643834 ( OCLC )

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THE SPONTANEOUSLY DIABETIC BB RAT: A MODEL OF
AUTOIMMUNITY AND IMMUNODEFICIENCY












By


MELISSA ELLEN ELDER


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY





UNIVERSITY OF FLORIDA


1982

























Dedicated to the memory of

Mr. Mellow













ACKNOWLEDGEMENTS


First and foremost, I wish to thank my parents,

Wayne and Nora Elder, for their lifelong love and support.

I would like to also especially acknowledge my sister,

Melanie Elder, for her love, understanding, and friendship.

I wish to likewise thank Phillip Ruiz for his affection and

help during my graduate education.

I wish to express my deep appreciation to Dr. Noel

Maclaren for his expert guidance and assistance during the

course of this research. The members of my committee,

Dr. Juan Scornik, Dr. Ammon Peck, Dr. Edward Wakeland,

Dr. Paul Klein and Dr. Christopher West, are also thanked

for their interaction, assistance and interest in this

project. Other members of the Pathology faculty to whom I

express special thanks for their help are Dr. William Riley,

Dr. Raul Braylan and Dr. Arthur Kimura. I am grateful to

the Department of Pathology for the financial support I

received during my graduate career.

I would like to express my gratitude to Lee Glancey,

Tom McConnell and Edith Rosenbloom for their invaluable,

technical assistance and support in the completion of these

studies. Their excellent work aided in the development and

completion of this project. My thanks also go to everyone

else associated with the Lab. The help of Crystal Grimes


iii








and Flo Jordan in the preparation of this manuscript and

other papers is deeply appreciated. I would also like to

thank the graduate students of the Department of Pathology

for their encouragement and friendship.

Finally, these acknowledgements would not be complete

without mentioning some of the special people whose friend-

ship and support have been important in the completion of

this endeavor: Joan Appleyard, Steve Noga, Dan Cook,

and Art Alamo.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS ............... .................... iii

COMMONLY USED ABBREVIATIONS.......................... vi

ABSTRACT..... ............................ .............. viii

INTRODUCTION ................ .......................... 1

Insulin-dependent Diabetes (IDD)................ 2
The BB Rat........................................ 15

SPECIFIC AIMS................ ............................. 18

MATERIALS AND METHODS............................... 21

RESULTS.......... .. ...... .... .. ........... ........ 32

DISCUSSION............... .... ... ...... .............. 149

The BB rat as a model of IDD and organ-
specific autoimmunity.......................... 149
The BB rat as a model of
immunodeficiency............................. ... 156

REFERENCES................... ........... .......... .. 168

BIOGRAPHICAL SKETCH................................. 183









Commonly Used Abbreviations


BB
BB/O
BB/W
BB x WF
BB x Lewis


OC
Con A
cpm


BioBreeding
BB rats obtained from Ottawa, Ontario
BB rats obtained from Worcester, Mass.
Cross of male BB rat with female WF rat
Cross of male BB rat with female Lewis rat


Degrees Centigrade
Concanavalin A
Counts per minute


HLA D region-associated antigen


Fl
F2
FCS

HLA

Ia
ICA
ICSA
IDD
Ig
IgG
IgM
IL 2


MLC
2-ME
MRC OX6

MRC OX8


n.d.

PBS
PCA
PHA
PWM

RT.1

S.D.
SMA


First filial generation
Second filial generation
Fetal calf serum


Major histocompatibility complex of man

Immune response-associated antigen
Islet cell antibodies (cytoplasmic)
Islet cell surface antibodies
Insulin-dependent diabetes
Immunoglobulin
Immunoglobulin Class G
Immunoglobulin Class M
Interleukin 2 or T cell growth factor

Mixed lymphocyte culture
2-mercaptoethanol
Monoclonal antibody which defines rat Ia-
positive cells
Monoclonal antibody which defines rat
cytotoxic/suppressor T cell subset

Not done

Phosphate-buffered saline
Gastric parietal cell autoantibodies
Purified phytohemagglutinin
Pokeweed motigen

Major histocompatibility complex of the rat

Standard deviation
Smooth muscle antibodies









WF Wistar Furth
W3/13 Monoclonal antibody which defines rat T
lymphocytes
W3/25 Monoclonal antibody which defines rat
helper T cell subset

x Irradiated


vii














Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


THE SPONTANEOUSLY DIABETIC BB RAT: A MODEL
OF AUTOIMMUNITY AND IMMUNODEFICIENCY


By


Melissa Ellen Elder


August, 1982


Chairman: Noel K. Maclaren, M.D.
Major Department: Pathology

The BB rat is presently the best available animal

model for human insulin-dependent diabetes (IDD). IDD in

the BB rat may result from autoimmunity since it is accom-

panied by lymphocytic inflammation of the pancreatic islets

and is preventable by immunosuppression. Antibodies to pan-

creatic islets (ICA) and other endocrine tissues in BB rats

were sought as evidence for an autoimmune etiology for IDD.

ICA were not detected, while smooth muscle and gastric par-

ietal cell autoantibodies (PCA) were frequently identified.

Although functional abnormalities of gastric parietal cells

were not noted, PCA-positive rats had evidence of lympho-

cytic gastritis. PCA appeared during the age period that


viii








the animals were developing IDD. Thus, the BB rat has an

autoimmune diathesis of which IDD may be only one result.

Immunocompetency of the BB rat was also studied.

Severe T lymphopenia was observed in all BB rats, irrespec-

tive of age or presence of IDD, while numbers of B cells

and immunoglobulin levels were normal. Both the numbers of

helper T cells and cytotoxic/suppressor T lymphocytes were

depressed, and an inversion of the ratio of helper T cells

to cytotoxic/suppressor T cells occurred in all BB rats with

maturity.

Concomitantly, profound impairments of T cell-mediated

immunity were noted to mitogenic stimulation and in allo-

responses. BB lymphocytes produced IL 2 normally; however,

irradiated WF cells and Con A supernatants did not restore

BB responses, suggesting that BB lymphocytes may have

defective responses to helper factors such as IL 2. In

contrast to BB peripheral T cells, BB and WF thymocytes re-

sponded equally well to mitogens. Whereas BB thymic histol-

ogy was normal, BB spleens and lymph nodes were severely

depleted of T lymphocytes. Thymocytotoxic autoantibodies

were also detected in many BB rats. These findings suggest

that the defect in T cell immunoresponsiveness may be post-

thymic or peripherally acquired. Although the BB rat is an

intriguing model of autoimmunity and immunodeficiency, no

clear relationship between the immunoincompetence and IDD

is known to date.














INTRODUCTION


Autoimmune diseases result from loss of self toler-

ance which leads to an immune response by the individual to

autologous antigens and subsequent cellular and tissue

destruction or other effects. Such diseases are separable

into two groups, i.e. organ specific disorders such as the

autoimmune endocrinopathies, and autoimmune diseases that

are systemic and not confined to any one organ such as

systemic lupus erythematosus.

A disease is generally considered to be an organ-

specific autoimmune disorder if there are mononuclear in-

filtrations of the affected organ or tissue, organ-specific

autoantibodies, and a tendency for more than one of these

diseases to occur simultaneously in individual patients (1).

Such organ-specific autoimmune diseases include chronic

lymphocytic thyroiditis, Graves' disease, Addison's disease,

acquired hypoparathyroidism and insulin-dependent diabetes

(IDD).

Most autoimmune endocrinopathies are associated with

disturbed frequencies of certain HLA antigens, especially

an increased frequency of the immune response gene HLA DR3

(1,2). HLA DR4 is also increased in IDD (1,2). For some









diseases of this group, such as chronic lymphocytic thyroid-

itis, however, no definitive HLA associations have been

found.

The presence of organ-specific autoantibodies in the

patient's serum does not mean the patient has clinical dis-

ease nor that the antibodies are actually the cause of tis-

sue damage. However, because the autoantibodies are very

specific and are found in low frequencies in the general

population, they are considered to be markers or indicators

of the presence of an autoimmune disease process occurring

in the patient, regardless of whether this process leads to

overt disease (3,4).

Proof of a primary role for autoimmunity in the patho-

geneses of these putative autoimmune endocrinopathies re-

mains difficult to obtain. Current etiologic concepts for

such autoimmune diseases most probably must include discus-

sion of the relationship between environmental triggers

and immune responsiveness (as defined by HLA DR antigens)

in the genetically predisposed individual.



Insulin-Dependent Diabetes (IDD)

It has been suggested that human IDD may result from

the autoimmune destruction of the insulin-secreting beta

cells of the pancreatic islets. Evidence to support an

autoimmune etiology for IDD has included observations of

mononuclear infiltrations in pancreatic islets of patients

who have died suddenly after onset of IDD (5,6). These









infiltrative lesions were predominantly composed of

lymphocytes, with few polymorphonuclear leukocytes, eosino-

phils or plasma cells present and are referred to as

"insulitis" (7,8). The frequency of insulitis in patients

with IDD is variable, with reports ranging from extremely

rare (9,10) to greater than 50% (8,11) in patients with re-

cent onset of IDD. Lymphocytic infiltrations of pancreatic

islets have not been demonstrated in patients with IDD of

greater than one year duration, in noninsulin-dependent

diabetics or in normal controls (12).



Islet Cell Autoantibodies in IDD. The presence of anti-

bodies to pancreatic islet cells in sera of patients with

IDD and polyendocrinopathies has been extensively described

(13,14). Cytoplasmic-reactive islet cell autoantibodies

(ICA), detectable by indirect immunofluorescence on normal

pancreatic tissue, react with all cell types of the pancre-

atic islets in addition to beta cells (15-17), which is in

contrast to the specific loss of beta cells seen in IDD.

ICA do not cross react with gastrointestinal cells secreting

hormones also found in pancreatic islets, such as glucagon

and somatostatin (2). These antibodies are exclusively of

the IgG class, usually fix complement and are believed to

react with a microsomal membrane lipoprotein found normally

in all islet cells (13,14,18-20). The frequency of ICA in

patients with IDD varies according to the patient's race, to

the time elapsed after clinical diagnosis of IDD, and has








been found to precede development of the clinical disease

(13,15,21,22). Neufeld et al. determined the frequency of

ICA to be approximately 74% in Caucasian children tested

within three months of onset of IDD, whereas ICA were

detected in less than 50% of patients three years after

diagnosis (15). However, 10-15% of IDD patients have been

noted to have persistent ICA for many years and have been

shown to have higher frequencies of associated thyroid,

gastric and adrenal autoimmune diseases as well as higher

frequencies of HLA B8-bearing haplotypes than patients with

IDD who become ICA-negative over similar periods (19,23).

In contrast, black insulin-dependent diabetics have only

about half the frequency of ICA in relation to duration of

IDD as do Caucasian patients with IDD, suggesting that much

of the IDD among black patients may be different from IDD

seen in Caucasian populations (15,24). In addition,

children who are ICA-negative at onset of IDD rarely become

positive for these antibodies later (2,3). ICA have also

been demonstrated in approximately 3-5% of nondiabetic

relatives of IDD probands (15,23) and in about 0.5% of

healthy controls (15,23).

Observations that ICA-positive family members of

patients with IDD and noninsulin-dependent diabetics with

ICA tend to become insulin-requiring with time (22,25,26)

suggest that these antibodies may be of clinical value in

predicting the subsequent development of IDD (22). However,

a causal relationship between ICA and IDD remains to be








proven. A direct role for ICA in the pathogenesis of IDD in

fact seems unlikely, especially since these antibodies react

with a shared antigen found in the cytoplasm of all islet

cell types. Furthermore, autoantibody molecules are not

normally considered to be able to cross membranes of living

cells to effect damage. In addition, experimental evidence

to suggest that ICA do not cause IDD includes findings that

transfers of ICA from human patients with IDD to mice have

not resulted in IDD in these animals (19,27), nor has placen-

tal passage of ICA resulted in the development of IDD in

newborn infants or even to affect neonatal insulin secretion

(19). Observations that ICA titers decrease with time after

clinical onset of IDD is probably related to the progressive

loss of the relevant antigen, whatever the disease

mechanism.



Islet Cell Surface Autoantibodies in IDD. Autoantibodies

reactive to antigens on pancreatic islet cell membranes

(ICSA) have also been demonstrated in sera of patients with

IDD (21,28-30). Using cell surface immunofluorescence tech-

niques or 125I-labeled protein A assays, ICSA have been

detected either by reaction with cultured human insulinoma

cells (29), beta cells isolated from dispersed rat or mouse

pancreatic islets (21,31,32) or human fetal pancreatic islet

cell cultures (33). Analagous to ICA, ICSA have been de-

tected in about 67% of children at onset of IDD and decrease

in frequency within the first year after diagnosis (34,35).








In contrast, ICSA are reported to be more common than ICA in

controls and seem to possibly occur independently of ICA in

patients with IDD (36).

ICSA in sera from patients with IDD have been shown to

be cytotoxic to mouse pancreatic islets in vitro (37), how-

ever some nondiabetic sera without ICSA were also

cytotoxic to these cells. Sera from IDD patients have also

been shown to have complement-mediated cytotoxic effects on

hamster islets (38) and rat islets (39), correlating with

the detection of ICSA in the serum. In addition, ICSA-

positive sera in the presence of complement have been dem-

onstrated to cause increased chromium release from labeled

rat islet cells (40,41) while sera containing ICA alone did

not have such effects (40). Difficulties with this study

include defects in the method of ICA detection, and findings

that 25% of ICSA-positive sera from nondiabetic first degree

relatives were also cytotoxic to islet cells.

There has been little convincing evidence to suggest

that ICSA react specifically with pancreatic beta cells.

Indeed in one study, cytotoxicity of sera with detectable

ICSA was shown to not be restricted to pancreatic beta

cells, but also affected a somatostatin-producing tumor line

(42). However, Dobersen and Scharff has recently demon-

strated the preferential lysis of rat beta cells with

minimal killing of other types of islet cells by ICSA-

containing diabetic sera using double-label immunofluores-

cence techniques (28). In addition, eleven of twenty-one








sera from diabetic patients with ICSA were shown to be able

to suppress glucose and theophylline-stimulated insulin

release but not glucagon release in vitro by dispersed mouse

islets (43). Paradoxically, ICSA have been demonstrated to

have a stimulatory effect on basal insulin release from

cultured mouse islets (44). These results are consistent

with the primary role for ICSA in beta cell destruction seen

in IDD proposed by some investigators (28), but much more

substantial evidence is needed.



Cell-Mediated Autoimmunity in IDD. No consistent general-

ized defects in cell-mediated immunity as defined in terms

of lymphocyte responsiveness to phytohemagglutinin (PHA) (45-

48) and in the enumeration of lymphocyte subpopulations

(45,49-51) appear to be present in well-treated patients

with IDD. However, poorly controlled diabetics have been

observed to have depressed mitogenic responses to PHA in

comparison with matched adequately treated IDD patients and

healthy controls (47,52) suggesting that the metabolic

derangements of IDD have an adverse effect on T lymphocyte

responsiveness. Other studies have indicated that specific

antipancreatic cell-mediated immunity may be observed in

patients with IDD (45,47). Nerup et al. demonstrated

significant inhibition of migration of leukocytes from

insulin-dependent diabetics in the presence of porcine

pancreas (53) or fetal calf pancreas (54,55) homogenates.

Abnormal migration inhibition was especially noted in those









patients with IDD for less than one year, but was also

demonstrated in some noninsulin-dependent diabetics.

Positive leukocyte migration inhibition in IDD patients has

also been observed using human pancreas homogenates (56) or

insulinoma extracts (57) as antigens. Concomittantly, de-

layed hypersensitivity skin reactions to porcine pancreatic

suspensions were seen by Nerup in patients with IDD who

exhibited inhibition of leukocyte migration using the same

antigen (53). In order to examine the possible role of cell-

mediated immunity to insulin and thus to beta cells in the

pathogenesis of IDD, MacCuish (47) and others (58) were able

to demonstrate significantly greater blastogenesis, as

measured by 3H-thymidine incorporation, by lymphocytes

from diabetic patients when cultured with bovine or porcine

insulin than by lymphocytes from control patients.

Huang and Maclaren were able to demonstrate specifi-

cally enhanced cytoadherence and cytotoxicity of human in-

sulinoma cells in vitro by peripheral blood lymphocytes from

children with IDD (59). Specific insulinoma-tumor cell

cytotoxicity, as measured by eosin exclusion, was seen both

with and without added patient sera, but was not obtained

using either lymphocytes from patients with systemic lupus

erythematosus or autoimmune thyroid disease, or with differ-

ent tumor lines as targets (59,60). Peripheral blood lymph-

ocytes from twenty-one out of thirty-three patients with IDD

were shown to inhibit insulin release by rat islets to glu-

cose and theophyllin (61), while no inhibition of insulin








secretion was noted by lymphocytes from noninsulin-depend-

ent diabetics or controls. Significantly increased levels

of circulating killer (K) cells, classified as low affinity

E-rosette forming cells, were also found in 57% of newly

diagnosed insulin-dependent diabetics (62). These levels

returned to normal within twelve months from diagnosis of

IDD. Raised K cell numbers were accompanied by signifi-

cantly enhanced levels of antibody-dependent cell-mediated

cytotoxicity activity to chromium-labeled human erythro-

cytes sensitized with antierythrocyte antibodies in many

IDD patients (63).

Several investigators have suggested that defects in

suppressor T cell activity were present in patients with IDD

(64,65). In one study, Concanavalin A (Con A)-activated

lymphocytes from patients with IDD poorly suppressed allo-

geneic mixed lymphocyte cultures when compared to suppressor

activity demonstrated by Con A activated lymphocytes from

controls (64). In contrast, Slater et al. recently found a

statistically significant increase in Con A activated

suppressor T cell activity in thirteen patients with IDD

(66). These measurements of cell-mediated immunity,

however, are difficult to analyze even in healthy people,

and biologically significant results with diabetic patients

are even more questionable because the effects and

complications of IDD itself affect lymphocyte function and

responsiveness.








In a provocative study, indium-labelled autologous

peripheral blood leukocytes from two of three newly diag-

nosed patients wtih IDD were observed by CAT scanning to

become distributed in the same patient's pancreas after

intravenous reinjection (67). No pancreatic localization

using the same procedure was noted in scans of patients with

other diseases. Whether the lymphocytes specifically homed

to the pancreas or were trapped there nonspecifically as a

consequence of inflammation, these results suggest the

presence of pancreatic insulitis in these patients.



Associated Autoimmune Diseases. Other organ-specific

autoimmunities are found with increased frequencies in IDD

(1,19,68,69). These diseases mainly involve the thyroid,

the adrenal and the parietal cells of the gastric mucosa.

In studies by Riley and colleagues of more than 500 chil-

dren with IDD, 17% of the patients had detectable thyroid

microsomal antibodies and 5% had overt thyroid disease in

comparison with less than 2% of the control population even

having thyroid antibodies (15). Adrenal autoantibodies and

Addison's disease were also more frequent in Caucasian chil-

dren with IDD, being found in 2% and 0.5% of insulin-depen-

dent diabetics respectively, compared to adrenal antibodies

being detected in 0.7% of matched controls (15,69). Final-

ly, gastric parietal cell autoantibodies were detected in

approximately 9% of patients with IDD and in only 1% of

matched controls (19). Children with other autoimmune








endocrinopathies in addition to IDD have even higher fre-

quencies of associated organ-specific autoantibodies (19).

For example, patients with both IDD and thyroid microsomal

antibodies have augmented incidences of adrenal antibodies

to about 6% (19). Organ-specific autoantibodies and auto-

immune disease are not found in increased frequencies in

noninsulin-dependent diabetics (19).



HLA Associations with IDD. Singal and Blajchman first

reported that IDD was associated with disturbed frequencies

of HLA antigens and noted an increase in HLA B15 in these

patients (70). Nerup and coworkers later documented

statistically significant increases in HLA B8 and HLA B15 in

patients with IDD in comparison with matched controls, while

no HLA differences were seen between controls and noninsulin

-dependent diabetics (71). Subsequent studies have both

confirmed and extended these observations (72-74). Cudworth

(75) and others (76) have postulated that there are two HLA

haplotypes associated with increased relative risks for

IDD: HLA Al (A30) B8 (B18) Cw3 DR3 and HLA A2 B15 (B40)

DR4. The primary association of IDD seems to be with the

HLA DR antigens and secondarily due to linkage disequilib-

rium with the HLA A and B antigens (2,72,73,77). HLA DR3

is found in 36-59% of IDD patients compared to between 11-

24% in normal controls (72), while HLA DR4 is seen in 32-

58% of insulin-dependent diabetics compared to 16-28% in the

general population (72). Patients with multiple autoimmune









endocrinopathies have an even higher frequency of HLA Al B8

DR3 haplotypes and especially HLA DR3, suggesting that a

gene predisposing for general organ-specific autoimmunity is

associated with HLA DR3 (2,45). The risk of developing IDD

is highest for HLA DR3/DR4 heterozygotes, implying the

possibility of the existence of at least two hereditary

susceptibility genes for IDD associated wtih HLA genes--one

associated with HLA DR3 and the other with HLA DR4 (34,45,

77,78). One haplotype has been suggested to possibly render

protection against IDD because it is decreased and virtually

absent in Caucasian patients with IDD: HLA A3 (All) B7 DR2

(2,75,77,78), although the low frequencies of HLA DR2-bear-

ing haplotypes in IDD may be due in part to the increased

frequencies of HLA DR3 and HLA DR4 (79). Black patients

with IDD as a whole do not have as significantly disturbed

frequencies of HLA DR3 and DR4 antigens as Caucasian child-

ren with IDD (24). However, those black diabetic children

who are positive for ICA seem to invariably type HLA DR3 or

HLA DR4 (24), which is probably due to the impact of Cauca-

sian IDD genes in the black genome by racial admixture (24).

Families with multiple members affected by IDD tend to show

an excess of individuals typing for HLA DR3 or HLA DR4. In

addition, several investigators (80-82) have observed

significantly disturbed frequencies of several complement

factor B alleles in insulin-dependent diabetics (2). The

mechanisms through which HLA antigens, especially the immune

response associated HLA DR antigens, affect susceptibility
to IDD remain to be elucidated.








Mechanism of Inheritance of IDD. The apparent linkage

between IDD and HLA has made it possible to analyze the

segregation of HLA haplotypes as markers for IDD in multi-

plex families with two or more siblings with IDD. Several

investigators have suggested IDD to be a recessive disease

(83,84), while others believe IDD to be dominant. In either

case, reduced penetrance would need to be invoked to explain

the segregation of IDD with HLA haplotypes (85,86). Indeed,

the penetrance or the percentage of people carrying the IDD

susceptibility gene(s) that actually have clinical IDD seems

to be quite low, approximating 15-30% in multiplex families

(86). Speilman has recently suggested a hypothesis of dif-

ferential susceptibility to IDD depending on dosage of IDD

susceptibility gene(s), rather than simple dominant or re-

cessive inheritance (87). Heterozygosity of IDD alleles

would result in significant susceptibility to IDD, but homo-

zygosity for the gene(s) would be associated with even

greater risk (penetrance) for the disease. However, because

of uncertainty as to the random frequency of IDD genes in

the general population, the crossover rates between HLA and

IDD gene loci (if not one and the same), and the probable

genetic heterogeneity of the disease, estimations of the

mode of transmission of IDD are difficult to make (19).



Environmental Factors in the Pathogenesis of IDD. As the

concordance for IDD in monozygotic twins has been shown

by Leslie and Pyke to be at most 50% (88), environmental








influences have been implicated as at least secondary

factors in the development of IDD (86). Support from animal

models for a viral role in the pathogenesis of IDD includes

evidence that encephalomyocarditis virus causes a diabetes-

like syndrome in susceptible SJL mice, associated with beta

cell loss and pancreatic insulitis (89).

There is limited direct evidence for such viral partic-

ipation in human IDD. Coxsackie B4 virus was isolated from

the pancreas of a child with fatal diabetic ketoacidosis

which proved capable of inducing insulitis and hyperglycemia

in some strains of mice (90). An epidemiological relation-

ship between annual cycles of infection with Coxsackie B4

virus in people and the seasonal incidence of IDD has also

been found (91), and a high frequency of IDD in children who

have suffered severe congenital rubella or mumps infection

has been reported (45,92). One study has suggested that the

frequency of IDD in patients after infections with these

viruses is positively associated with HLA B8 and thus by

linkage disequilibrium with HLA DR3 (93). If confirmed,

such findings would suggest a relationship between the

immune response of an individual to a virus and the

development of IDD. Several investigators suggest that

these viruses do not usually cause IDD, but instead may

trigger IDD by a stress effect in the susceptible predia-

betic individual with preexisting insulinopenia. To this

effect, it is notable that no epidemics of IDD have been

reported in several large registry studies in the United

States, London and Denmark.








The BB Rat

The spontaneously diabetic BB rat was first recognized

in 1974 in an outbred colony of Wistar rats at the BioBreed-

ing Laboratories (94). These rats have subsequently been

bred for the IDD phenotype. BB animals which have been

formally inbred for seven to twelve generations develop

spontaneous severe IDD at about 70-120 days of age, which is

characterized by insulinopenia, marked hyperglycemia,

ketoacidosis, weight loss and an absolute requirement for

exogeneous insulin (95). The BB rat most closely resembles

human IDD of all animal models known to date. IDD develops

in genetically susceptible male and female rats with equal

frequencies (95,96) and is thought to be inherited either as

a single autosomal recessive gene with reduced penetrance

(94) or as multiple genes.

Moderate insulitis is seen in the pancreatic islets of

BB rats at the time of diagnosis of IDD, resembling the pan-

creatic lesions seen in recently diagnosed human patients

with IDD (96,97). Insulitis has also been observed in non-

diabetic animals (94). Immunochemical staining of pancre-

atic islets from diabetic BB rats for insulin content re-

veals depletions of beta cells, especially marked in pan-

creases from BB rats with longstanding IDD (98,99). Such

pancreatic islets are comprised almost exclusively of glu-

cagon, somatostatin and pancreatic polypeptide-secreting

cells (94).









The IDD seen in the BB rats is probably not due to

recognized infectious agents, since animals raised in a

gnotobiotic environment, such that they did not develop

antibodies to any bacteria, viruses or parasites, did not

have decreased incidences of IDD (100). However, this study

was based on results from only one litter and, in any event,

does not rule out the possibility of a role for a vertically

transmitted virus in the etiology of IDD in these rats.

The typical insulitis lesions and the genetic predispo-

sition for IDD suggest that IDD in the BB rat may be the

result of beta cell autoimmunity. Evidence to support such

a hypothesis includes recent findings of ICSA in twelve of

fourteen diabetic BB rats using a 125-labelled protein A

assay (101). Other data include observations of the

reduction in frequency of IDD in susceptible BB rats

after administration of antilymphocyte serum (102),

neonatal thymectomy (103,104) or bone marrow reconstitu-

tion (105). However, the experimental designs were ques-

tionable since the studies were performed between groups of

litters rather than within litters. Because the incidence

of IDD varies from 0-60% in any one litter to the next, the

observed decrease in frequency of IDD, but not total IDD

prevention, may instead be due to litter assignment rather

than treatment. In addition, no research group has yet been

able to successfully transfer IDD immunologically from BB

rats to nondiabetic recipients.








Less direct evidence for an involvement of autoimmunity

in the pathogenesis of IDD in the BB rat includes recent

findings by Colle and coworkers of a genetic linkage between

IDD and the rat major histocompatibility complex RT.1 in F2

animals produced by initial matings of male BB rats with IDD

and RT.1 incompatible female Lewis rats (106). BB rats have

also been shown to have decreased numbers of circulating T

lymphocytes (107) and to be extremely susceptible to

opportunistic infections. As is the case with human IDD,

more evidence is needed in order to prove an autoimmune

etiology for the loss of pancreatic beta cells and thus IDD

in the BB rat.















SPECIFIC AIMS


From the previously mentioned findings, IDD in the BB

rat and in humans is thought to result from pancreatic beta

cell autoimmunity. However, much more evidence is required

to substantiate this hypothesis. In order to better under-

stand the etiology and genetics of IDD in the BB rat and its

similarities to human IDD, the following studies were per-

formed.

1. Identification of organ-specific autoantibodies in

BB rat sera. Several of such antibodies occur with

increased frequencies in human IDD and their pres-

ence in BB rats would support a role for autoimmu-

nity in the strain.

a. Autoantibodies to the cells of the pancreatic

islets, the thyroid, the adrenal gland, and the

gastric mucosa were sought in BB rats from an

early age before the development of IDD in or-

der to determine whether a correlation between

IDD onset and the appearance of autoantibodies

existed.

b. Clinical evidence of disease was determined in

those rats with organ-specific autoantibodies.








c. Crosses between BB and WF rats were performed

in order to indicate the inheritance of auto-

antibodies and their relationship to IDD.

2. Evaluation of the immune system of the BB rat.

These studies were done in the hopes of identifying

abnormalities which could both predispose to auto-

immune disease and explain the increased suscepti-

bility of these animals to infection. Both

diabetic and nondiabetic BB rats were studied.

a. The various circulating leukocyte populations

and major lymphocyte subsets were counted in BB

rats and compared to numbers observed in WF and

BB x WF Fl animals.

b. The ability of the immune system of BB rats to

function in vivo was determined by observing

how well these rats could reject skin grafts

involving both major and minor histocompati-

bility differences.

c. The ability of BB lymphocytes to function in

vitro was tested by the responses of lympho-

cytes to mitogens or to allogeneic cells in

mixed leukocyte cultures (MLCs).

d. The possible presence of increased suppressor

activity in BB rats and the ability of BB

lymphocytes to produce and respond to helper

factors such as interleukin 2 (IL 2) were

determined.








e. The proliferative responses of BB and WF thymo-

cytes to mitogens were compared.

f. Autoantibodies to thymocytes were sought in

sera from BB rats.

g. Histological examination of thymuses, spleens,

and lymph nodes from BB rats were made.

h. Gamma globulin levels were measured in BB rats

as a gross indication of B lymphocyte function.

3. Attempts to transfer IDD with pancreas extracts and

cells from spleens and lymph nodes from BB rats to

nondiabetic BB, WF and BB x WF Fl rats were

performed. Positive findings would thus provide

convincing proof of an autoimmune etiology for IDD

in the BB rat.

4. Evaluation of the genetics of IDD and autoanti-

bodies in the BB rat.

a. Different mating combinations were performed

between BB rats with and without IDD and/or

autoantibodies in order to observe the modes of

inheritance of these autoimmune parameters.

b. Crosses between male BB rats with IDD and both

female WF and Lewis rats were performed in

order to observe the relationships between IDD,

autoantibodies, and the rat major histocompati-

bility complex, RT.1.














MATERIALS AND METHODS


Animals. One hundred and fifty BB rats of both sexes and

of varying ages in addition to Wistar Furth (WF) rats

(Charles River Laboratories, Wilmington, MA) and Lewis rats

(Charles River) were used in these studies. Initially, 40

BB rats from 15 litters were obtained from Dr. Pierre

Thibert (Animal Resources Division, Health Protection

Branch, Ottawa, Ontario) and 28 BB rats from 3 litters were

obtained from Dr. Arthur Like (University of Massachusetts,

Worcester, MA). The animals obtained from Dr. Thibert and

Dr. Like are referred to as BB/O and BB/W rats

respectively. The remaining 82 BB rats were bred in our

laboratory from the original animals obtained above. Male

BB rats with IDD were also mated with inbred WF or Lewis

females. Male and female Fl progeny were then interbred to

produce F2 rats.

The animals were given Purina Rodent Chow 5001 and

water ad libitum, with light regulation at 0730 and 1830

hours. Diabetic rats were maintained on PZI insulin given

between 1500 and 1700 hours daily by subcutaneous injections

into axillary skin folds at doses sufficient to sustain

weights, minimize polyuria and avoid clinical hypoglycemic

episodes.








Detection of IDD and Sera Collection. All BB rats had

blood and urinary glucose levels determined weekly. Blood

samples were drawn from the periorbital venous sinus using

heparinized capillary tubes while the rats were under light

ether anesthesia. After clotting, serum glucose levels were

run in duplicate on a Beckman II glucose analyzer (Beckman

Instruments, Inc., Fullerton, California). Sera were

collected weekly and then stored at -200C until testing for

autoantibodies were made. Rats were considered to have IDD

if the animals had glycosuria or serum glucose levels above

250 mg/dl. For some studies, intraperitoneal glucose toler-

ance tests (1.75 g/kg) were performed after an overnight

fast on BB rats aged 6-9 months who had not developed overt

IDD.



Handling and Preservation of Tissues for Autoantibody
Detection.

Normal WF rat tissues were used for detection of organ-

specific autoantibodies. After removal, the tissues (thy-

roid, pancreas, adrenal and stomach) were cubed and immedi-

ately snap frozen in isopentane cooled in a mixture of dry

ice and acetone and then stored at -800C. Four micrometer

tissue sections were cut out on a SLEE HR Mark II cryostat

(Slee Medical Equipment Ltd., London, England) at -200C,

placed on slides and air-dried. The slides were either used

immediately or stored for no more than one month at -800C.








Tissue Histology. Thymuses, spleens, lymph nodes,

stomachs and pancreases were removed from BB, WF and BB x WF

Fl rats and placed in a 10% formalin solution. The final

paraffin-embedded tissue sections were stained with hematox-

ylin and eosin before histological examination.



Isolation of Peripheral Blood Mononuclear Cells. One to 3

ml blood samples were withdrawn from the periorbital venous

sinus or tail vein into 3 ml EDTA vacutainer tubes (Becton-

Dickinson, Rutherford, NJ) while the rats were under ether

anesthesia. The blood was diluted with phosphate-buffered

saline (PBS), layered over a Ficoll-Hypaque gradient (24:10

v/v; 9% Ficoll 400, Pharmacia, Piscataway, NJ: 34% Hypaque

M, Winthrop Laboratories, New York, NY), and the mononuclear

cells (PBL) isolated at the interface after centrifugation

at 2200 rpm for 13-15 minutes. Any remaining erythrocytes

were lysed by exposure to an ammonium chloride-Tris buffer

solution for 3 minutes at 370C. The PBL were then washed

twice in PBS before adjustment to the desired cell concen-

trations in either PBS for T cell subset determinations or

in complete medium: RPMI 1640 (GIBCO, Grand Island, NY)

supplemented with 50 pg/ml gentamycin (Schering, Kenilworth,

NJ), 5% heat-inactivated fetal calf serum (FCS) (GIBCO), and

5 x 10-5M 2-mercaptoethanol (2-ME) (Bio-Rad, Richmond, CA)

for mitogen and MLC assays. Viability of cells was measured

by trypan blue exclusion.








Isolation of Splenic Lymphocytes. The spleens were

removed asceptically from anesthesized rats, minced, pressed

through nylon mesh screens and resuspended in cold PBS. Any

cell clumps were dispersed by repeatedly aspirating the

suspensions through a series of varying gauge (19,21,23,25)

needles, after which the cell suspensions were centrifuged

at 1600 rpm for 10 minutes at 200C. Erythrocytes were

removed as stated previously. After washing the pellets

twice in PBS, the cells were resuspended in either PBS for

transfer experiments or in complete medium for in vitro

assays.



Preparation of Purified Splenic Lymphocytes. For some

experiments, spleen cells were purified of Fc receptor-

bearing T cells and Ig-positive B cells and monocytes by

passage through rabbit immunoglobulin (Ig)-antirat Ig-coated

glass bead columns, prepared according to the protocol of

Wigzell et al. (108). After passage through the columns,

the remaining cells were washed in PBS and resuspended in

complete medium. The purified spleen cell populations were

observed to be 98% W3/13 monoclonal antibody reactive cells

with less than 2% MRC OX8-positive cells and B lymphocytes

present. (See below under leukocyte populations and T cell

subsets for discussion of monoclonal antibodies).



Preparation of Thymocytes. Thymocyte suspensions were

prepared using the above method for splenic lymphocytes, but








were resuspended in RPMI 1640 without FCS or 2-ME when used

in microcytotoxicity assays.



Preparation of Pancreas and Peritoneal Cavity Suspensions.

Pancreases were removed aseptically, rinsed in PBS, minced

and pressed through nylon mesh screens. The suspension was

then centrifuged at 1600 rpm for 10 minutes at 200C and re-

suspended in 1 ml of PBS. Peritoneal cells were obtained by

rinsing the open cavity with PBS and withdrawing the fluid

by pipette. This procedure was repeated several times until

the fluid removed from the peritoneal cavity was clear. The

suspension was then spun and resuspended in 1 ml of PBS.



Production of Con A Supernatants. Spleen cells from both

BB and WF rats at concentrations of 4-5 x 106 cells/ml

were incubated with 5 Vg/ml Con A in complete medium for

16-20 hours at 370C in 25 cm2 tissue culture flasks

(Corning, Medfield, MA). The contents of the flasks were

then centrifuged at 2000 rpm for 10 minutes, the cell-free

supernatants (Con A sups) removed and filtered through

0.45 pm filters (Sybron/Nalge, Rochester, NY) and stored at

-700 C for future use.



Detection of Organ-Specific Autoantibodies. BB and WF

rat sera were applied undiluted to slides of air-dried,

unfixed pancreatic or adrenal tissue and at 1:4 dilution and








at 1:10 dilution to thyroid and stomach sections respect-

ively. Optimal results (lowest background and highest sen-

sitivity) were obtained when these specified dilutions were

used on the various tissues. After incubation with rat sera

for 30 minutes in the dark, the slides were washed three

times in PBS. Rabbit antirat IgG-fluorescein isothiocya-

nate conjugate (Cappel Laboratories, Cochranville, PA) was

then added at 1:60 final dilution to the slides and

incubated for 60 minutes in the dark. After washing in PBS,

the slides were dried, covered with glycerol and glass slips

and read under a Leitz Dialux 20 ultraviolet glass micro-

scope fitted with an HB-100 mercury lamp and KP490, TK510

and K515 filters (E. Leitz, Inc., Rockleigh, NJ). All

results were read double blind with control negative and

antibody-positive sera in each batch. Sere were considered

to have autoantibodies if positive immunofluoresence of the

tissue was observed. Sera were tested for the presence of

autoantibodies at least monthly after the rats were 40 days

of age.



Measurement of Gastric Acidity and Serum Iron and Vitamin
B12 Levels.

Serum vitamin B12 levels were measured by radioimmunoassay

(Diagnostic Products, Los Angeles, CA). Serum iron levels

and iron binding capacities were measured by the ACA-III

autoanalyzer (Dupont Inc., Wilmington, DL). Gastric acidity

after pentagastrin administration (0.06 mg/kg body weight)









was determined by gastric aspirates during the fasting state

and was quantitated by color changes of pH indicator paper.



Transfer Studies. Spleen, mesenteric lymph nodes and

peritoneal suspensions from BB rats 1 to 10 days after onset

of IDD were injected into the tail veins of anesthesized

recipient nondiabetic BB, WF or BB x WF Fl rats using 25

gauge butterflys (Deseret, Sandy, UT). Pancreas suspensions

were given intraperitoneally. In some experiments, the

recipients were given 300-350 rads of irradiation 24 hours

before the transfer. Blood glucose levels were measured on

sera from recipient rats on day 0, day 3 and every week

thereafter for at least 90 days. Autoantibodies to the

cells of the pancreatic islets, the thyroid gland, and the

gastric mucosa were also sought monthly in sera recipient

rats. After approximately 90-120 days, the recipients were

sacrificed and the pancreases removed for histological

examination.



Determination of Leukocyte Populations and Rat T Cell
Subsets.

White blood cell counts were determined on whole blood after

1:200 dilution in acetic acid using a hemacytometer, and

differentials were made from slides of Wright-Giemsa stained

cells.

Monoclonal antisera to the various T cell subpopula-

tions were kindly provided by Dr. Alan Williams (Medical








Research Council Cellular Immunology Unit, Oxford, England).

The specificities of the antibodies were known to be as

follows: W3/13 all T lymphocytes, W3/25 helper T cells,

MRC OX8 cytotoxic/suppressor T cells, and MRC OX6 Ia-

positive cells (109). Aliquots of 1-2 x 106 PBL were

incubated at 40C for 30 minutes with 1:20 dilutions of these

antisera in addition to a 1:15 dilution of rabbit antirat Ig

(Accurate Chemicals, Westbury, NY) for determination of the

numbers of B cells and monocytes, and PBS or normal rat

serum alone as a control. After 2 washings with cold PBS,

all PBL aliquots were incubated with a 1:20 dilution of a

fluorescein isothiocynate conjugated goat antimouse IgG

(Cappel Laboratories) for 30 minutes at 40C, washed in cold

PBS, and resuspended in 30 Ils of PBS-glycerol. Slides were

then prepared and were observed for positive immunofluore-

scence under the ultraviolet microscope.



Detection of Thymocytotoxic Autoantibodies. Two p1

samples of sera from both diabetic and nondiabetic BB

animals of varying ages, WF rats and Lewis rats were applied

undiluted or at 1:2 dilution to 72 well microcytotoxicity

trays (Falcon, Oxnard, CA). A modification of the microcy-

totoxicity method of Amos was used for determination of

thymocytotoxic autoantibodies (110). One pl samples of

either a BB or WF thymocyte suspension at a concentration of

2 x 106 cells/ml were added to each well and incubated

with the various sera for 30 minutes at room temperature.








The cells were then washed in PBS and incubated with 4 pls

of a 1:10 dilution of guinea pig serum (Dutchland Labora-

tories, Denver, PA) for 60 minutes at room temperature.

After washing and staining with a 1% trypan blue solution,

the plates were read for determination of positive

cytotoxicity (greater than 50% cell death in any well).



Skin Graftings. Sections of ear pinnae were removed from

anesthesized nondiabetic BB, WF or Lewis rats, split in

half, and washed in sterile PBS. Sections of skin conform-

ing to the shapes of the ear grafts were removed from the

sides of shaved, anesthesized BB or WF rats. After washing

the wounds with sterile PBS, the ear grafts were placed onto

the prepared areas with the hairless sides down and lightly

sprayed with a plastic dressing (Aeroplast, Parke-Davis,

Greenwood, SC). Gauze bandages were wrapped around the rats

and left in place for one week and then removed. The grafts

were considered to have taken if no macroscopic evidence of

necrosis was apparent at this time. Skin grafted rats were

then followed for evidence of rejection by daily inspection.



Mitogen Assays. In most mitogen or MLC experiments, only

nondiabetic BB rats were used due to the possible effects of

hyperglycemia on lymphocyte responsiveness. However, a few

experiments were performed using well controlled diabetic BB

rats in order to see if similar results would be obtained.

Unseparated or purified (rabbit Ig antirat Ig treated)








splenic lymphocytes or PBL from nondiabetic or well con-

trolled diabetic BB and WF rats at various cell concentra-

tions ranging from 0.3-2 x 105 cells/well were cultured in

round-bottom microtiter plates (Costar, Cambridge, MA) with

several mitogen concentrations. Pokeweed mitogen (PWM;

1-25 yg/ml) (Sigma, St. Louis, MO), PHA (0.025-1%) (Difco,

Detroit, MI), Con A (0.5-10 yg/ml) (Miles-Yeda, Rehovoth,

Israel) and WF Con A sup (0.1/ml/well) were used for a total

volume of 0.2 ml of complete medium in each well. After 48

hours incubation at 370C in 5% C02, the cultures were

pulsed with 1.0 pCi/well of 3H-thymidine (Schwarz/Mann,

Spring Valley, NY, specific activity of 6 Ci/mM) and

harvested 18 hours later onto filter paper with a 24-line

cell harvester (Otto Hiller, Madison, WI). The filters were

then air-dried, placed into vials containing scintillation

fluid, and counted in a LKB Model 8100 liquid scintillation

counter (LKB Instruments, Rockville, MD). In several

experiments, 0.5 x 105 spleen cells irradiated with 3000

rads from a 137Cs source (Gammator Model M) were also

added to some wells. Some 0.5 x 105 or 1 x 105 BB and

WF thymocytes/well were also incubated with 0.1 pg/ml and 1

pg/ml Con A and 0.1 ml/well WF Con A sup for 48 hours at 370

C, pulsed with 1.0 pCi/well of 3H-thymidine and harvested

18 hours later.



Mixed Leukocyte Cultures (MLCs). MLCs were performed in

round-bottom microtiter plates, with each well containing








0.25-1 x 105 unseparated or purified responder spleen

cells and 0.5-3 x 105 irradiated (3000 rads) unseparated

spleen cells as stimulators. In addition, third-party

irradiated or nonirradiated unseparated spleen cells at a

concentration of either 0.25 x 105 or 0.5 x 105 cells

were added to some wells with a final volume in each well

always of 0.2 ml of complete medium. The plates were incu-

batd at 370C in 5% CO2 for 5 days, pulsed with 1.0 pCi/

well of 3H-thymidine as previously described, and

harvested 18 hours later. Also, 0.1 ml of WF Con A sup was

also added in some cases to wells containing either

responders alone or both responder and stimulator cells.



Interleukin 2 (IL 2) Assay. Levels of IL 2 in Con A sups

from both BB and WF rats were determined by the stimulatory

activity of these samples on a murine IL 2-dependent cyto-

toxic T cell line (anti-EL4, generously provided by Dr.

Shiro Shimuzu, University of Florida). Some 20 x 103 cyto-

toxic T cells in 0.1 ml of complete medium were incubated at

370C for 22 hours with 0.1 ml of various Con A sups and 6

dilutions (50%-1.5%) of a reference murine Con A sup. The

cultures were then pulsed with 0.5 PCi/well of 3H-thymi-

dine and harvested 6 hours later.



Measurement of Gamma Globulin Levels. Gamma globulin

levels were determined on 10 pl serum samples from BB rats

with and without IDD, and WF rats using the Beckman micro-

zone serum protein electrophoresis system.














RESULTS


Age at Onset and Frequency of IDD. The frequency of IDD

was determined in the original 20 BB/O and 28 BB/W rats.

The incidence of IDD was found to be 80% (16/20) in BB/O

rats, while IDD developed in 75% (21/28) of BB/W animals

(Table 1). However, two female BB/O rats greater than 285

days of age and one male BB/W rat who had reached 180 days

of age without developing overt IDD, had one hour peak

glucose levels of 513 mg/dl, 671 mg/dl and 298 mg/dl

respectively, after glucose loading. Peak glucose levels

seen in three control rats did not exceed 220 mg/dl after

glucose loading (data not shown). Thus, these three BB rats

were considered to have noninsulin-dependent diabetes.

No significant sex difference was seen in the frequency

of IDD in either BB/O or BB/W animals. The time period for

onset of IDD in the BB/O rats was from 90 to 120 days of age

(Figure 1), while the BB/W rats had a broader age range for

onset of IDD of between 70 and 161 days (Figure 2). All BB

rats were studied beyond 180 days of age, which exceeded the

critical age span for development of the disease in these

rats. Mild to moderate insulitis was seen in pancreases

from BB rats at onset of IDD (Figure 3). None of the















TABLE 1

IDD AND AUTOANTIBODIES IN BB/O, BB/W,
WF AND BB x WF Fl HYBRID RATS


Rats


IDD %


Autoantibodies
ICA % PCA % SMA %


BB/O


(n=20)


BB/W


(n=28)


BB x WF Fl


(n=50)


(n=30)


aOne rat had equivocal PCA. All rats ascertained for the
above were studied up to and beyond 6 months of age.

Thyroid colloid autoantibodies.


TCA %













FIGURE 1. Frequencies of insulin-dependent diabetes (IDD),
gastric parietal cell antibodies (PCA) and smooth muscle
antibodies (SMA) in 20 BB/O rats with age.






o---o PCA
1001 o--0 SMA
90 IDD
80



m 60
50
m
wiX,;t:::::-----
40

30
10


2240

60 80 0 120 140 160 180 220 4
AGE (days)



























0




0
03















00









rj






CO
H


















5 ~
o ^3





o---o PCA
--a SMA
IDD


AGE (days)


100


180














FIGURE 3. Section of pancreas from a BB rat at onset of
IDD. The islet in the center has disorganized architecture
and lymphocytic infiltration (black arrow). A normal beta
cell is indicated by a clear arrow.

























rjvw*A-.,4L








control WF animals or BB x WF Fl rats, which were followed

for at least 180 days after birth, developed IDD or

pancreatic insulitis (Table 1), indicating that IDD in the

BB rat is not due to a single dominant gene.



Presence of Organ-Specific Autoantibodies. ICA and other

organ-specific autoantibodies were sought in BB/O and BB/W

rats because these antibodies are characteristically found

in human IDD, are evidence for an autoimmune etiology for

IDD in the BB rat, and can be used as markers for animals

with autoimmune tendencies. ICA were never detected in any

BB rats, regardless of age or duration of IDD. To rule out

the possibility that ICA were present but were directed

against diabetic antigens, ICA were also sought on pancre-

atic sections from both nondiabetic BB rats and BB rats at

onset of IDD. However, no such autoantibodies were identi-

fied. In addition, ICA were not demonstrated in BB rat sera

when using fluorescein conjugated antirat Ig (all classes)

instead of antirat IgG in case ICA of the IgM class were

present. Thyroid microsomal autoantibodies and adrenal

autoantibodies were never found in any BB animals.

However, autoantibodies to the parietal cells of the

gastric mucosa (PCA) (Figure 4), thyroid colloid antigens

and smooth muscle (SMA) (Figure 5) were demonstrated in the

sera of a considerable number of BB/O and BB/W rats (Table

1). PCA were detected in the sera of 35% (7/20) of the BB/O

rats and in the sera of of 68% (19/28) of the BB/W animals.














FIGURE 4. Positive indirect immunofluorescence staining of
the parietal cells of rat gastric funds.





42














FIGURE 5. Positive indirect immunofluorescence staining of
smooth muscle in rat gastric funds.






44








The intensity of immunofluorescence of the PCA-positive sera

tended to increase with duration of PCA. Except for an

equivocal result from the serum of one WF rat, none of the

WF or BB x WF Fl animals had demonstrable PCA in their

serum. As was the case with IDD, PCA did not seem to have a

dominant mode of inheritance. Thyroid colloid autoanti-

bodies were of low immunofluorescence intensity and were

less frequent than PCA, being found in only 5% (1/20) and

18% (5/28) of the sera from BB/O rats and BB/W rats,

respectively. None of the control animals had autoanti-

bodies to thyroid colloid. SMA were demonstrated in 55%

(11/20) of BB/O rats, in 61% (17/28) of BB/W rats, and in 9%

(7/80) of WF and BB x WF Fl animals. The presence of SMA

was unrelated to the presence of IDD or PCA in the BB rats

(Table 2).

Some 71% (5/7) of the BB/O rats with PCA and 79%

(15/19) of the BB/W rats with PCA had IDD (Table 2).

However, two PCA-positive BB animals (one BB/O and one BB/W)

without clinical evidence of IDD had abnormal glucose

tolerance tests as previously mentioned. Thus, only 15%

(4/26) of the BB rats with detectable PCA had no discernible

IDD confirmed by normal glucose tolerance tests.

The appearance of PCA in the serum usually shortly

preceded the onset of IDD in BB rats that developed the

disease. The frequency of PCA in the BB rats was seen to

increase at an age coincident with the development of IDD

and did not further rise after the critical age span for












TABLE 2

RELATIONSHIPS BETWEEN IDD, PCA AND SMA
IN BB/O, BB/W AND WF RATS


Rats Studied Total PCA % SMA %


I. Total BB rats with IDD 37 54 57

Total BB rats without IDD 11 54 64



BB/O rats with IDD 16 31 50

BB/O rats without IDD 4 50 75



BB/W rats with IDD 21 71 62

BB/W rats without IDD 7 57 57


Rats Studied


Total


IDD %


SMA %


II. Total

Total


BB rats with PCA

BB rats without PCA


BB/O rats with PCA

BB/O rats without PCA



BB/W rats with PCA

BB/W rats without PCA


42

100








onset of IDD in these rats had passed (Figures 1 and 2).

Although BB/W rats were susceptible to IDD for a longer age

range than BB/O animals, the marked increase in the

frequency of PCA in BB/W rats between 70 and 110 days of age

mirrored the increase in PCA frequency observed in BB/O rats

between 90 and 113 days of age. The frequency of SMA in BB

rats increased with age and, unlike PCA, did not closely

parallel the development of IDD in BB/O rats. The frequency

of SMA, however, increased in parallel with PCA in the BB/W

animals.

Functional abnormalities of the gastric parietal cells

were sought in BB rats with PCA as evidence that gastric

autoimmunity may result in clinical disease. Achlorhydria

was not demonstrated in the gastric aspirates of BB rats

with PCA, the pH values of which ranged from 2 to 3.

Vitamin B12 levels were measured in sera from 14 BB rats

with PCA, 14 PCA-negative BB rats, and 7 control WF rats.

No statistically significant differences were seen in serum

vitamin B12 levels between the three groups as evaluated by

Student's t-test (Table 3). Comparison of serum iron levels

and total iron binding capacities between 14 BB rats with

PCA, 11 BB rats without PCA and 7 control WF animals also

revealed no significant differences (Table 3).

Histological examinations of stomach sections from 12

BB rats with PCA were performed in order to see if lympho-

cytic infiltration suggestive of autoimmunity were present.

All sections revealed mild to moderate lymphocytic









TABLE 3

SERUM VITAMIN B12 AND IRON LEVELS IN BB AND WF RATS


BB rats with PCA (n=14)a BB rats without PCAa (n=14) WF rats (n=7)


x = 472 + 38 (244-710) x = 466 + 44 (109-759) x = 473 + 49 (275-666)


Vitamin B12


BB rats with PCA (n=14) BB rats without PCA (n=ll) WF rats (n=7)


Iron x = 223 + 13 ( 84-352) x = 210 + 21 (131-326) x = 234 + 33 ( 84-372)

Total iron binding capacity x = 492 + 17 (328-620) x = 474 + 36 (355-565) x = 519 + 78 (285-742)

% iron binding x = 45 + 2 ( 33- 78) x = 48 + 5 ( 35- 60) x = 51 + 6 ( 26- 70)

aThe BB rats used are from the group of 48 animals designated as either BB/O (20 rats) or as BB/W (28 rats).
All results are expressed as the mean + 1 standard error of the mean.
bSera from 11 BB rats without PCA were used to determine serum iron levels, however only sera from 9 BB rats
without PCA had measurements for iron binding capacity and percent iron binding performed.








infiltrations of the gastric mucosa with some loss of normal

mucosal cells and increased fibrosis (Figure 7), in

comparison with gastric funds obtained from WF and PCA-

negative BB rats (Figure 6). However, severe atrophy of the

gastric mucosa was not found in any of the tissues studied.

In two BB rats with PCA for the longest periods of approxi-

mately 7 months, degrees of squamous metaplasia of the

gastric mucosa were seen in sections taken well below the

junction between the proximal stomach and the funds (Figure

8). Only one of these rats had IDD. No stomach sections

from spleen BB rats without PCA or 5 control WF rats

revealed inflammatory lesions of the gastric mucosa.



Characterization of Peripheral Leukocyte Populations. Due

to observations of increased susceptibility to opportunistic

infections (especially of the respiratory tract) in both

diabetic and nondiabetic BB rats, and indications of both

pancreatic and gastric autoimmunities in this strain, immuno-

logical studies of these animals were performed.

Increased percentages and absolute numbers of

peripheral blood polymorphonuclear leukocytes (PMNs) were

observed in all BB rats regardless of the presence of IDD,

in comparison with WF and Fl hybrid rats (p <0.0025, Table

4), perhaps reflective of the increased rate of infections

among these animals. In contrast, all 16 BB rats with IDD

and all 32 nondiabetic rats, ranging in age from 25 to 400

days, were observed to have significantly decreased absolute














FIGURE 6. Section of normal BB rat gastric funds stained
with hematoxylin and eosin. Organized rows of parietal
cells (black arrow) are present.














'" r -


hm~~














FIGURE 7. Hematoxylin and eosin stained section of gastric
funds from a BB rat with PCA showing lymphocytic
infiltration of the mucosa (black arrow).






53








oil.mm iu














FIGURE 8. Hematoxylin and eosin stained section of gastric
funds from a BB rat with PCA for 7 months. Lymphocytic
infiltration, fibrosis and squamous metaplasia (black arrow)
of the mucosa are present.






55

























Leukocyt


PMNs/mn'

%PM^1s


Lymphocs

% Lymphn


Monocyt

% Monoc


Bosinoph

% Eosinc


TABLE 4

ENUMERATION OF PERIPHERAL BLOOD LEUKOCYTES IN


Animals

BB Rats With NonDiabetic BB
IDD (n=16) Rats (n=32)


tes/mn3 5673 + 1840a'b 5655 + 1761b


3 2770 + 1132c 2225 + 1015c

57 + 11c 34 + 11C


tes/mn3 2805 + 1071c'e 3403 + 1041b

cytes 47 + 15c'd 60 + 12b


es/mn3 228 + 140 187 + 113

ytes 4+ 4 4+ 2


lils/mn3 46 + 45 136 + 309

)phils 2+ 4 2+ 4


BB, WF AND BB x WF Fl RATS


Studied


WF Rats (n=28) Fl


9680 + 2858 108


1334 + 923 12'

14 + 7


7740 + 2317 911

82 + 7


277 + 201 4

3+ 1


31 + 58

1+ 3


Rats


43 +


75 +

12 +


05 +

34 +


32 +

4+


0

0


(n=8)


1521


503

3


1084

4


251

2


I


I









Table 4-extended.

a Each value is stated as mean + S.D.
b
p <0.0005 by Student's t test when compared to WF and Fl rats.

Sp <0.0025 when compared to WF and Fl rats.
d
p <0.0025 when aorpared to BB rats without IDD.

p <0.01 when ccapared to BB rats without IDD.








numbers of both total peripheral blood leukocytes and

lymphocytes, when compared to 28 control WF rats and 8 Fl

hybrid animals (p <0.0005). The lymphopenia was more

striking than the leukopenia, reflecting significant

complementary decreases in the percentage of lymphocytes

seen in BB rats from that observed in WF and BB x WF Fl rats

(p <0.0005). Significant differences were also seen in both

the percentages and absolute numbers of lymphocytes in

diabetic BB rats when compared to BB rats without IDD (p

<0.01).

Due to the severe lymphopenia observed in all BB rats,

analyses of lymphocyte subpopulations were next performed.

Irrespective of age or the presence of IDD, increased

percentages (p <0.0005) but similar absolute numbers of Ia-

positive cells (MRC OX6 monoclonal antibody reactive) and Ig-

positive cells were found in BB rats in comparison with WF

rats and BB x WF Fl rats (Table 5). In contrast, the

absolute numbers of peripheral T lymphocytes (W3/13 mono-

clonal antibody reactive) were significantly lower in all BB

rats when compared to WF and Fl animals (p <0.001). Corre-

spondingly significant depressions of both absolute numbers

(p <0.005) and percentages (p <0.025) of W3/25-positive

cells (helper T cells) were observed in all BB rats,

independent of age or IDD, in comparison with WF and Fl

animals. Increased percentages (p <0.005) but decreased

absolute numbers (p <0.005) of cytotoxic/ suppressor T

lymphocytes (MRC OX8 monoclonal antibody reactive) were also












TABLE 5

LYMPHOCYTE SUBSETS IN BB, WF AND BB


x WF Fl RATS


Animals Studied

BB Rats With BB Rats Without
IDD (n=8) IDD (n=15) WF Rats (n=12) Fl Rats (n=4)


Leukocytes/mn3

Lynphocytes/mrm3


W3/13+ Cells/mn3

% W3/13+ Cells


W3/25+ Cells/mmn3

% W3/25' Cells


MRC OX8+ Cells/rm3

% MRC OXB+ Cells


MRC X6m+ Cells/mm3

% MRC OX6+ Cells


4837

2986


2060

70


1323

37


1650

46.5


1351

43.5


2373a,b

b991
991


817c

14


688C

15c


729c

9d


462

15b


4921

2847


2069

71


1097

38


1258

44


1377

47


1754b

943b


818c

10


501c

13d


460

12b


10154

8296


6034

74


4243

50


3431

38


1592

18


3858

2714


1421

9


1035

9


902

7


649

4


9475

7441


5809

79


3840

47.5


3358

41


1260

16.5


2306

1840


1051

8


1343

2


1327

2


247

2







Table 5-extended.

Ig Cells/mn3

% Ig+ Cells


1158 + 446

36.5 + 10b


1384 +

47 +


559

14b


1324 + 509

16 + 4


1370 + 899

17 + 8


aEach value is stated as mean + S.D.

b
p <0.0005 by Student's t test when compared to WF rats.

p <0.005 by Student's t test when compared to WF rats.

p <0.025 by Student's t test when compared to WF rats.
ep 01 by Student's t test when pared to WF rats.
p <0.01 by Student's t test when compared to WF rats.








observed in all BB rats when compared to WF control and BB x

WF Fl rats. Numbers of T lymphocytes in both lymphocyte

subsets of Fl hybrid animals tended to range between those

of WF and BB rats. However, there were no significant

differences between WF and BB x WF Fl values.

An inversion of the W3/25-positive subset to MRC OX8-

positive subset ratio to less than 1.0 (mean 0.7 + 0.2) also

occurred in BB rats between 75 to 115 days of age, which was

not influenced by the presence of IDD (p <0.001, Figure 9).

In younger BB rats with and without IDD, the mean W3/25-

positive subset to MRC OX8-positive subset ratio was similar

to the mean ratio seen in WF rats at all ages studied (1.2 +

0.2 versus 1.3 + 0.1).



Presence of Thymocytotoxic Autoantibodies. As a possible

explanation for the extremely decreased numbers of T lympho-

cytes present in BB rats, autoantibodies to BB or WF thymo-

cytes were sought in these animals. As shown in Table 6,

many unabsorbed sera from both diabetic and nondiabetic BB

rats had demonstrable thymocytotoxic autoantibodies,

especially when the sera were tested at 1:2 dilution.

Although a few WF sera were also antibody positive, signifi-

cantly more BB sera had antibodies to thymocytes (p <0.05).

Only sera giving reactions of greater than 50% cytotoxicity

were considered to be positive for these autoantibodies.



Depressed Ability to Reject Allografts. Because BB rats

had both severely decreased numbers of peripheral T lympho-













FIGURE 9. A plot of the ratio of the W3/25-positive subset
(helper T lymphocytes) to the MRC OX8-positive subset
(cytotoxic/ suppressor T cells) versus age of the BB rats.
N designates nondiabetic BB rats and D designates BB rats
with IDD.








2.8
1.9
1.8
1.7
O
I- 1.6
S1.5 N
S1.4
1.3
1.2 NDN
o1.l N ND
u1.8
S.9 NN N
.8 N D
m *7 N ND
.6 D ND
.5 D N
.4 N
.3
.2
.1

58 188 150 288 258 388 35 400 88

AGE (days)















TABLE 6

THYMOCYTOTOXIC AUTOANTIBODIES IN BB AND WF RAT SERA


Autoantibody
Positive


BB rats with IDD





BB rats without IDD


undiluted

1:2 dilution


25%

32%


undiluted

1:2 dilution


16%

47%


(16/65)

(10/31)b



(6/38)

(8/17)a


WF rats undiluted

1:2 dilution


ap <0.05 by Chi-square

bp 0.01 when compared
p <0.01 when compared


6% (1/18)

9% (2/23)


analysis when compared to WF rats.

to WF rats.


Sera


--








cytes and circulating thymocytotoxic autoantibodies, the

ability of T lymphocytes from BB rats to function normally

in vivo was studied. Both BB and WF rats are thought to

share the rat major histocompatibility complex RT.lu

genotype, while Lewis rats have the RT.11 haplotype (106-

111). Lewis skin grafts would thus be expected to be

rejected by both BB and WF rats in less than 14 days.

However, as seen in Table 7, nondiabetic BB rats rejected

Lewis allografts significantly more slowly than expected and

in comparison with WF controls (p <0.0005). Furthermore, WF

skin grafts were not rejected by nondiabetic BB rats, while

rejections of BB allografts by WF rats occurred in 17 + 3

days (p <0.0005), suggesting the presence of multiple minor

histocompatibility differences between BB and WF rats. BB

rats were thus extremely deficient in their ability to

reject grafts across both major and minor histocompatibility

barriers, and this defect was not dependent on the presence

of IDD.



Mitogen Responsiveness. Since the skin graft results

suggested that BB rats have defective in vivo T cell-

mediated immune responses, the ability of T lymphocytes from

BB rats to respond to mitogens in vitro was next studied.

PWM, PHA and Con A, which are primarily T cell mitogens in

the rat (112-114), were used. Dose-response curves were

initially prepared for each mitogen using varying concentra-

tions of each mitogen and of WF spleen cells (0.3-2 x 105

















TABLE 7

ALLOGRAFT REJECTION BY BB AND WF RATS


Recipients Donor Graft Survival (Days)a


8 BB rats without IDD Lewis 30 + 4


6 WF rats Lewis 12 + 2


6 BB rats without IDD WF > 90


6 WF rats BB 17 + 3


aThe period of graft survival was measured from day of
graft placement to day of complete graft rejection. Each
value is expressed as mean + S.D.







cells/well) as responders (Figures 10-12). Optimal stimu-

lation indices were obtained from these curves and

subsequent experiments using 1.0 pg/ml and 5.0 pg/ml PWM,

0.5% and 1.0% PHA, and 1.0 Vg/ml and 5.0 pg/ml Con A in each

well. WF cells responded well to these mitogens at all cell

concentrations tested, but subsequent response comparisons

with BB cells were made using spleen cell or PBL concentra-

tions at 0.3 x 105, 1 x 105 and 2 x 105 cells/well.

The results were not dependent on the BB cell concentrations

used or on the presence of IDD in the BB rats.

Utilizing spleen cell concentrations of 0.3 x 105

cells/well (Figure 13), 1 x 105 cells/well (Figure 14),

and 2 x 105 cells/well (p <0.0005, Figure 15), BB splenic

lymphocytes showed markedly diminutive responses to PWM in

comparison to WF splenic lymphocytes at the same mitogen

concentration. Similar relative results were obtained when

using 0.3 x 105 PBL/well (Figure 16) or 1 x 105 PBL/well

(p <0.01, Figure 17). However, the level of proliferation

of WF lymphocytes to PWM using 0.3 x 105 PBL/well was

quite low, suggesting that insufficient numbers and/or types

of cells were present for optimal proliferation. Both BB

and WF proliferative responses to PWM (also PHA and Con A)

were higher using -spleen cells rather than PBL as responders

but background counts also tended to be higher. PWM

responses of BB lymphocytes did increase with increasing

cell numbers per well as expected. However, BB results

comparable to those by WF cells were only observed when













FIGURE 10. Proliferative responses (cpm) of increasing
concentrations of WF splenic lymphocytes to 3 concentrations
of PWM measured on day 3. The following spleen cell
concentrations were used: 0.2 x 105 cells/well (open
circle), 0.4 x 105 cells/well (open triangle), 0.6 x 105
cells/well (open square), 0.8 x 105 cells/well (open
hexagon), 1.0 x 105 cells/well (closed circlet, 1.2 x
105 cells/well (closed triangle) and 1.6 x 105 cells/
well (closed square).

















15I




120




99






30


39


8 5 10 25


PHM Concentration (pg/ml)













FIGURE 11. Proliferative responses (cpm) of increasing
concentrations of WF splenic lymphocytes to 5 concentrations
of PHA measured after 3 days. These spleen cell concentra-
tions were used: 0.2 x 105 cells/well (open circle), 0.4
x 105 cells/well (closed circle), 0.6 x 105 cells/well
(open triangle), 0.8 x 105 cells/well (closed triangle),
1.0 x 105 cells/well (open square 1.2 x 105 cells/
well (closed square) and 1.6 x 10 cells/well (open
hexagon).


















168


3Ba
I

c se

Q.


PHR Concentration (%)


a .025 .05 .1 .2 .4













FIGURE 12. Proliferative responses (cpm) of increasing
concentrations of WF splenic lymphocytes to 3 concentrations
of Con A measured on day 3. The following spleen cell con-
centrations were used: 0.2 x 105 cells/well (open
circle), 0.4 x 105 cells/well (open triangle), 0.6 x 105
cells/well (open square), 0.8 x 105 cells/well (open
hexagon), 1.0 x 100 cells/well (closed circle), 1.2 x
105 cells/well (closed triangle) and 1.6 x 105
cells/well (closed square).











































0 1.0 5.0 10.8

Con R Concentrations (ug/ml)













FIGURE 13. Responses (cpm) of splenic lymphocytes at 0.3 x
105 cells/well from 2 nondiabetic BB rats (striped bars)
and 2 WF rats (open bars) to 1 pg/ml PWM and 4 pg/ml PWM.
Each value is mean of triplicate cultures + S.D.





















60




48





m 36


x


Q_ 24




12


1 ug/ml PWM


0 ug/ml PWM


5 mg/ml PWM














FIGURE 14. Proliferative responses (cpm) of splenic lympho-
cytes at 1 x 105 cells/well from 2 nondiabetic BB rats
(striped bars) and 3 WF rats (open bars) to 2 concentrations
of PWM. Each value is mean of triplicate cultures + S.D.
















238




184


8 ug/ml PWM


5 ag/ml PNM


1 pg/ml PWM













FIGURE 15. Responses (cpm) of splenic lymphocytes at 2 x
105 cells/well from 4 nondiabetic BB rats (striped bars)
and 4 WF rats (open bars) to 10 pg/ml PWM. Each value is
mean of triplicate cultures + S.D.


















220




176


18 Mg/ml PHM


80 0g/ml PWM













FIGURE 16. Responses (cpm) of PBL at 0.3 x 105 cells/well
from 1 diabetic BB rat (first striped bar), 2 nondiabetic
BB rats (remaining striped bars) and 3 WF rats (open bars)
to 1 g/ml PWM and 5 pg/ml PWM. Each value is mean of
triplicate cultures + S.D.













































9 jg/ml PWM


1 pg/ml PM


5 Mg/ml PHM














FIGURE 17. PWM responses (cpm) of PBL at 1.0 x 105
cells/well from 3 nondiabetic BB rats (striped bars) and 5
WF rats (open bars). Each value is mean of triplicate
cultures + S.D.




























0~ug/mI PWM 1.0 pg / ml PWM 5.0 ~g/ml PWM


l00r


801


60-


40


20

? ..^r~ 0,riir~ 7


Og/ml PWM


1.0 Ajg / mi PWM


5.0 ug/mi PWM


ii








using 2 x 105 BB spleen cells/well and 0.3 x 105 WF

spleen cells/well.

Lymphocytes from BB rats also responded poorly to

various concentrations of PHA at 0.3 x 105 spleen cells/

well (Figure 18), 1 x 105 spleen cells/well (Figure 19),

and 2 x 105 spleen cells/well (p <0.05, Figure 20), in

comparison with WF lymphocytes. Although similar results

were obtained when using 1 x 105 PBL/well (p <0.01, Figure

21), low levels of proliferation with no significant

differences between WF and BB PHA responses at 0.3 x 105

PBL/well (Figure 22) were noted, most likely due to

insufficient numbers of cells as stated previously.

Although lymphocytes from BB rats were most responsive

to Con A of the mitogens used, WF cells still responded

significantly better at all of the following cell concen-

trations: 0.3 x 105 spleen cells/well (Figure 23),

1 x 105 spleen cells/well (Figure 24), 1 x 105 PBL/well

(p <0.01, Figure 25) and 2 x 105 spleen cells/well (p

<0.005, Figure 26). As was true for PWM and PHA, WF

lymphocytes at 0.3 x 105 PBL/well responded poorly to Con

A and thus no significant differences were observed at this

cell concentration between BB and WF proliferative responses

(Figure 27). However, in one experiment, splenic

lymphocytes from a single BB rat at 2 x 105 cells/well

were able to mount a significant response to Con A

comparable to responses by WF spleen cells. However, BB

lymphocytes from the same animal minimally responded to Con














FIGURE 18. PHA responses (cpm) of splenic lymphocytes at
0.3 x 105 cells/well from 2 nondiabetic BB rats (striped
bars) and 2 WF rats (open bars). Each value is mean of
triplicate cultures + S.D.


















110




88


8I H 8.5 PHA -


ex PHR


8.5% PHA














FIGURE 19. Proliferative responses (cpm) of splenic lympho-
cytes at 1 x 105 cells/well from 2 nondiabetic BB rats
(striped bars) and 2 WF rats (open bars) to 0.5% and 1.0%
PHA. Each value is mean of triplicate cultures + S.D.
















258




288




' 158

x
X

0..188
U




58


8.5% PHR


8% PHRF


1.8% PHR














FIGURE 20. Comparison of proliferative responses (cpm) of
splenic lymphocytes at 2 x 105 cells/well from 2 non-
diabetic BB rats (striped bars) and 2 WF rats (open bars)
to 5 concentrations of PHA. Each value is mean of
triplicate cultures + S.D.















288




168


8 PH .825% PHA .85% PHR


.1% PHR .2% PHR .4% PHR














FIGURE 21. Proliferative responses (cpm) of PBL at 1 x
105 cells/ well from 1 diabetic BB rat (first striped
bar), 2 nondiabetic BB rats (remaining striped bars) and 5
WF rats (open bars) to 2 PHA concentrations. Each value is
mean of triplicate cultures + S.D.




Full Text
FIGURE 11. Proliferative responses (cpm) of increasing
concentrations of WF splenic lymphocytes to 5 concentrations
of PHA measured after 3 days. These spleen cell concentra
tions were used: 0.2 x 10* cells/well (open circle), 0.4
x 10^ cells/well (closed circle), 0.6 x 10^ cells/well
(open triangle), 0.8 x 10^ cells/well (closed triangle),
1.0 x 105 cells/well (open square), 1.2 x 10^ cells/
well (closed square) and 1.6 x 10* cells/well (open
hexagon).


24
Isolation of Splenic Lymphocytes. The spleens were
removed asceptically from anesthesized rats, minced, pressed
through nylon mesh screens and resuspended in cold PBS. Any
cell clumps were dispersed by repeatedly aspirating the
suspensions through a series of varying gauge (19,21,23,25)
needles, after which the cell suspensions were centrifuged
at 1600 rpm for 10 minutes at 20C. Erythrocytes were
removed as stated previously. After washing the pellets
twice in PBS, the cells were resuspended in either PBS for
transfer experiments or in complete medium for in vitro
assays.
Preparation of Purified Splenic Lymphocytes. For some
experiments, spleen cells were purified of Fc receptor
bearing T cells and Ig-positive B cells and monocytes by
passage through rabbit immunoglobulin (Ig)-antirat Ig-coated
glass bead columns, prepared according to the protocol of
Wigzell et al. (108). After passage through the columns,
the remaining cells were washed in PBS and resuspended in
complete medium. The purified spleen cell populations were
observed to be 98% W3/13 monoclonal antibody reactive cells
with less than 2% MRC OX8-positive cells and B lymphocytes
present. (See below under leukocyte populations and T cell
subsets for discussion of monoclonal antibodies).
Preparation of Thymocytes. Thymocyte suspensions were
prepared using the above method for splenic lymphocytes, but


Table 4extended.
Each value is stated as mean + S.D.
p <0.0005 by Student's t test when compared to WF and FI rats.
0
p <0.0025 when compared to WF and FI rats.
^ p <0.0025 when compared to BB rats without IE.
0
p <0.01 when compared to BB rats without IDD.


29
The cells were then washed in PBS and incubated with 4 yls
of a 1:10 dilution of guinea pig serum (Dutchland Labora
tories, Denver, PA) for 60 minutes at room temperature.
After washing and staining with a 1% trypan blue solution,
the plates were read for determination of positive
cytotoxicity (greater than 50% cell death in any well).
Shin Graftings. Sections of ear pinnae were removed from
anesthesized nondiabetic BB, WF or Lewis rats, split in
half, and washed in sterile PBS. Sections of skin conform
ing to the shapes of the ear grafts were removed from the
sides of shaved, anesthesized BB or WF rats. After washing
the wounds with sterile PBS, the ear grafts were placed onto
the prepared areas with the hairless sides down and lightly
sprayed with a plastic dressing (Aeroplast, Parke-Davis,
Greenwood, SC). Gauze bandages were wrapped around the rats
and left in place for one week and then removed. The grafts
were considered to have taken if no macroscopic evidence of
necrosis was apparent at this time. Skin grafted rats were
then followed for evidence of rejection by daily inspection.
Mitogen Assays. In most mitogen or MLC experiments, only
nondiabetic BB rats were used due to the possible effects of
hyperglycemia on lymphocyte responsiveness. However, a few
experiments were performed using well controlled diabetic BB
rats in order to see if similar results would be obtained.
Unseparated or purified (rabbit Ig antirat Ig treated)


7
sera from diabetic patients with ICSA were shown to be able
to suppress glucose and theophylline-stimulated insulin
release but not glucagon release in vitro by dispersed mouse
islets (43). Paradoxically, ICSA have been demonstrated to
have a stimulatory effect on basal insulin release from
cultured mouse islets (44). These results are consistent
with the primary role for ICSA in beta cell destruction seen
in IDD proposed by some investigators (28), but much more
substantial evidence is needed.
Cell-Mediated Autoimmunity in IDD. No consistent general
ized defects in cell-mediated immunity as defined in terms
of lymphocyte responsiveness to phytohemagglutinin (PHA) (45-
48) and in the enumeration of lymphocyte subpopulations
(45,49-51) appear to be present in well-treated patients
with IDD. However, poorly controlled diabetics have been
observed to have depressed mitogenic responses to PHA in
comparison with matched adequately treated IDD patients and
healthy controls (47,52) suggesting that the metabolic
derangements of IDD have an adverse effect on T lymphocyte
responsiveness. Other studies have indicated that specific
antipancreatic cell-mediated immunity may be observed in
patients with IDD (45,47). Nerup et al. demonstrated
significant inhibition of migration of leukocytes from
insulin-dependent diabetics in the presence of porcine
pancreas (53) or fetal calf pancreas (54,55) homogenates.
Abnormal migration inhibition was especially noted in those


9
secretion was noted by lymphocytes from noninsulin-depend
ent diabetics or controls. Significantly increased levels
of circulating killer (K) cells, classified as low affinity
E-rosette forming cells, were also found in 57% of newly
diagnosed insulin-dependent diabetics (62). These levels
returned to normal within twelve months from diagnosis of
IDD. Raised K cell numbers were accompanied by signifi
cantly enhanced levels of antibody-dependent cell-mediated
cytotoxicity activity to chromium-labeled human erythro
cytes sensitized with antierythrocyte antibodies in many
IDD patients (63).
Several investigators have suggested that defects in
suppressor T cell activity were present in patients with IDD
(64,65). In one study, Concanavalin A (Con A)-activated
lymphocytes from patients with IDD poorly suppressed allo
geneic mixed lymphocyte cultures when compared to suppressor
activity demonstrated by Con A activated lymphocytes from
controls (64). In contrast, Slater et al. recently found a
statistically significant increase in Con A activated
suppressor T cell activity in thirteen patients with IDD
(66). These measurements of cell-mediated immunity,
however, are difficult to analyze even in healthy people,
and biologically significant results with diabetic patients
are even more questionable because the effects and
complications of IDD itself affect lymphocyte function and
responsiveness.


159
The MLC is considered to be an in vitro correlate of
the process which occurs in vivo during allograft rejection.
The responding cells are helper T cells which proliferate
after recognition of allodeterminants on the stimulator
cell. The proliferative response is determined primarily by
differences at the B locus of the rat major histocompatibil
ity complex RT.l (130-132). However, proliferation can also
be measured to differences at other loci within the RT.l
complex and to minor histocompatibility differences
(133,134).
As was demonstrated by the transplant studies, T lymph
ocytes from BB rats failed to proliferate adequately in MLCs
to either allogeneic Lewis or WF stimulating cells. Because
lymphocytes from WF animals were able to respond to
irradiated BB cells in MLCs, these results indicate, as did
the skin graft results, that significant histocompatibility
differences exist between BB and WF rats. Yet, BB lympho
cytes were totally unable to respond both in vivo and in
vitro to such differences. As was stated previously for the
allograft and mitogen findings, the severe T lymphopenia
especially of the helper T cells, probably contributed to
the defective ability of lymphocytes from BB rats to
proliferate in the presence of allogeneic cells. Most
likely, the numbers of BB monocytes and accessory cells were
sufficient for adequate proliferative responses by BB T
cells.


86
0* PHfi
0.5* PHfi
1.035 PHfi


FIGURE 14. Proliferative responses (cpm) of splenic lympho
cytes at 1 x 1()5 cells/well from 2 nondiabetic BB rats
(striped bars) and 3 WF rats (open bars) to 2 concentrations
of PWM. Each value is mean of triplicate cultures + S.D.


39


BIOGRAPHICAL SKETCH
Melissa Ellen Elder was born on August 28, 1955, at
Nellis Air Force Base, Nevada. She graduated from Wagner
High School at Clark Air Base, Phillippines, in June, 1973.
She attended Agnes Scott College in Decatur, Georgia, for
two years and then transferred to Florida State University
in Tallahassee, Florida, where she graduated in June, 1977,
with Bachelor of Science degrees in both chemistry and
biological sciences. After working in industry for one year
as a research analytical chemist, Melissa entered the grad
uate program of the Department of Pathology in the Univer
sity of Florida College of Medicine. She is currently a
candidate for the Ph.D. degree specializing in immunology.
In August, 1982, Melissa will enter the University of Flor-
da School of Medicine.
183


174
63. Sensi, M., Pozzilli, P., Gorsuch, A. N., Bottazzo, G.,
and Cudworth, A. G. 1981. Increased killer cell
activity in insulin dependent (Type I) diabetes melli-
tus. Diabetologia 20:106.
64. Fairchild, R. S., Kyner, J., and Abdou, N. I. 1980.
Suppressor cell dysfunction in insulin-dependent
diabetes. Diabetes 29 (Suppl. 2):52.
65. Horowitz, S. D., Borcherding, W., and Bargman, C. J.
1978. Suppressor T cell function in diabetes mellitus.
Lancet 2:1290.
66. Slater, L. M., Murray, S. L., Kershnar, A., and Mosier,
M. A. 1980. Immunological suppressor cell activity
in insulin dependent diabetes. J. Clin. Lab
Immunol. 3:105.
67. Kaldany, A., Hill, T., Wentworth, S., Brink, S. J.,
D'Elea, J. A., Clause, M., and Soeldner, J. S. 1982.
Trapping of peripheral blood lymphocytes in the
pancreas of patients with acute-onset insulin-dependent
diabetes mellitus. Diabetes 31:463.
68. Irvine, W., Clark, B., Scarth, L., Cullen, D. R., and
Duncan, L. J. P. 1970. Thyroid and gastric auto
immunity in patients with diabetes mellitus. Lancet
2:163.
69. Riley, W., Maclaren, N., and Neufeld, M. 1980. Adren
al autoantibodies and Addison's disease in insulin
dependent diabetes mellitus. J. Pediatrics 97:191.
70. Singal, P. P. and Blajchman, M. A. 1972. Histocom-
patability (HL-A) antigens, lymphocytotoxic antibodies
and tissue antibodies in patients with diabetes melli
tus. Diabetes 22:429.
71. Nerup, J., Platz, P., Andersen, 0. 0., Christy, M.,
Lyngsoe, J., Poulsen, J. E., Ryder, L. P., Nielsen,
L. S., Thomsen, M., and Svejgaard, A. 1974. HL-A
antigens and diabetes mellitus. Lancet 2:864.
72. Christy, M., Green, A., Christau, B., Kromann, H.,
Nerup, J., Platz, P., Thomsen, M., Ryder, N., and
Svejgaard, A. 1979. Studies of the HLA system and
insulin-dependent diabetes mellitus. Diabetes Care:
209.
73. Solow, H., Hidalgo, R., and Singal, D. P. 1979.
Juvenile-onset diabetes: HLA-A, -B, -C, and -DR
alloantigens. Diabetes 28:1.


53


84
using 2 x 10^ BB spleen cells/well and 0.3 x 10^ WF
spleen cells/well.
Lymphocytes from BB rats also responded poorly to
various concentrations of PHA at 0.3 x 10^ spleen cells/
well (Figure 18), 1 x 10^ spleen cells/well (Figure 19),
and 2 x 10^ spleen cells/well (p <0.05, Figure 20), in
comparison with WF lymphocytes. Although similar results
were obtained when using 1 x 10^ PBL/well (p <0.01, Figure
21), low levels of proliferation with no significant
differences between WF and BB PHA responses at 0.3 x 10^
PBL/well (Figure 22) were noted, most likely due to
insufficient numbers of cells as stated previously.
Although lymphocytes from BB rats were most responsive
to Con A of the mitogens used, WF cells still responded
significantly better at all of the following cell concen
trations: 0.3 x 10^ spleen cells/well (Figure 23),
1 x 10^ spleen cells/well (Figure 24), 1 x 10^ PBL/well
(p <0.01, Figure 25) and 2 x 10^ spleen cells/well (p
<0.005, Figure 26). As was true for PWM and PHA, WF
lymphocytes at 0.3 x 10^ PBL/well responded poorly to Con
A and thus no significant differences were observed at this
cell concentration between BB and WF proliferative responses
(Figure 27). However, in one experiment, splenic
lymphocytes from a single BB rat at 2 x 10^ cells/well
were able to mount a significant response to Con A
comparable to responses by WF spleen cells. However, BB
lymphocytes from the same animal minimally responded to Con


FIGURE 30. Hematoxylin and eosin stained section of a
thymus from a WF rat at 21 days of age.


y U03 [ui/Brt g
y uo3 pu/Brt \
y 1103 iu/Brf g
CPM (Xl0"


172
42. Eisenbarth, G. S.f Morris, M. A., and Scearce, R. M.
1981. Cytotoxic antibodies to cloned rat-islet-cells
in serum of patients with diabetes mellitus. J. Clin.
Invest. 67:403.
43. Sai, P., Boitard, C., Debray-Sachs, M., Pouplard, A.,
Assan, R., and Hamburger, J. 1981. Complement-fixing
islet-cell antibodies from some diabetic patients alter
insulin release in vitro. Diabetes 30:1051, 1981.
44. Nielsen, J. H., Eff, C., Deckert, T., and Anderson, A.
1981. Stimulatory effect of serum from diabetic pa
tients on insulin release from mouse pancreatic islets
maintained in tissue culture. Diabetologia 20:60.
45. Galbraith, R. M. 1979. Immunological aspects of
diabetes mellitus. CRC Press, Boca Raton, FL. p. 13.
46. MacCuish, A. C., Urbaniak, S. J., Campbell, C. J.,
Duncan, L. J. P., and Irvine, W. J. 1974. Phytohemag
glutinin transformation and circulating lymphocyte
populations in insulin-dependent diabetic patients.
Diabetes 23:708.
47. MacCuish, A. C., Jordan, J., Campbell, C. J., Duncan,
L. J. P., and Irvine, W. J. 1975. Cell-mediated immu
nity in diabetes mellitus. Diabetes 24:36.
48. Ragab, A. H., Hazlett, B., and Cowan, H. D. 1972.
Response of peripheral blood lymphocytes from patients
with diabetes mellitus to phytohemagglutinin and
candida albicans antigen. Diabetes 21:906.
49. Hann, S., Kaye, R., and Falkner, B. 1976. Subpopula
tions of peripheral blood lymphocytes in juvenile
diabetes. Diabetes 25:101.
50. Cattaneo, R., Saibene, V., and Pozza, G. 1976. Peri
pheral T-lymphocytes in juvenile-onset diabetics (JOD)
and in maturity-onset diabetes (MOD). Diabetes 25:
223.
51. Muller, R., Kolb, H., Kuschak, D., Jorgens, V., and
Gries, F. 1980. Analysis of T-lymphocyte subpopula
tions in juvenile-onset diabetics. Clin. Exp.
Immunol. 39:130.
52. Brody, J., and Merlie, K. 1970. Metabolic and bio
synthetic features of lymphocytes from patients with
diabetes mellitus-similarities to lymphocytes in chron
ic lymphatic leukemia. Br. J. Haematol. 19:193.


33
TABLE 1
IDD AND AUTOANTIBODIES IN BB/O, BB/W,
WF AND BB x WF FI HYBRID RATS
Rats
Autoantibodies ,
IDD % ICA % PCA % SMA % TCA %D
BB/O 80 0 35 55 5
(n=20)
BB/W 75 0 68 61 18
(n=28)
BB x WF FI 0 0 0 21 0
(n=50)
WF 0 0 0a 7 0
(n=30)
aOne rat had equivocal PCA. All rats ascertained for the
above were studied up to and beyond 6 months of age.
^Thyroid colloid autoantibodies.


and Flo Jordan in the preparation of this manuscript and
other papers is deeply appreciated. I would also like to
thank the graduate students of the Department of Pathology
for their encouragement and friendship.
Finally, these acknowledgements would not be complete
without mentioning some of the special people whose friend
ship and support have been important in the completion of
this endeavor: Joan Appleyard, Steve Noga, Dan Cook,
and Art Alamo.
IV


TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
COMMONLY USED ABBREVIATIONS vi
ABSTRACT viii
INTRODUCTION 1
Insulin-dependent Diabetes (IDD) 2
The BB Rat 15
SPECIFIC AIMS 18
MATERIALS AND METHODS 21
RESULTS 32
DISCUSSION 149
The BB rat as a model of IDD and organ-
specific autoimmunity 149
The BB rat as a model of
immunodeficiency 156
REFERENCES 168
BIOGRAPHICAL SKETCH 183
v


FIGURE 20. Comparison of proliferative responses (cpm) of
splenic lymphocytes at 2 x 10^ cells/well from 2 non
diabetic BB rats (striped bars) and 2 WF rats (open bars)
to 5 concentrations of PHA. Each value is mean of
triplicate cultures + S.D.


105
A at lower cell concentrations. Although sharp decreases in
responsiveness to PWM, PHA and Con A were observed in many
older WF rats who were greater than 250 days of age, no BB
responses exceeded even the proliferative responses observed
in older WF animals.
Effect of Irradiated or Nonirradiated Allogeneic Ceils on
Mitogenic Responses.
In order to determine whether the lack of significant
responses by BB lymphocytes to mitogens was due to increased
suppressor activity, irradiated or nonirradiated BB spleen
cells were added to WF responders, and the subsequent mito
genic responses measured. To examine the possibility of in
sufficient levels of necessary lymphokines or helper factors
as a cause of BB unresponsiveness, the effects of the
addition of irradiated WF cells on BB mitogenic responses
were also studied. Irradiated WF cells, although unable to
proliferate, can secrete helper factors such as IL 2
(115,116). The presence of IL 2 or T cell growth factor is
necessary for lymphocytes to proliferate in response to
mitogens or to allogeneic cells in MLCs (117-120).
Experiments were performed with similar results using
either PWM, PHA or Con A and either 1 x 10^ PBL/well or
0.5 x 10^ spleen cells/well as responders. Proliferation
of WF splenic lymphocytes to varying concentrations of Con A
(0.5 4.0 pg/ml) was not overall significantly suppressed
by the addition of either 0.5 x 10^ irradiated or
0.5 x 105 nonirradiated BB spleen cells, nor were the


FIGURE 27. Comparison of responses (cpm) of PBL at 0.3 x
10^ cells/well from diabetic BB rat (first striped bar), 2
nondiabetic BB rats (remaining striped bars) and 3 WF rats
(open bars) to 1 yg/ml Con A and 5 yg/ml Con A. Each value
is mean of triplicate cultures + S.D.


FIGURE 28. Proliferative activities (cpm) of Con A
supernatants from 13 diabetic and nondiabetic BB rats and 9
WF rats on a murine IL 2-dependent cytotoxic T cell line.
Activities of 6 dilutions of a control murine Con A
supernatant are also indicated. Plotted in increasing
order, the dilutions used: 1.5%, 3.1%, 6.3%, 12.5%, 25% and
50%. Lines indicating mean + 1 S.D. of IL 2 activity of
Con A sups from each group are shown.


FIGURE 4. Positive indirect immunofluorescence staining of
the parietal cells of rat gastric fundus.




TABLE 19extended
5 9/10 3 nays 3 BB x WF Fix 2
1
6 7/8 7 Days 3 BB x WF Fix 1
1
1 BB w/o It 1
Spleen + Lymph nodes 70 x 10
Spleen + Lymph nodes 70 x 10
+ Pancreas
Spleen + Lymph nodes 60 x 10
Spleen + Lymph nodes 60 x 10
Pancreas
Spleen + Lymph nodes 60 x 10
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
3 CellB fran spleen, mesenteric lymph nodes and peritoneal cavity were injected into tail veins of recipients. Pancreas suspension was
given intraperltoneally.
b Hyperglycemia defined as blood glucose levels greater than 250 mj/dl.
c Pancreases were removed from recipients 90-120 days after receiving donor cells.
'' ICR, TMR, PCA and SMR were sought in recipient rats.
e x designates recipients Irradiated with 300-350 rads 24 hours before transfer.
^ Blood gluaose levels were run on recipients on day 0, day 3 and every 7 days after for at least 90 days.
^ ICR, TFW or PCR were never detected in any BB rats. Ftaur rats positive for autoantibodies had SMR.
144


RESULTS
Age at Onset and Frequency of IDD. The frequency of IDD
was determined in the original 20 BB/0 and 28 BB/W rats.
The incidence of IDD was found to be 80% (16/20) in BB/O
rats, while IDD developed in 75% (21/28) of BB/W animals
(Table 1). However, two female BB/O rats greater than 285
days of age and one male BB/W rat who had reached 180 days
of age without developing overt IDD, had one hour peak
glucose levels of 513 mg/dl, 671 mg/dl and 298 mg/dl
respectively, after glucose loading. Peak glucose levels
seen in three control rats did not exceed 220 mg/dl after
glucose loading (data not shown). Thus, these three BB rats
were considered to have noninsulin-dependent diabetes.
No significant sex difference was seen in the frequency
of IDD in either BB/O or BB/W animals. The time period for
onset of IDD in the BB/O rats was from 90 to 120 days of age
(Figure 1), while the BB/W rats had a broader age range for
onset of IDD of between 70 and 161 days (Figure 2). All BB
rats were studied beyond 180 days of age, which exceeded the
critical age span for development of the disease in these
rats. Mild to moderate insulitis was seen in pancreases
from BB rats at onset of IDD (Figure 3). None of the
32


161
serve as a second signal which is necessary for the
continuous proliferation of activated T cells (116,135,136).
The latter has been supported by evidence that T lymphocytes
require IL 2 for proliferation after activation to mitogens
and to alloantigens (117,120,137). Thus, the inability of T
lymphocytes to respond to or to produce IL 2 could result in
serious defects in normal T cell function. The Mrl/Mp-
lpr/lpr mouse which develops both autoimmune and lymphopro-
liferative diseases has absent IL 2 activity (138).
With a few exceptions, irradiated WF cells, which were
still capable of secreting helper factors such as IL 2, did
not enhance mitogenic or MLC responses of BB lymphocytes.
BB cells also did not proliferate as well as lymphocytes
from WF rats to known IL 2-containing Con A sups, nor did
the addition of these conditioned media augment prolifera
tive responses of BB lymphocytes to Con A or to allogeneic
cells in MLCs. However, after rabbit Ig-antirat Ig treat
ment, T cells from BB animals did respond better to Con A
sup, but still to a much less degree than lymphocytes from
WF rats. This positive effect is probably explained by the
removal of suppressor cells.
Lymphocytes from BB rats were able to produce IL 2,
although in many cases, not as well as WF animals. This
finding suggests that sufficient amounts of IL 2 were
normally present in BB mitogen and MLC reaction wells, and
especially after the addition of irradiated WF cells or Con
A sup. It is thus probable that BB lymphocytes are


FIGURE 16. Responses (cpm) of PBL at 0.3 x 10^ cells/well
from 1 diabetic BB rat (first striped bar), 2 nondiabetic
BB rats (remaining striped bars) and 3 WF rats (open bars)
to 1 yg/ml PWM and 5 yg/ml PWM. Each value is mean of
triplicate cultures + S.D.


73


141
TABLE 18
SERUM GAMMA GLOBULIN LEVELS IN BB AND WF RATS
Sera
% Gamma Globulins in Serum3
10 BB
rats with IDD 5.4 + 1.7
7 BB
rats without IDD 4.4 + 2.8
7 WF
rats 5.2 + 1.5
a
Each value is mean + S.D


20
e. The proliferative responses of BB and WF thymo
cytes to mitogens were compared.
f. Autoantibodies to thymocytes were sought in
sera from BB rats.
g. Histological examination of thymuses, spleens,
and lymph nodes from BB rats were made.
h. Gamma globulin levels were measured in BB rats
as a gross indication of B lymphocyte function.
3. Attempts to transfer IDD with pancreas extracts and
cells from spleens and lymph nodes from BB rats to
nondiabetic BB, WF and BB x WF FI rats were
performed. Positive findings would thus provide
convincing proof of an autoimmune etiology for IDD
in the BB rat.
4. Evaluation of the genetics of IDD and autoanti
bodies in the BB rat.
a. Different mating combinations were performed
between BB rats with and without IDD and/or
autoantibodies in order to observe the modes of
inheritance of these autoimmune parameters.
b. Crosses between male BB rats with IDD and both
female WF and Lewis rats were performed in
order to observe the relationships between IDD,
autoantibodies, and the rat major histocompati
bility complex, RT.l.


175
74. Galbraith, R. M., and Fudenberg, H. H. 1977. Auto
immunity in chronic active hepatitis and diabetes
mellitus. Clin. Immunol. Immunopathol. 8:116.
75. Cudworth, A. G. 1978. Type I diabetes mellitus.
Diabetologia 14:281.
76. Rotter, J. I., and Rimoin, D. L. 1978. Heterogeneity
in diabetes mellitusUpdate 1978: Evidence for fur
ther genetic heterogeneity within juvenile-onset
insulin-dependent diabetes mellitus. Diabetes 27:599.
77. Nerup, J., Christy, M., Kromann, H., Andersen, 0. 0.,
Platz, P., Ryder, L. P., Thomsen, M., and Svejgaard,
A. 1980. Genetic susceptibility and resistance to
insulin-dependent diabetes mellitus (IDDM). In
Immunology of Diabetes. W. J. Irvine (Ed.). Teviot
Scientific Publications, Edinburgh. p. 55.
78. lloren, J., Herva, E., Tiilikainen, A. 1978. HLA-Dw2
as a marker of resistance against juvenile diabetes
mellitus. Tissue Antigens 11:144.
79. Sachs, J. A., Cudworth, D., Jaraquemada, D., Gorsuch,
A. N., and Festenstein, H. 1980. Type I diabetes and
the HLA-D locus. Diabetologia 18:41.
80. Raum, D., Ayer, C. A., Stein, R., and Gabbay, K. H.
1979. Genetic marker for insulin-dependent diabetes
mellitus. Lancet 1:1208.
81. Cudworth, A. G., Usher, N., and Woodrow, J. C. 1977.
Factor B phenotypes in "juvenile-onset" diabetes.
Diabetologia 13:388.
82. Kirk, R. L., Theophilus, J., Whitehouse, S., Court, J.,
and Zimmet, P. 1979. Genetic susceptibility to dia
betic mellitus: The distribution of properdin factor
B(Bf) and glyoxalase (GLO) phenotypes. Diabetes 28:
949.
83. Suciu-Foca, N., Nicholson, J. F., Reemstma, K., and
Rubinstein, P. 1977. The HLA system and the genetics
of Juvenile Diabetes Mellitus. Diabete et Metabolisme
3:193.
84. Rubinstein, P., Ginsberg-Fellner, F., and Falk, C. T.
1980. Type I diabetes: A recessive disease? In
Immunology of Diabetes. W. J. Irvine (Ed..). Teviot
Scientific Publications, Edinburgh. p. 109.


TABLE 14extended
Cx
(0.25)
24407 4
3655
n.d,

(0.5)
31223 4
4232
51176 4
934

(1.0)
43031 4
20594
70704 4
1492
"
(2.0)
n.d
04221 4
3507
Ax
(0.25)

Cx
(0.25)
14591 4
7000
n.d
"
f
*
(0.5)
27071 4
4755
53414 4
3772
"
f

(1.0)
49403 4
8270
103099 4
3443
-


(2.0)
n.d

8B840 4
5132
A
(0.25)

Cx
(0.25)
14897 4
4901
n.d


(0.5)
30920 4
4272
59194 4
10402



(1.0)
39400 4
5883
05855 4
7407

f
M
(2.0)
n.d

99303 4
5110
Cbn
A aup
63083 4
10030
50015 4
6453
*

Cx
(0.25)
60078 4
613
n.d
f
"
(0.5)
44642 4
3533
103600 4
3360

-
(1.0)
51319 4
1684
106304 4
1574

-
(2.0)
n.d
.
149534 4
2526
C Lewie (0.25)
-
16769 4 3309
2912 4
690
Ax
(0.5)
20601 4 1282
26483 4
4564
M
(1.0)
n.d.
31363 4
7018
Bx
(0.5)
19700 4 3614
24212 4
3213
*
(1.0)
n.d.
7000 4
1012
M
(2.0)
n.d.
15153 4
592
a Rabbit Ig antirat Ig treated apleen celia were ueed aa reapondera at 0.25 x 10^ cells/well.
* Unaeparated apleen celia were uaed aa atijiulatora at 0.2S-2.0 x 105 cella/well.
Each value is expressed aa mean of triplicate cultures S.D.
0.1 ml of VF Cbn A sup was added to each well.
e
Not (tone
118


77
0 yg/ml PWM
1 jug/m 1 PWM
5 yg/ml PWM


TABLE 10
III. EFFECT OF ADDITION OF IRRADIATED OR NONIRRADIATED ALLOGENEIC CELLS ON
MITOGENIC RESPONSES OF BB AND WF PERIPHERAL BLOOD LYMPHOCYTES
Counts Per Minute3
Responder (cells/well) FWM Concentration (yg/ml) Con A Concentration (yg/ml)
0
1.0
5.0
5.0
A = BB
1.0 x
io5
2096
+
17
2518 + 141
4062 + 597
23550 + 4566
B = WF
1.0 x
105
1353
+
273
25952 + 3302
24088 + 3851
202255 + 6199
A + Bxb
1.0 x
105
each
2331
+
437
4812 + 523
3958 + 31
n.d.
B + Ax
1.0 x
105
each
1957
+
219
n.d.
n.d.
245270 + 1882
Ax 1.0
x 105
n.c
i.C
n.d.
n.d.
2037 + 1630
Bx 1.0
x 105
1984
+
444
2968 + 661
825 + 171
n.d.
Each value is expressed as mean of triplicate cultures + 1 S.D.
x designates irradiated cells,
not done
109


153
but this period of observation might have been insufficient
to develop more striking gastric lesions and/or serum
vitamin B12 and iron deficiency states.
No sex difference was noted in the frequencies of PCA
in either BB/O or BB/W rats, which is in contrast to PCA in
human IDD where females are more commonly affected. PCA
first appeared and rapidly increased in frequency during the
age span that the BB rats were most susceptible to the
development of IDD and did not further increase with age
after this critical period. Thus, it appears that BB rats
with IDD and gastric autoimmunity have onsets of these
disease processes at the same time of life. However, the
rates of progression of these individual diseases to
clinical states obviously vary, as no clinical disease state
of the gastric parietal cells occurred. In studies by Riley
and Maclaren of human IDD, thyroid microsomal autoantibodies
and PCA were shown in the majority of cases to be present at
the time of onset of IDD (127). Thus, these findings in the
BB rat are of great interest, since they suggest a similar
ity between BB rats and human insulin-dependent diabetic
patients, and indicate an underlying defect in tolerance
occurring at an early age in both humans and BB rats with
IDD.
As was the case with IDD, no WF, BB x WF FI, or BB x
Lewis FI rats were positive for PCA, suggesting that PCA
were not the result of a single dominant gene. Assuming
that PCA represents some genetic predisposition, it is of


13
Mechanism of Inheritance of IDD. The apparent linkage
between IDD and HLA has made it possible to analyze the
segregation of HLA haplotypes as markers for IDD in multi
plex families with two or more siblings with IDD. Several
investigators have suggested IDD to be a recessive disease
(83,84), while others believe IDD to be dominant. In either
case, reduced penetrance would need to be invoked to explain
the segregation of IDD with HLA haplotypes (85,86). Indeed,
the penetrance or the percentage of people carrying the IDD
susceptibility gene(s) that actually have clinical IDD seems
to be quite low, approximating 15-30% in multiplex families
(86). Speilman has recently suggested a hypothesis of dif
ferential susceptibility to IDD depending on dosage of IDD
susceptibility gene(s), rather than simple dominant or re
cessive inheritance (87). Heterozygosity of IDD alleles
would result in significant susceptibility to IDD, but homo
zygosity for the gene(s) would be associated with even
greater risk (penetrance) for the disease. However, because
of uncertainty as to the random frequency of IDD genes in
the general population, the crossover rates between HLA and
IDD gene loci (if not one and the same), and the probable
genetic heterogeneity of the disease, estimations of the
mode of transmission of IDD are difficult to make (19).
Environmental Factors in the Pathogenesis of IDD. As the
concordance for IDD in monozygotic twins has been shown
by Leslie and Pyke to be at most 50% (88), environmental


FIGURE 2. Frequencies of IDD, PCA and SMA in 28 BB/W rats
with age.


FIGURE 1. Frequencies of insulin-dependent diabetes (IDD),
gastric parietal cell antibodies (PCA) and smooth muscle
antibodies (SMA) in 20 BB/0 rats with age.


19
c. Crosses between BB and WF rats were performed
in order to indicate the inheritance of auto
antibodies and their relationship to IDD.
2. Evaluation of the immune system of the BB rat.
These studies were done in the hopes of identifying
abnormalities which could both predispose to auto
immune disease and explain the increased suscepti
bility of these animals to infection. Both
diabetic and nondiabetic BB rats were studied.
a. The various circulating leukocyte populations
and major lymphocyte subsets were counted in BB
rats and compared to numbers observed in WF and
BB x WF FI animals.
b. The ability of the immune system of BB rats to
function in vivo was determined by observing
how well these rats could reject skin grafts
involving both major and minor histocompati
bility differences.
c. The ability of BB lymphocytes to function in
vitro was tested by the responses of lympho
cytes to mitogens or to allogeneic cells in
mixed leukocyte cultures (MLCs).
d. The possible presence of increased suppressor
activity in BB rats and the ability of BB
lymphocytes to produce and respond to helper
factors such as interleukin 2 (IL 2) were
determined.


2
diseases of this group, such as chronic lymphocytic thyroid
itis, however, no definitive HLA associations have been
found.
The presence of organ-specific autoantibodies in the
patient's serum does not mean the patient has clinical dis
ease nor that the antibodies are actually the cause of tis
sue damage. However, because the autoantibodies are very
specific and are found in low frequencies in the general
population, they are considered to be markers or indicators
of the presence of an autoimmune disease process occurring
in the patient, regardless of whether this process leads to
overt disease (3,4).
Proof of a primary role for autoimmunity in the patho
geneses of these putative autoimmune endocrinopathies re
mains difficult to obtain. Current etiologic concepts for
such autoimmune diseases most probably must include discus
sion of the relationship between environmental triggers
and immune responsiveness (as defined by HLA DR antigens)
in the genetically predisposed individual.
Insulin-Dependent Diabetes (IDD)
It has been suggested that human IDD may result from
the autoimmune destruction of the insulin-secreting beta
cells of the pancreatic islets. Evidence to support an
autoimmune etiology for IDD has included observations of
mononuclear infiltrations in pancreatic islets of patients
who have died suddenly after onset of IDD (5,6). These


TABLE 11extended.
A
(0.5) 4 Cx
(1.0)
170577 4 30519
17260 4 1770
4675 4 651

M
(2.0)
140302 4 5404
9460 4 430
n.d.
D
(0.5) 4 Cx
(l.n)
n.d
n.d.
15753 4 967
Cbn
A sup
n.d.
143070 4 56
00324 4 6000
-
4 Ax
(1.0)
n.d.
n.d.
05104 4 1157

Cx
(1.0)
n.d.
145223 4 4213
106705 4 7900
M
"
(2.0)
n.d.
142652 4 11720
n.d.
C Ms (0.5)
-
n.d.
1100 4 300
6455 4 2605
Ax
(0.5)
n.d.
n.d.
19993 4 643
-
(1.0)
n.d.
6715 4 142
15440 4 2610

(2.0)
n.d.
10508 4 2126
n.d.
Bx
(1.0)
n.d.
19560 4 212
11695 4 1350

(2.0)
n.d.
15939 4 434
n.d.
Dx
(1.0)
n.d.
n.d.
17366 4 2015
Ax
(0.5) 4 Bx
(1.0)
n.d.
n.d.
14243 4 530
A
(0.5) 4
(1.0)
n.d.
n.d.
17594 4 1763
Dx
(0.5) 4 Bx
(1.0)
n.d.
n.d.
20026 4 2659
D
(0.5) 4 "
(1.0)
n.d.
n.d.
22160 4 2602
D BBf
-
n.d.
n.d.
004 4 177
Ax
(1.0)
n.d.
n.d.
1253 4 132
Bx
(1.0)
n.d.
n.d.
035 4 105
Cx
(1.0)
n.d.
n.d.
625 4 255
Bx
(0.5) 4 Cx
(1.0)
n.d.
n.d.
926 4 250
8 Ml responders were
used at 0.5
x 105 cells/well.
All stimulators
were
used at 0.5
-2.0 x
10^ cells/wel1.
r Each value in expressed as mean of triplicate oil tures S.P.
^ not Vme
0.1 ml of VW Obn A sup added to relevant wells.
^In expprlinaU 1, spleens from 2 BH rats were used
111


FIGURE 31. Section of spleen from a nondiabetic BB rat
stained with hematoxylin and eosin. Normal B cell-dependent
areas (black arrow) are present, but a virtual absence of T
cell-dependent areas around arterioles (clear arrow) are
seen.


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE SPONTANEOUSLY DIABETIC BB RAT: A MODEL
OF AUTOIMMUNITY AND IMMUNODEFICIENCY
By
Melissa Ellen Elder
August, 1982
Chairman: Noel K. Maclaren, M.D.
Major Department: Pathology
The BB rat is presently the best available animal
model for human insulin-dependent diabetes (IDD). IDD in
the BB rat may result from autoimmunity since it is accom
panied by lymphocytic inflammation of the pancreatic islets
and is preventable by immunosuppression. Antibodies to pan
creatic islets (ICA) and other endocrine tissues in BB rats
were sought as evidence for an autoimmune etiology for IDD.
ICA were not detected, while smooth muscle and gastric par
ietal cell autoantibodies (PCA) were frequently identified.
Although functional abnormalities of gastric parietal cells
were not noted, PCA-positive rats had evidence of lympho
cytic gastritis. PCA appeared during the age period that
vm


166
unrelated to IDD in the BB rat. It may instead be a second
serious disorder that occurs independently of autoimmunity
in these animals.
Since lymphopenia and defects in peripheral T cell
functions were present in all BB rats studied, regardless of
age or the presence of IDD, it is difficult to visualize how
a solely T cell-mediated autoimmune process could cause
pancreatic beta cell destruction, resulting in IDD. The
participation of humoral immunity, antibody-dependent cell-
mediated cytotoxicity, and natural killer cell activity may
instead by required for the development of IDD. Interest
ingly, Mycoplasma neurolytica and Mycoplasma pulmonis
have recently been demonstrated to be potent B cell mitogens
in the rat (114,150). Because BB rats have chronic
infections of Mycoplasma mainly involving the respiratory
tract, these results suggest the possibility that polyclonal
B cell activation may occur in these animals. This
activation of B cells could result in the production of
autoantibodies to pancreatic islet cells, which either alone
or with the participation of K cells, could be cytotoxic to
beta cells, leading to IDD. Thus, the inability to transfer
IDD with lymphocytes from diabetic BB rats could be
explained as being due to the immunoincompetency of the
transferred T cells or their minimal role in the pathogene
sis of IDD in these animals.
In summary, the BB rat is severely immunocompromised
with major defects in the T cell compartment. This


TABLE 21
RESULTS OF MATINGS OF MALE BB
DIABETIC
RATS WITH
BOTO FEMALE WF AND
LEWIS RATS
Matings
Offspring
Male BB
Female
Total
Litters
Total
Rats
IDD+ (no.).
PCA+ (no.)
4/2 IDD+ PGA"
WF
1
11
0
0
1/1 IDD+ PGA+
WF
1
10
0
0
5/2 IDD+ PCA+
WF
1
9
0
0
12/3 IDD+ PGA-
WF
1
12
0
0
21/2 IDD+ PGA+
WF
1
8
0
0
7/1 IDD+ PGA+
Lewis
2
14
0
0
1/1 x WF
1/1 x WF
1
3
0
0
1/1 x WF
4/2 x WF
1
1
0
0
5/2 x WF
5/2 x WF
2
0
0
0
12/3 x WF
12/3 x WF
1
4
oa
oa
7/1 x Lewis
7/1 x Lewis
9
57
oa
oa
Not all the F2 rats were yet past the age period for susceptibility to IDD and PCA.
148


151
variable penetrance in both BB rats with IDD and in human
insulin-dependent diabetics.
The identification of ICA provided major support for
an autoimmune etiology for human IDD. In contrast to
human patients with IDD, ICA were not found in the sera of
any BB rats at anytime before, during or after onset of the
disease. These autoantibodies were also not detected on
pancreas sections from BB rats at the onset of IDD,
indicating that antibodies against IDD-specific cytoplasmic
antigens were not in fact produced in these animals.
However, these studies have not excluded the presence of
autoantibodies reactive with pancreatic beta cell surface
antigens (ICSA) in BB rats, which indeed have recently been
reported (101). It is not yet known whether ICSA were
complement fixing or cytotoxic in BB rats. ICA were not
demonstrated by indirect immunofluorescence using antirat
total immunoglobulins in addition to antirat IgG as second
antibodies, suggesting that ICA of the IgM class were also
not produced by these rats. Similarly, ICA of the IgM class
have not been identified in human patients with IDD (124).
Thus, no readily apparent marker for the disease process
resulting in IDD is available in the BB rat to identify
susceptible prediabetic animals, although detection of ICSA,
albeit cumbersome to detect, has this potential.
In contrast to reports in human IDD (15), neither
thyroid microsomal nor adrenocortical autoantibodies were
detected in any BB rats. The absence of ICA, thyroid


8
patients with IDD for less than one year, but was also
demonstrated in some noninsulin-dependent diabetics.
Positive leukocyte migration inhibition in IDD patients has
also been observed using human pancreas homogenates (56) or
insulinoma extracts (57) as antigens. Concomittantly, de
layed hypersensitivity skin reactions to porcine pancreatic
suspensions were seen by Nerup in patients with IDD who
exhibited inhibition of leukocyte migration using the same
antigen (53). In order to examine the possible role of cell-
mediated immunity to insulin and thus to beta cells in the
pathogenesis of IDD, MacCuish (47) and others (58) were able
to demonstrate significantly greater blastogenesis, as
measured by ^H-thymidine incorporation, by lymphocytes
from diabetic patients when cultured with bovine or porcine
insulin than by lymphocytes from control patients.
Huang and Maclaren were able to demonstrate specifi
cally enhanced cytoadherence and cytotoxicity of human in
sulinoma cells in vitro by peripheral blood lymphocytes from
children with IDD (59). Specific insulinoma-tumor cell
cytotoxicity, as measured by eosin exclusion, was seen both
with and without added patient sera, but was not obtained
using either lymphocytes from patients with systemic lupus
erythematosus or autoimmune thyroid disease, or with differ
ent tumor lines as targets (59,60). Peripheral blood lymph
ocytes from twenty-one out of thirty-three patients with IDD
were shown to inhibit insulin release by rat islets to glu
cose and theophyllin (61), while no inhibition of insulin


FIGURE 23. Con A responses of splenic lymphocytes at 0.3 x
10^ cells/well from 2 nondiabetic BB rats (striped bars)
and 2 WF rats (open bars). Each value is mean of triplicate
cultures + S.D.


TABLE 3
SERUM VITAMIN B12 AND IRON LEVELS IN BB AND WF RATS
BB rats with PGA (n=14)a BB rats without PCAa (n=14) WF rats (n=7)
Vitamin B12 x = 472 + 38 (244-710) x = 466 + 44 (109-759) x = 473 + 49 (275-666)
BB rats with PGA (n=14) BB rats without PCA (n=ll) WF rats (n=7)
Iron x = 223 + 13 ( 84-352) x = 210 + 21 (131-326)b x = 234 + 33 ( 84-372)
Total iron binding capacity x = 492 + 17 (328-620) x = 474 + 36 (355-565) x = 519 + 78 (285-742)
% iron binding x = 45 + 2 ( 33- 78) x = 48 + 5 ( 35- 60) x = 51 + 6 ( 26- 70)
a The BB rats used are fran the group of 48 animals designated as either BB/0 (20 rats) or as BB/W (28 rats).
All results are expressed as the mean + 1 standard error of the mean.
b Sera fran 11 BB rats without PGA were used to determine serum iron levels, however only sera from 9 BB rats
without PCA had measurements for iron binding capacity and percent iron binding performed.


17
Less direct evidence for an involvement of autoimmunity
in the pathogenesis of IDD in the BB rat includes recent
findings by Colle and coworkers of a genetic linkage between
IDD and the rat major histocompatibility complex RT.1 in F2
animals produced by initial matings of male BB rats with IDD
and RT.l incompatible female Lewis rats (106). BB rats have
also been shown to have decreased numbers of circulating T
lymphocytes (107) and to be extremely susceptible to
opportunistic infections. As is the case with human IDD,
more evidence is needed in order to prove an autoimmune
etiology for the loss of pancreatic beta cells and thus IDD
in the BB rat.


TABLE 15
II. MLC RESPONSES OF PURIFIED SPLEEN CELLS FROM BB, WF AND LEWIS RATS
Responder Stimulator .
(Celle/Well) (Cells/Well)
Grants Per Mlnutec
btpt. I fcrpt. 1 bpt. ] KpFTT
A BB
(0.5)
-
2211

1130
715

21
961 4 147
2545 4
245
Bn
(0.5)
2115

612
1239

188
1485 536
1721 4
117
-
(1.0)
2129

474
1269

714
2051 4 12
2119 4
288
-
(2.0)
1415

444
1218

978
5256 4 951
2011 4
450
Cm
(0.5)
2484

68
2118

lie
1872 4 991
n.d

(1.0)
2662

221
2152

254
2712 4 162
1521 4
511
-
(2.0)
2166

20
4114

922
5651 4 101
2881 4
112
Bn
(0.5) 4
Cn
(0.5)
1972

48
7077

291
11590 4 1016
n.d
-

-
(1.0)
5511

449
4409

1652
1918 4 1095
11148 4
2095
-

-
(2.0)
1802

14
4129
t
851
1576 4 560
15658 4
6228
Cbn
d
h sup
57258

9127
52189

480
45222 4 8189
59514 4
7818
-
Cl
(0.5)
14192

1611
51501

1269
n.d."
n.d
-


(1.0)
21884

1999
25489

1518
n.d.
66912 4
11072
*

-
(2.0)
8718

2117
9112
t
91
n.d.
50281 4
9291
B vr
0.5
-
12991

986
9610

1810
4287 4 1647
29968 4
1971
Rx
(0.5)
25070

1726
40611

9684
22175 4 2846
54508 4
7151
M
(10)
11849

1616
26650 4
0081
49987 4 6894
17502 4
1167
M
(2.0)
64484

6997
19810

1277
28011 4 1026
40601 4
8772
A
(0.5)
51077

5661
45757

6211
18119 4 6409
71014 4
1559
Cn
(0.5)
41811

5665
86202

1080
41161 4 4821
n.d

-
(1.0)
85191

1762
107759

2685
40619 4 6791
181268 4
11894
-
(2.0)
116212

5950
117505

7199
59841 4 9125
84645 4
6379
119


WF
W3/13
W3/25
x
Wistar Furth
Monoclonal antibody which defines rat T
lymphocytes
Monoclonal antibody which defines rat
helper T cell subset
Irradiated
vii


51


170
22. Irvine, W. J., Gray, R. S., and McCallum, C. J. 1976.
Pancreatic islet-cell-antibody as a marker for a symp
tomatic and latent diabetes and prediabetes. Lancet
2:1097.
23. Irvine, W. J., McCallum, C. J., Gray, R. S., Campbell,
C. J., Duncan, L. J. P., Farquhar, J. W., Vaughan, H.,
and Morris, P. J. 1977. Pancreatic islet-cell anti
bodies in diabetes mellitus correlated with the dura
tion and type of diabetes, coexistent autoimmune
disease and HLA type. Diabetes 26:138.
24. Maclaren, N., Riley, W., Rosenbloom, E., Elder, M.,
Spillar, R., and Cuddeback, J. 1982. The heterogene
ity of black insulin dependent diabetes. Diabetes 31
(Suppl. 2):257.
25. Lendrum, R., Walker, G., Cudworth, A. G., Theophanides,
C., Pyke, D. A., Bloom, A., and Gamble, D. R. 1976.
Islet-cell antibodies in diabetes mellitus. Lancet
2:1273.
26. Irvine, W. J., McCallum, C. J., Gray, R. S., and
Duncan, L. J. P. 1977. Clinical and pathogenic sig
nificance of pancreatic-islet-cell antibodies in
diabetes treated with oral hypoglycemic agents.
Lancet 1:1025.
27. Neufeld, M., McLaughlin, J., Maclaren, N., Rosenbloom,
A., and Donnelly, W. 1979. Failure to transfer dia
betes from man to mouse. N. Engl. J. Med. 301:665.
28. Dobersen, M. J., and Scharff, J. E. 1982. Preferen
tial lysis of pancreatic B-cells by islet cell surface
antibodies. Diabetes 31:459.
29. Maclaren, N., Huang, S., and Fogh, J. 1975. Antibody
to cultured human insulinoma cells in insulin-dependent
diabetes. Lancet 1:997.
30. Lernmark, A., Freedman, F., Kanatsuna, T., Patzeit, C.,
Rubenstein, A. H., and Steiner, D. F. 1980. Islet
cell surface antibodies and diabetes mellitus. In
Immunology of Diabetes (Ed.) W. J. Irvine. Teviot
Scientific Publications. Edinburgh. p. 155.
31. Lernmark, A., Sehlin, J., Taljedal, I., Kromann, H.,
and Nerup, J. 1978. Possible toxic effects of normal
and diabetic patient serum on pancreatic B-cells.
Diabetologia 14:25.


FIGURE 21. Proliferative responses (cpm) of PBL at 1 x
105 cells/ well from 1 diabetic BB rat (first striped
bar), 2 nondiabetic BB rats (remaining striped bars) and 5
WF rats (open bars) to 2 PHA concentrations. Each value is
mean of triplicate cultures + S.D.


75


FIGURE 32. Hematoxylin and eosin stained section of spleen
from a WF rat. Both B cell areas (black arrow) and T cell-
dependent areas (clear arrow) around arterioles are
present.


163
cells by BB lymphocytes after neonatal bone marrow
reconstitution (139). However, these enhanced proliferative
responses by T cells from BB rats were still less than half
as great as responses by WF and Lewis cells, indicating that
further factors contributing to the T lymphocyte immunode
ficiency may be present in the peripheral circulation.
In contrast to defects in T lymphocyte function, gamma
globulin levels were similar in both BB and WF animals.
This result suggests that perhaps the functions as well as
the numbers of B lymphocytes are unimpaired in the BB rat.
Clearly, more specific indicators of B cell function such as
IgG production and plaque-forming assays need to be per
formed before this supposition can be proven. A summary of
the findings on the BB rat is shown in Table 22.
Most T lymphocyte immunodeficiencies seen in man and
animals involve to some degree the B cell limb of the immune
system. The BB rat appears to have immunological defects
primarily of T cells. Both the nude mouse (140,141) and the
nude rat (142-144) have similarities with the BB rat, such
as deficient responses to mitogens, to allogeneic cells in
MLCs, and to IL 2. However, the obvious difference with
these athymic animals is the presence of what seems to be a
relatively normal, functioning thymus in the BB rat.
Increased incidences of lymphomas and lymphoprolifera-
tive disorders are found in many immunodeficiency and auto
immunity syndromes (145). Such a relationship is well
illustrated in New Zealand black/white FI hybrid mice which


157
production of antihapten antibodies, and the initiating cell
in graft versus host reactions (128). MRC 0X8-positive
cells are considered to be responsible for suppression of
allograft rejection (128). The T lymphopenia in BB rats
involved both major T cell subpopulations, however, W3/25-
positive cells were affected more severely than MRC 0X8-
positive cells. An inversion of the ratio of the W3/25-
positive subset to the MRC 0X8-positive subset to less than
one occurred in all BB rats with increasing age and was
probably a reflection of the specific decrease in
circulating helper T cells. Normally, as observed in
younger BB rats and WF controls, the numbers of helper T
lymphocytes were greater than the numbers of cytotoxic/
suppressor T cells. Interestingly, this ratio inversion
occurred during the same age period (90 to 120 days of age)
that the BB rats were most susceptible to the development of
IDD and PCA. This finding suggests that the abnormality in
immunological regulation acquired by BB animals with
relatively increased cytotoxic/suppressor T cells may partly
contribute to or be the result of the development of IDD and
PCA. However, even BB rats who did not develop overt auto
immunity had decreased numbers of helper T cells and
cytotoxic/suppressor T lymphocytes and inverted W3/25-
positive subset to MRC 0X8-positive subset ratios.
Concurrently with the severe depressions of both major
T cell subsets and the abnormal susceptibility of these
animals to opportunistic infections, severe defects in both


3
infiltrative lesions were predominantly composed of
lymphocytes, with few polymorphonuclear leukocytes, eosino
phils or plasma cells present and are referred to as
"insulitis" (7,8). The frequency of insulitis in patients
with IDD is variable, with reports ranging from extremely
rare (9,10) to greater than 50% (8,11) in patients with re
cent onset of IDD. Lymphocytic infiltrations of pancreatic
islets have not been demonstrated in patients with IDD of
greater than one year duration, in noninsulin-dependent
diabetics or in normal controls (12).
Islet Cell Autoantibodies in IDD. The presence of anti
bodies to pancreatic islet cells in sera of patients with
IDD and polyendocrinopathies has been extensively described
(13,14). Cytoplasmic-reactive islet cell autoantibodies
(ICA), detectable by indirect immunofluorescence on normal
pancreatic tissue, react with all cell types of the pancre
atic islets in addition to beta cells (15-17), which is in
contrast to the specific loss of beta cells seen in IDD.
ICA do not cross react with gastrointestinal cells secreting
hormones also found in pancreatic islets, such as glucagon
and somatostatin (2). These antibodies are exclusively of
the IgG class, usually fix complement and are believed to
react with a microsomal membrane lipoprotein found normally
in all islet cells (13,14,18-20). The frequency of ICA in
patients with IDD varies according to the patient's race, to
the time elapsed after clinical diagnosis of IDD, and has


TABLE 19
SUMMARY OF TRANSFER EXPERIMENTS
Transferred Cells a
Exp*:.
BB Rat Duration No. of Nixrfcer of Cells Nimtoer with b fAmtoer witlj
Donor of IDD Recipient Rats Cell Source Transferred to Each Hyperglycemia Insulitis
1
4/1
Onset
8 WF
3
Spleen
10.5 x 10b
2
Lyrgph nodes
5 x 106
2
Peritoneal cavity
4 x 106
1
No cells
2
2/1
Onset
8 WF
3
Spleen
33 x 106
2
Lynpjh nodes
3 x 106
2
Peritoneal cavity
3 x 106
1
No cells
3 1/2 Onset 8 WFxe 3
2
2
1
Spleen
13.5 x
106
0
Lynpjh nodes
5 x
106
0
Peritoneal cavity
3 x
106
0
No cells
0
0
0
0
0
7/11 9 Days 1 BB x WF Fix 1
0
Hirber with ^
Autoantibodles
0
0
0
1
0
1
0
0
0
1
0
4
Pancreas
0
0
143


This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate Council, and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Lo-C.
Noel K. Maclaren, M.D., Chairman
Professor of Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
VufwJ
H, M.D.
Scorni
Associate Professor of Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
'NUmjimv :isr, tg.
Ammon B. Peck, Ph.D.
Assistant Professor of Pathology


FIGURE 10. Proliferative responses (cpm) of increasing
concentrations of WF splenic lymphocytes to 3 concentrations
of PWM measured on day 3. The following spleen cell
concentrations were used: 0.2 x 10^ cells/well (open
circle), 0.4 x 10^ cells/well (open triangle), 0.6 x 10^
cells/well (open square), 0.8 x 10^ cells/well (open
hexagon), 1.0 x 10^ cells/well (closed circle), 1.2 x
10^ cells/well (closed triangle) and 1.6 x 10* cells/
well (closed square).


TABLE 4
ENUMERATION OF PERIPHERAL BLOOD LEUKOCYTES IN BB, WF AND BB x WF FI RATS
Animals Studied
BB Rats With
NonDiabetic BB
IDD (n=16)
Rats (n=32)
WF Rats (n=28)
FI Rats (n=8)
3
Leukocytes /mm
5673
+
1840a,b
5655
+
1761b
9680
+
2858
10843 +
1521
3
FMNs/nm
2770
+
1132C
2225
+
1015C
1334
+
923
1275 +
503
%PMNs
57
+
11C
34
+
11C
14
+
7
12 t
3
3
Lymphocytes /irm
2805
+
1071'6
3403
+
1041b
7740
+
2317
9105 +
1084
% Lymphocytes
47
+
15c,d
60
+
12b
82
+
7
84 +
4
3
Monocytes/ntn
228
+
140
187
+
113
277
+
201
432 +
251
% Monocytes
4
+
4
4
+
2
3
+
1
4 t
2
3
Bosinophils/ntn
46
+
45
136
+
309
31
+
58
0
% Eosinophils
2
+
4
2
+
4
1
+
3
0


67
cells/well) as responders (Figures 10-12). Optimal stimu
lation indices were obtained from these curves and
subsequent experiments using 1.0 yg/ml and 5.0 yg/ml PWM,
0.5% and 1.0% PHA, and 1.0 yg/ml and 5.0 yg/ml Con A in each
well. WF cells responded well to these mitogens at all cell
concentrations tested, but subsequent response comparisons
with BB cells were made using spleen cell or PBL concentra
tions at 0.3 x 10^, 1 x 10^ and 2 x 10^ cells/well.
The results were not dependent on the BB cell concentrations
used or on the presence of IDD in the BB rats.
Utilizing spleen cell concentrations of 0.3 x 10^
cells/well (Figure 13), 1 x 10^ cells/well (Figure 14),
and 2 x 10^ cells/well (p <0.0005, Figure 15), BB splenic
lymphocytes showed markedly diminutive responses to PWM in
comparison to WF splenic lymphocytes at the same mitogen
concentration. Similar relative results were obtained when
using 0.3 x 10^ PBL/well (Figure 16) or 1 x 10^ PBL/well
(p <0.01, Figure 17). However, the level of proliferation
of WF lymphocytes to PWM using 0.3 x 10^ PBL/well was
quite low, suggesting that insufficient numbers and/or types
of cells were present for optimal proliferation. Both BB
and WF proliferative responses to PWM (also PHA and Con A)
were higher using spleen cells rather than PBL as responders
but background counts also tended to be higher. PWM
responses of BB lymphocytes did increase with increasing
cell numbers per well as expected. However, BB results
comparable to those by WF cells were only observed when


FIGURE 26. Responses (cpm) of splenic lymphocytes at 2 x
1C>5 cells/well from 4 nondiabetic BB rats (striped bars)
and 4 WF rats (open bars) to 5 yg/ml Con A. Each value is
mean of triplicate cultures + S.D.


65
cytes and circulating thymocytotoxic autoantibodies, the
ability of T lymphocytes from BB rats to function normally
in vivo was studied. Both BB and WF rats are thought to
share the rat major histocompatibility complex RT.1U
genotype, while Lewis rats have the RT.l1 haplotype (106-
111). Lewis skin grafts would thus be expected to be
rejected by both BB and WF rats in less than 14 days.
However, as seen in Table 7, nondiabetic BB rats rejected
Lewis allografts significantly more slowly than expected and
in comparison with WF controls (p <0.0005). Furthermore, WF
skin grafts were not rejected by nondiabetic BB rats, while
rejections of BB allografts by WF rats occurred in 17 + 3
days (p <0.0005), suggesting the presence of multiple minor
histocompatibility differences between BB and WF rats. BB
rats were thus extremely deficient in their ability to
reject grafts across both major and minor histocompatibility
barriers, and this defect was not dependent on the presence
of IDD.
Mitogen Responsiveness. Since the skin graft results
suggested that BB rats have defective in vivo T cell-
mediated immune responses, the ability of T lymphocytes from
BB rats to respond to mitogens in vitro was next studied.
PWM, PHA and Con A, which are primarily T cell mitogens in
the rat (112-114), were used. Dose-response curves were
initially prepared for each mitogen using varying concentra
tions of each mitogen and of WF spleen cells (0.3-2 x 10^


TABLE 20
FREQUENCIES OF IDD AND PCA IN SOME MATING COMBINATIONS OF BB RATS
BB Matings3 Offspring
Male
Female
Total Litters
Total Rats
IDD+ (no.)
PCA+ (no.)
IDD PCA 7
IDD+ PCA+//-
8
20
10
6
IDD+ PCA+/
IDD- PCA+/-
9
42
15
14
IDD- PCA+/-
IDD- PCA+/
3
22
3
2C
IDD+/_d PCA+
IDD+/- PCA+
8
36
8
5
IDD 7 PCA
IDD+//- PCA-
7
21
9
7
IDD+/ PCA"
IDD+//_ PCA+
2
6
3
4
IDD+/- PCA-
IDD+/_ PCA-
3
21
8
6
IDD PCA
IDD PCA
+ -
1
2
1
0
IDD PCA
IDD PCA
1
8
3
4
IDD PCA
IDD PCA
-
-
-
-
IDD- PCA+
IDD- PCA+
3
22
3
2C
146


FIGURE 18. PHA responses (cpm) of splenic lymphocytes at
0.3 x 10^ cells/well from 2 nondiabetic BB rats (striped
bars) and 2 WF rats (open bars). Each value is mean of
triplicate cultures + S.D.


PERCENTAGE OF BB RATS
U>
Ul


15
The BB Rat
The spontaneously diabetic BB rat was first recognized
in 1974 in an outbred colony of Wistar rats at the BioBreed-
ing Laboratories (94). These rats have subsequently been
bred for the IDD phenotype. BB animals which have been
formally inbred for seven to twelve generations develop
spontaneous severe IDD at about 70-120 days of age, which is
characterized by insulinopenia, marked hyperglycemia,
ketoacidosis, weight loss and an absolute requirement for
exogeneous insulin (95). The BB rat most closely resembles
human IDD of all animal models known to date. IDD develops
in genetically susceptible male and female rats with equal
frequencies (95,96) and is thought to be inherited either as
a single autosomal recessive gene with reduced penetrance
(94) or as multiple genes.
Moderate insulitis is seen in the pancreatic islets of
BB rats at the time of diagnosis of IDD, resembling the pan
creatic lesions seen in recently diagnosed human patients
with IDD (96,97). Insulitis has also been observed in non
diabetic animals (94). Immunochemical staining of pancre
atic islets from diabetic BB rats for insulin content re
veals depletions of beta cells, especially marked in pan
creases from BB rats with longstanding IDD (98,99). Such
pancreatic islets are comprised almost exclusively of glu
cagon, somatostatin and pancreatic polypeptide-secreting
cells (94).


64
TABLE 6
THYMOCYTOTOXIC AUTOANTIBODIES IN BB AND WF RAT SERA
Autoantibody
Sera Positive
BB
rats
with IDD
undiluted
25%
(16/65)
1:2 dilution
32%
(10/31)
BB
rats
without IDD
undiluted
16%
(6/38)
1:2 dilution
47%
(8/17)
WF
rats
undiluted
6%
(1/18)
1:2 dilution
9%
(2/23)
ap <0.05 by Chi-square analysis when compared to WF rats,
p <0.01 when compared to WF rats.


106
mitogenic responses of BB lymphocytes enhanced by the
addition of 0.5 x 10^ irradiated WF spleen cells (Table
8). These latter results were, however, somewhat equivocal
about the inhibitory effects of added BB cells on WF
responses. Similar findings were obtained with PWM and PHA
using 0.5 x 10^ spleen cells/well (Table 9), and with PWM
and Con A at 1.0 x 10^ PBL/well (Table 10). Thus, the
inability of BB lymphocytes to respond to mitogenic
stimulation does not seem to be solely related to either
increased suppressor activity or to deficient levels of
lymphokines necessary for proliferation in BB reactions.
Lack of MLC Responses. Proliferative responses by lympho
cytes from BB rats to allogeneic cells in MLCs were also
studied for comparison with the skin graft findings.
Initially, the responses of 0.5 x 10^ unseparated spleen
cells from BB and WF rats to 1 x 10^ and 2 x 10^
irradiated Lewis stimulators were compared. As revealed in
three representative experiments presented in Table 11,
lymphocytes from BB rats were unable to proliferate to allo
geneic Lewis spleen cells in MLCs, while WF lymphocytes
responded well to Lewis stimulator cells. The addition of
IL 2-containing WF Con A sups or irradiated WF spleen cells
did not enhance the allogeneic responses of BB lymphocytes
to Lewis stimulators. Thus, these findings as well as the
mitogen results, suggest that even in the presence of helper
factors such as IL 2, BB lymphocytes cannot mount immune


90
3Z PHR
225% PHR .05* PHR
1% PHR
2% PHR
4% PHR


TABLE 12
I. MITOGENIC RESPONSES OF PURIFIED SPLEEN CELTS
C
Cfcxints Per Minute
Responder
(Cells/WeU)a
Mitogen
Expt. 1
Expt. 2
Expt. 3
Expt.
4
Expt.
5
Unseparated RB
(0.5)
-
451 + 132
711 + 131
n.d.
1970
+
258
973
+
106
1 ug/ml Con A
n.d.e
2095 + 526
n.d.
24831
+
1306
9656
1928
" + Con A supd
n.d.
n.d.
n.d.
7266
+
328
6233
+
738
Con A supd
273 + 197
873 + 157
n.d.
7857
+
92
6760
+
285
1% PUA
n.d.
n.d.
n.d.
3593
+
657
1609
+
166
Purifiedb EB
(0.5)
-
n.d.
1932 + 272
4773 + 259
1814
+
181
1941
+
216
1 ug/ml Con A
n.d.
8284 + 1864
n.d.
41505
+
1040
52251
+
749
" + Con A sup
n.d.
n.d.
n.d.
22533
+
1654
22678
+
2092
Con A sup
n.d.
39289 + 8603
11206 + 634
16521
+
740
28486
+
511
1% PUA
n.d.
n.d.
n.d.
9507
+
1413
7819
+
450
Unseparateil WF
(0.5)
-
515 + 96
n.d.
n.d.
27091
+
898
4295
+
245
1 ug/ml Con A
n.d.
n.d.
n.d.
362930
+
35611
153008
4-
26
" + Con A sup
n.d.
n.d.
n.d.
370641
+
21075
153850
4-
7718
Con A sup
18838 + 2455
n.d.
n.d.
278114
+
33833
132875
4-
7014
1% PUA
n.d.
n.d.
n.d.
162233
-1-
4713
48518
4-
1847
114


FIGURE 24. Comparison of proliferative responses (cpm) of
splenic lymphocytes at 1 x 10^ cells/well from 2 non
diabetic BB rats (striped bars) and 3 WF rats (open bars) to
1 yg/ml Con A and 5 yg/ml Con A. Each value is mean of
triplicate cultures + S.D.


167
immunodeficiency may be a culmination of circulating T
lymphopenia resulting from the presence of thymocyotoxic
autoantibodies and increased suppressor activity. However,
mature T cells from BB rats may also be deficient in their
ability to function normally, including being able to
respond to helper factors such as IL 2. The relationship
between these separate findings of an autoimmune diathesis
and profound immunoincompetence in the BB rat remains to be
elucidated.


152
microsomal antibodies, and adrenal antibodies in diabetic BB
rats is in striking contrast to human IDD, and might support
the hypothesis that IDD in the BB rat may not be the same
disease or have the same pathogenesis as human IDD.
However, in similarity with human IDD, PCA were
detected in a considerable number of BB rats, although no BB
rats with PCA had functional abnormalities of the gastric
parietal cells such as achlorhydria. Human PCA-positive
diabetic patients with achlorhydria are known to absorb
protein-bound vitamin B12 poorly and be susceptible to
vitamin B12 deficiences (125). However, in comparison to
the serum vitamin B12 levels measured in BB rats without PCA
and in control WF rats, serum vitamin B12 levels were not
significantly lower in BB rats with PCA, probably because
the animals had normal amounts of hydrochloric acid and
sufficient amounts of intrinsic factor. Achlorhydria has
also been shown to decrease the absorption of iron (126).
No PCA-positive BB rats had significantly lower serum iron
levels than BB rats without detectable PCA or control WF
animals.
Notwithstanding, the presence of PCA was associated
with histological evidence of chronic lymphocytic gastritis,
squamous metaplasia, and degrees of loss of the normal
gastric mucosal cells. It remains possible that BB rats may
develop atrophic gastritis, which is seen in some human
patients with PCA (127), at a greater age than the animals
in this study. These rats were followed for nine months,


10
In a provocative study, indium-labelled autologous
peripheral blood leukocytes from two of three newly diag
nosed patients wtih IDD were observed by CAT scanning to
become distributed in the same patient's pancreas after
intravenous reinjection (67). No pancreatic localization
using the same procedure was noted in scans of patients with
other diseases. Whether the lymphocytes specifically homed
to the pancreas or were trapped there nonspecifically as a
consequence of inflammation, these results suggest the
presence of pancreatic insulitis in these patients.
Associated Autoimmune Diseases. Other organ-specific
autoimmunities are found with increased frequencies in IDD
(1,19,68,69). These diseases mainly involve the thyroid,
the adrenal and the parietal cells of the gastric mucosa.
In studies by Riley and colleagues of more than 500 chil
dren with IDD, 17% of the patients had detectable thyroid
microsomal antibodies and 5% had overt thyroid disease in
comparison with less than 2% of the control population even
having thyroid antibodies (15). Adrenal autoantibodies and
Addison's disease were also more frequent in Caucasian chil
dren with IDD, being found in 2% and 0.5% of insulin-depen-
dent diabetics respectively, compared to adrenal antibodies
being detected in 0.7% of matched controls (15,69). Final
ly, gastric parietal cell autoantibodies were detected in
approximately 9% of patients with IDD and in only 1% of
matched controls (19). Children with other autoimmune


TABLE 17extended
WF
0.5 x 105
1.0 x 105
-
n.d.
5806 + 2465
1887
+
592
0.1 yg/ml
Con
A
n.d.
n.d.
5303
+
1170
0.1 yg/ml
Con
A +
sup
n.d.
n.d.
96032
+
6500
1.0 yg/ml
Con
A
n.d.
156783 + 11178
37747
+
2108
1.0 yg/ml
Con
A +
sup
n.d.
327965 + 14647
136274
+
1854
sup
n.d.
n.d.
109054
+
5122
-
1707 + 453
7287 + 1054
8080
+
1629
0.1 yg/ml
Con
A
n.d.
n.d.
5393
+
813
0.1 yg/ml
Con
A +
sup
n.d.
n.d.
150478
+
14030
1.0 yg/ml
Con
A
3892 + 518
205707 + 7302
60307
+
6737
1.0 yg/ml
Con
A +
sup
27281 + 6875
318668 + 8665
179770
+
3557
sup
12235 + 506
n.d.
180830
+
642
Con A responses of thymocytes were measured on day 3. Each value is mean of triplicate cultures + S.D.
0.1 ml of WF Gon A sup was added to each well.
Not done
128


REFERENCES
1. Neufeld, M., Maclaren, N., and Blizzard, R. 1980.
Autoimmune polyglandular syndromes. Pediatr. Annals
9:43.
2. Cudworth, A. G., Bottazzo, G. F., and Doniach, D.
1980. Genetic and immunological factors in Type I
Diabetes. In Immunology of Diabetes (Ed.)
W. J. Irvine. Teviot Scientific Publications,
Edinburgh, p. 67.
3. Irvine, W. J., Gray, R. S., and Steel, J. M. Islet
cell antibody as a marker for early stage Type I dia
betes mellitus. In Immunology of Diabetes (Ed.)
W. J. Irvine. Teviot Scientific Publications.
Edinburgh, p. 117.
4. Maclaren, N. K. 1977. Viral and immunological basis
of juvenile diabetes. Amer. J. Diseases Children.
131:1149.
5. Gepts, W. 1976. Islet cell changes suggesting a pos
sible immune etiology of human diabetes mellitus.
Acta Endocr. Copenhagen, 83 (Suppl. 205):95.
6. Gepts, W., and Demey, J. 1978. Islet-cell survival
determined by morphology. Diabetes 527 (Suppl. 1):
251.
7. Von Meyenburg, H. 1940. Uber "insulitis" bei dia
betes. Schweiz, Med. Wochenschr. 21:554.
8. Gepts, W. 1965. Pathologic anatomy of the pancreas
in juvenile diabetes mellitus. Diabetes 14:619.
9. Ogilvie, R. F. 1964. The endocrine pancreas in human
and experimental diabetes. In Ciba Foundation Collo-
quia on Endocrinology. The Aetiology of Diabetes
Mellitus and its Complications. M. P. Cameron and
M. O'Connor (Eds.). London: Churchill. 15:49.
10.Doniach, I., and Morgan, A. G. 1973. Islets of
Langerhans in juvenile mellitus. Clin. Endocr. 2:233.
168


FIGURE 17. PWM responses (cpm) of PBL at 1.0 x 10^
cells/well from 3 nondiabetic BB rats (striped bars) and 5
WF rats (open bars). Each value is mean of triplicate
cultures + S.D.


49
infiltrations of the gastric mucosa with some loss of normal
mucosal cells and increased fibrosis (Figure 7), in
comparison with gastric fundus obtained from WF and PCA-
negative BB rats (Figure 6). However, severe atrophy of the
gastric mucosa was not found in any of the tissues studied.
In two BB rats with PCA for the longest periods of approxi
mately 7 months, degrees of squamous metaplasia of the
gastric mucosa were seen in sections taken well below the
junction between the proximal stomach and the fundus (Figure
8). Only one of these rats had IDD. No stomach sections
from spleen BB rats without PCA or 5 control WF rats
revealed inflammatory lesions of the gastric mucosa.
Characterization of Peripheral Leukocyte Populations. Due
to observations of increased susceptibility to opportunistic
infections (especially of the respiratory tract) in both
diabetic and nondiabetic BB rats, and indications of both
pancreatic and gastric autoimmunities in this strain, immuno
logical studies of these animals were performed.
Increased percentages and absolute numbers of
peripheral blood polymorphonuclear leukocytes (PMNs) were
observed in all BB rats regardless of the presence of IDD,
in comparison with WF and FI hybrid rats (p <0.0025, Table
4), perhaps reflective of the increased rate of infections
among these animals. In contrast, all 16 BB rats with IDD
and all 32 nondiabetic rats, ranging in age from 25 to 400
days, were observed to have significantly decreased absolute


PERCENTAGE OF BB RATS
IOO
90-
80-
70-
60-
50-
40-
30-
20-
10-
20
00 PC A
SMA
* IDD
AGE (days)
U>


61
observed in all BB rats when compared to WF control and BB x
WF FI rats. Numbers of T lymphocytes in both lymphocyte
subsets of FI hybrid animals tended to range between those
of WF and BB rats. However, there were no significant
differences between WF and BB x WF FI values.
An inversion of the W3/25-positive subset to MRC 0X8-
positive subset ratio to less than 1.0 (mean 0.7 + 0.2) also
occurred in BB rats between 75 to 115 days of age, which was
not influenced by the presence of IDD (p <0.001, Figure 9).
In younger BB rats with and without IDD, the mean W3/25-
positive subset to MRC 0X8-positive subset ratio was similar
to the mean ratio seen in WF rats at all ages studied (1.2 +
0.2 versus 1.3 + 0.1).
Presence of Thymocytotoxic Autoantibodies. As a possible
explanation for the extremely decreased numbers of T lympho
cytes present in BB rats, autoantibodies to BB or WF thymo
cytes were sought in these animals. As shown in Table 6,
many unabsorbed sera from both diabetic and nondiabetic BB
rats had demonstrable thymocytotoxic autoantibodies,
especially when the sera were tested at 1:2 dilution.
Although a few WF sera were also antibody positive, signifi
cantly more BB sera had antibodies to thymocytes (p <0.05).
Only sera giving reactions of greater than 50% cytotoxicity
were considered to be positive for these autoantibodies.
Depressed Ability to Reject Allografts. Because BB rats
had both severely decreased numbers of peripheral T lympho-


150
almost universally associated with HLA DR3 and/or HLA DR4-
bearing haplotypes. The mode of transmission of IDD in man
is currently thought to be complex; however, with the proba
bility of polygenic inheritance as modified by environmental
factors. In observations reported here, no BB x WF FI rats
or BB x Lewis FI animals developed IDD, indicating that IDD
in the BB rat, as is the case with IDD in Caucasian
populations, is not dominantly inherited as a single gene.
The incidences of IDD in the offspring of matings between
two diabetic BB rats or two nondiabetic BB animals could be
explained by inheritance as a single autosomal recessive
gene with low penetrance or as multiple genes. Only the F2
animals produced by crosses of BB x WF FI rats or BB x Lewis
FI rats can provide definitive evidence for a recessive mode
of inheritance for IDD in the BB rat. At the time of this
writing, most F2 animals in this laboratory were not old
enough to develop IDD. However, Colie et al. (123) has
recently found that a few BB x Lewis F2 rats developed IDD,
while other rats only developed pancreatic insulitis,
suggesting that IDD may result from the interaction of at
least two genes. The F2 rats that developed IDD all had at
least one RT.1U (BB) bearing haplotype. Because only four
F2 rats have actually developed IDD in Colle's findings to
date, more extensive results are needed before the mode of
inheritance of IDD in the BB rat is actually proven. Not
withstanding, the studies to date support a polygenic inher
itance involving major histocompatibility complex genes and


FIGURE 6. Section of normal BB rat gastric fundus stained
with hematoxylin and eosin. Organized rows of parietal
cells (black arrow) are present.


FIGURE 8. Hematoxylin and eosin stained section of gastric
fundus from a BB rat with PCA for 7 months. Lymphocytic
infiltration, fibrosis and squamous metaplasia (black arrow
of the mucosa are present.


165
develop diverse nonorgan-specific autoantibodies, defects in
cell-mediated immunity, and malignant lymphomas (145-147).
The BB rat appeared to have an increased frequency of
lymphomas, all of which seem to be composed of B lymphocytes
(data not shown in results). Such a finding might be
expected, since most lymphomas are of B cell origin and
because the numbers of T cells were so severely reduced in
these animals.
Unlike other models for immunodeficiency associated
with autoimmunity such as New Zealand black/white FI mice
(147), primary human hypogammaglobulinemia (148) and ataxia-
telangiectasia syndromes (148,149), the BB rat appears to be
unique in exhibiting T lymphocyte immunoincompetence in
association with IDD. The disorder that appears to be most
comparable to the BB rat is Type I autoimmune polyglandular
syndrome seen in humans (1). The affected patients develop
autoimmunity to most endocrine glands, albeit except the
pancreatic islets, early in life. In addition, these
patients have functional defects in peripheral T lymphocytes
manifested by their development of overwhelming infections
with Candida albicans and chronic mucocutaneous
moniliasis.
Clearly, such severe defects in immunocompetence as
observed in BB rats, are not found in human patients with
IDD. However, more subtle immunological abnormalities may
be present in human diabetic patients that have not yet been
diagnosed. The T cell immunodeficiency may also be


88
0% PHfl
0.5% PHfl
1.0% PHfl


FIGURE 19. Proliferative responses (cpm) of splenic lympho
cytes at 1 x 1C)5 cells/well from 2 nondiabetic BB rats
(striped bars) and 2 WF rats (open bars) to 0.5% and 1.0%
PHA. Each value is mean of triplicate cultures + S.D.


11
endocrinopathies in addition to IDD have even higher fre
quencies of associated organ-specific autoantibodies (19).
For example, patients with both IDD and thyroid microsomal
antibodies have augmented incidences of adrenal antibodies
to about 6% (19). Organ-specific autoantibodies and auto
immune disease are not found in increased frequencies in
noninsulin-dependent diabetics (19).
HLA Associations with IDD. Singal and Blajchman first
reported that IDD was associated with disturbed frequencies
of HLA antigens and noted an increase in HLA B15 in these
patients (70). Nerup and coworkers later documented
statistically significant increases in HLA B8 and HLA B15 in
patients with IDD in comparison with matched controls, while
no HLA differences were seen between controls and noninsulin
-dependent diabetics (71). Subsequent studies have both
confirmed and extended these observations (72-74). Cudworth
(75) and others (76) have postulated that there are two HLA
haplotypes associated with increased relative risks for
IDD: HLA Al (A30) B8 (B18) Cw3 DR3 and HLA A2 B15 (B40)
DR4. The primary association of IDD seems to be with the
HLA DR antigens and secondarily due to linkage disequilib
rium with the HLA A and B antigens (2,72,73,77). HLA DR3
is found in 36-59% of IDD patients compared to between 11-
24% in normal controls (72), while HLA DR4 is seen in 32-
58% of insulin-dependent diabetics compared to 16-28% in the
general population (72). Patients with multiple autoimmune


132



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182
149. Ammann, A. J., and Hong, R. 1971. Autoimmune pheno
mena in ataxia telangiectasis. J. Pediatrics 78:821
150. Naot, Y., Siman-Tov, R., and Ginsburg, H. 1979.
mitogenic activity of Mycoplasma Pulmonis. II.
Studies on the biochemical nature of the mitogenic
factor. Eur. J. Immunol. 9:149.


28
Research Council Cellular Immunology Unit, Oxford, England).
The specificities of the antibodies were known to be as
follows: W3/13 all T lymphocytes, W3/25 helper T cells,
MRC 0X8 cytotoxic/suppressor T cells, and MRC 0X6 la-
positive cells (109). Aliquots of 1-2 x 10^ PBL were
incubated at 4C for 30 minutes with 1:20 dilutions of these
antisera in addition to a 1:15 dilution of rabbit antirat Ig
(Accurate Chemicals, Westbury, NY) for determination of the
numbers of B cells and monocytes, and PBS or normal rat
serum alone as a control. After 2 washings with cold PBS,
all PBL aliquots were incubated with a 1:20 dilution of a
fluorescein isothiocynate conjugated goat antimouse IgG
(Cappel Laboratories) for 30 minutes at 4C, washed in cold
PBS, and resuspended in 30 yls of PBS-glycerol. Slides were
then prepared and were observed for positive immunofluore
scence under the ultraviolet microscope.
Detection of Thymocytotoxic Autoantibodies. Two yl
samples of sera from both diabetic and nondiabetic BB
animals of varying ages, WF rats and Lewis rats were applied
undiluted or at 1:2 dilution to 72 well microcytotoxicity
trays (Falcon, Oxnard, CA). A modification of the microcy
totoxicity method of Amos was used for determination of
thymocytotoxic autoantibodies (110). One yl samples of
either a BB or WF thymocyte suspension at a concentration of
2 x 10^ cells/ml were added to each well and incubated
with the various sera for 30 minutes at room temperature.


44


23
Tissue Histology. Thymuses, spleens, lymph nodes,
stomachs and pancreases were removed from BB, WF and BB x WF
FI rats and placed in a 10% formalin solution. The final
paraffin-embedded tissue sections were stained with hematox
ylin and eosin before histological examination.
Isolation of Peripheral Blood Mononuclear Cells. One to 3
ml blood samples were withdrawn from the periorbital venous
sinus or tail vein into 3 ml EDTA vacutainer tubes (Becton-
Dickinson, Rutherford, NJ) while the rats were under ether
anesthesia. The blood was diluted with phosphate-buffered
saline (PBS), layered over a Ficoll-Hypaque gradient (24:10
v/v; 9% Ficoll 400, Pharmacia, Piscataway, NJ: 34% Hypaque
M, Winthrop Laboratories, New York, NY), and the mononuclear
cells (PBL) isolated at the interface after centrifugation
at 2200 rpm for 13-15 minutes. Any remaining erythrocytes
were lysed by exposure to an ammonium chloride-Tris buffer
solution for 3 minutes at 37C. The PBL were then washed
twice in PBS before adjustment to the desired cell concen
trations in either PBS for T cell subset determinations or
in complete medium: RPMI 1640 (GIBCO, Grand Island, NY)
supplemented with 50 yg/ml gentamycin (Sobering, Kenilworth,
NJ), 5% heat-inactivated fetal calf serum (FCS) (GIBCO), and
5 x 10^M 2-mercaptoethanol (2-ME) (Bio-Rad, Richmond, CA)
for mitogen and MLC assays. Viability of cells was measured
by trypan blue exclusion.


the animals were developing IDD. Thus, the BB rat has an
autoimmune diathesis of which IDD may be only one result.
Immunocompetency of the BB rat was also studied.
Severe T lymphopenia was observed in all BB rats, irrespec
tive of age or presence of IDD, while numbers of B cells
and immunoglobulin levels were normal. Both the numbers of
helper T cells and cytotoxic/suppressor T lymphocytes were
depressed, and an inversion of the ratio of helper T cells
to cytotoxic/suppressor T cells occurred in all BB rats with
maturity.
Concomitantly, profound impairments of T cell-mediated
immunity were noted to mitogenic stimulation and in allo-
responses. BB lymphocytes produced IL 2 normally; however,
irradiated WF cells and Con A supernatants did not restore
BB responses, suggesting that BB lymphocytes may have
defective responses to helper factors such as IL 2. In
contrast to BB peripheral T cells, BB and WF thymocytes re
sponded equally well to mitogens. Whereas BB thymic histol
ogy was normal, BB spleens and lymph nodes were severely
depleted of T lymphocytes. Thymocytotoxic autoantibodies
were also detected in many BB rats. These findings suggest
that the defect in T cell immunoresponsiveness may be post-
thymic or peripherally acquired. Although the BB rat is an
intriguing model of autoimmunity and immunodeficiency, no
clear relationship between the immunoincompetence and IDD
is known to date.
IX


SPECIFIC AIMS
From the previously mentioned findings, IDD in the BB
rat and in humans is thought to result from pancreatic beta
cell autoimmunity. However, much more evidence is required
to substantiate this hypothesis. In order to better under
stand the etiology and genetics of IDD in the BB rat and its
similarities to human IDD, the following studies were per
formed .
1. Identification of organ-specific autoantibodies in
BB rat sera. Several of such antibodies occur with
increased frequencies in human IDD and their pres
ence in BB rats would support a role for autoimmu
nity in the strain.
a. Autoantibodies to the cells of the pancreatic
islets, the thyroid, the adrenal gland, and the
gastric mucosa were sought in BB rats from an
early age before the development of IDD in or
der to determine whether a correlation between
IDD onset and the appearance of autoantibodies
existed.
b. Clinical evidence of disease was determined in
those rats with organ-specific autoantibodies.
18


TABLE 14
I. MDC RESPONSES OF PURIFIED SPLEEN CELLS FROM BB, WF AND LEIAIS RATS
Oounta Per HinuteC
RespoiKler
(Cell./Wei 1 (*
Stimulator .
(Calla/Well)
Expt.
1
Expt.
2
A BB (0.25)
-
676
94
445 4
72
Bx (0.25)
1240 4
124
1217 4
117
" (0.5)
906 4
121
919 4
196
* (1.0)
B12 4
175
1092 4
217
(2.0)
n.d
e
1154 4
229
Cx (0.25)
664 4
105
n.d
* (0.5)
1421 4
211
671 4
165
" (10)
1127 4
217
1215 4
215
" (2.0)
n.d
n.d
Bx (0.25) *
Cx
(0.25)
2907 4
192
n.d
"
M
(0.5)
2221 4
626
5719 4
816
*
M
(10)
2518 4
698
1560 4
725
"
M
(2.0)
n.d
n.d
Oon A up*
14041 4
11216
10897 4
2116
"
Cx
(0.25)
21776 4
8211
n.d
"
M
(0.5)
29994 4
4554
20912 4
1920
-
(10)
26999 4
4176
7521 4
1911
"
(2.0)
n.d
n.d.
B WF (0.25)
-
7218 4
14-28
1291 4
689
Ax (0.25)
12942 4
167
4162 4
200
" (0.5)
18121 4
596
10118 4
1819
(1.0)
5921 4
1217
16478 4
1207
(2.0)
n.d
n.d
A (0.25)
9676 4
82
7497 4
1959
117


AGE (days)
W3/25 SUBSET to MRC 0X8 SUBSET RATIO
ro
ts
9


173
53. Nerup, J., Andersen, 0. 0., Bendixen, G., Egeberg, J.,
and Poulsen, J. E. 1971. Antipancreatic cellular
hypersensitivity in diabetes mellitus. Diabetes 20:
424.
54. Nerup, J., Andersen, 0. 0., Bendixen, G., Egeberg, J.,
and Poulsen, J. E. 1973. Anti-pancreatic, cellular
hypersensitivity in diabetes mellitus: Antigenic
activity of fetal calf pancreas and correlation with
clinical type of diabetes. Acta Allergol. 28:223.
55. Nerup, J., Andersen, 0. 0., Bendixen, G., Egeberg, J.,
and Poulsen, J. E. 1974. Cellular hypersensitivity
to islet antigen(s) different from insulin in diabetes
mellitus. In Immunity and Autoimmunity in Diabetes
Mellitus. P. A. Bastenie and W. Gepts (Eds.).
Excerpta Medica, Amsterdam. p. 107.
56. MacCuish, A. C., Jordan, J., Campbell, C. J., Duncan,
L. J. P., and Irvine, W. J. 1974. Cell-mediated
immunity to human pancreas in diabetes mellitus.
Diabetes 23:693.
57. Irvine, W. J., MacCuish, A. C., Campbell, C. J., and
Duncan, L. J. P. 1976. Organ-specific cell-mediated
auto-immunity in diabetes mellitus. Acta Endocr.
(suppl.)205:65.
58. Faulk, W. P., Girard, J. P., and Welscher, H. D. 1975.
Cell-mediated immunity to insulin and its polypeptide
chains in insulin-treated diabetics. Int. Arch.
Allergy Appl. Immunol. 48:364.
59. Huang, S., and Maclaren, N. 1976. Insulin-dependent
diabetes: A disease of auto-aggression. Science
192:64.
60. Maclaren, N. K., and Huang, S. W. 1980. In vitro
antipancreatic cell-mediated immunity. In Immunology
of Diabetes (Ed.) W. J. Irvine. Teviot Scientific
Publications. Edinburgh. p. 185.
61. Boitard, C., Debray-Sachs, M., Pouplard, A., and
Assan, R. 1980. Inhibition of insulin release by
lymphocytes from diabetic patients in vitro. Diabet-
ologia 19:259.
62. Pozzilli, P., Gorsuch, A., Sensi, M., Bottazzo, G. F.,
and Cudworth, A. B. 1979. Evidence for raised K-cell
levels in type-I-Diabetes. Lancet 2:173.


FIGURE 12. Proliferative responses (cpm) of increasing
concentrations of WF splenic lymphocytes to 3 concentrations
of Con A measured on day 3. The following spleen cell con
centrations were used: 0.2 x 10^ cells/well (open
circle), 0.4 x 10^ cells/well (open triangle), 0.6 x 10^
cells/well (open square), 0.8 x 10^ cells/well (open
hexagon), 1.0 x 10^ cells/well (closed circle), 1.2 x
10^ cells/well (closed triangle) and 1.6 x 10^
cells/well (closed square).


176
85. Maclaren, N., Rosenbloom, A., McLaughlin, J., Bruck,
E., Lezotte, D. 1981. Genetics of IDD in multiplex
families. IRCS Med. Sci. 9:631.
86. Spielman, R. S., Baker, L., and Zmijewski, C. M. 1979.
Inheritance of susceptibility to juvenile onset dia
betes. Prog. Clin. Biol. Res. 32:567.
87. Spielman, R. S., Baker, L., and Zmijewski, C. M. 1980.
Gene dosage and susceptibility to insulin-dependent
diabetes. Ann. Hum. Genet., London. 44:135.
88. Leslie, R. D. G., and Pyke, D. A. 1980. Identical
twins in diabetes. In Immunology of Diabetes.
W. J. Irvine (Ed.). Teviot Scientific Publications.
Edinburgh. p. 101.
89. Craighead, J. E. 1975. The role of viruses in the
pathogenesis of pancreatic disease and diabetes
mellitus. Prog. Med. Virol. 19:161.
90. Yoon, J. W., Austin, M., Onodera, T., and Notkins, A.
1980. Virus-induced diabetes mellitus: Isolation of
a virus from the pancreas of a child with diabetic
ketoacidosis. N. Engl. J. Med. 300:1173.
91. Gamble, D. R., Taylor, K. W., and Cumming, H. 1973.
Coxsackie viruses and diabetes mellitus. Br. Med. J.
4:260.
92. Menser, M. A., Forrest, J. M., and Bransby, R. A.
1978. Rubella infection and diabetes mellitus. Lan
cet 1:57.
93. Menser, M. A., Forrest, J. M., Honeyman, M. C., and
Burgess, J. A. 1974. Diabetes, HLA antigens and con
genital rubella. Lancet 2:1508.
94. Like, A. A., Butler, L., Williams, R. M., Appel, M. C.,
Uleringer, E. J., and Rossini, A. A. 1982. Spontan
eous autoimmune diabetes mellitus in the BB rat.
Diabetes 31 (suppl.):7.
95. Nakhooda, F., Like, A., Chappel, C., Murray, F., and
Marliss, E. B. 1976. The spontaneously diabetic
Wistar rat: Metabolic and morphologic studies. Dia
betes 26:100.
96. Nakhooda, A. F., Like, A. A., Chappel, C., Wei, C-N.,
and Marliss, E. B. 1978. The spontaneously diabetic
Wistar rat (the "BB" rat): Studies prior to and during
development of the overt syndrome. Diabetologia
14:199.


130


FIGURE 34. Hematoxylin and eosin stained section of lymph
node from a WF rat. B cell follicles (black arrow) and T
cell areas (clear arrow) are indicated.


TABLE 13
II. MITOGENIC RESPONSES OF PURIFIED SPLEEN CELLS
Responder
(Cells/Well x 105)
Mitogen
Expt.
1
Expt. 2
Unseparated BB 1.0
-
676 +
102
n. d.
1 yg/ml Con
A
9661 +
4321
n. d.
Con A supc
n .d
d

n. d.
Purified9
BB 1.0
-
1787 +
525
4053 + 587
1 yg/ml Con
A
39289 +
8603
n. d.
Con A sup
n. d

21277 + 1699
Purified9
WF 1.0
-
13934 +
433
351 + 157
1 yg/ml Con
A
230554 +
21032
n. d.
Con A sup
358594 +
9869
44952 + 10247
Purified spleen cells were passed through rabbit Ig-antirat Ig columns.
Each value is expressed as mean of triplicate cultures + S.D.
0.1 ml of WF Con A sup added to each well.
d
Not done
116


12
endocrinopathies have an even higher frequency of HLA Al B8
DR3 haplotypes and especially HLA DR3, suggesting that a
gene predisposing for general organ-specific autoimmunity is
associated with HLA DR3 (2,45). The risk of developing IDD
is highest for HLA DR3/DR4 heterozygotes, implying the
possibility of the existence of at least two hereditary
susceptibility genes for IDD associated wtih HLA genesone
associated with HLA DR3 and the other with HLA DR4 (34,45,
77,78). One haplotype has been suggested to possibly render
protection against IDD because it is decreased and virtually
absent in Caucasian patients with IDD: HLA A3 (All) B7 DR2
(2,75,77,78), although the low frequencies of HLA DR2-bear-
ing haplotypes in IDD may be due in part to the increased
frequencies of HLA DR3 and HLA DR4 (79). Black patients
with IDD as a whole do not have as significantly disturbed
frequencies of HLA DR3 and DR4 antigens as Caucasian child
ren with IDD (24). However, those black diabetic children
who are positive for ICA seem to invariably type HLA DR3 or
HLA DR4 (24), which is probably due to the impact of Cauca
sian IDD genes in the black genome by racial admixture (24).
Families with multiple members affected by IDD tend to show
an excess of individuals typing for HLA DR3 or HLA DR4. In
addition, several investigators (80-82) have observed
significantly disturbed frequencies of several complement
factor B alleles in insulin-dependent diabetics (2). The
mechanisms through which HLA antigens, especially the immune
response associated HLA DR antigens, affect susceptibility
to IDD remain to be elucidated.


140


16
The IDD seen in the BB rats is probably not due to
recognized infectious agents, since animals raised in a
gnotobiotic environment, such that they did not develop
antibodies to any bacteria, viruses or parasites, did not
have decreased incidences of IDD (100). However, this study
was based on results from only one litter and, in any event,
does not rule out the possibility of a role for a vertically
transmitted virus in the etiology of IDD in these rats.
The typical insulitis lesions and the genetic predispo
sition for IDD suggest that IDD in the BB rat may be the
result of beta cell autoimmunity. Evidence to support such
a hypothesis includes recent findings of ICSA in twelve of
fourteen diabetic BB rats using a 12^-labelled protein A
assay (101). Other data include observations of the
reduction in frequency of IDD in susceptible BB rats
after administration of antilymphocyte serum (102),
neonatal thymectomy (103,104) or bone marrow reconstitu
tion (105). However, the experimental designs were ques
tionable since the studies were performed between groups of
litters rather than within litters. Because the incidence
of IDD varies from 0-60% in any one litter to the next, the
observed decrease in frequency of IDD, but not total IDD
prevention, may instead be due to litter assignment rather
than treatment. In addition, no research group has yet been
able to successfully transfer IDD immunologically from BB
rats to nondiabetic recipients.


154
interest to assess whether this predisposition is the same
one that leads to destruction of the beta cells of the
pancreas and IDD. Two findings suggest that the genes for
PCA and IDD might be separate. The frequencies of PCA in
BB/O and BB/W rats was different (although not significantly
so) despite similar rates of IDD. Furthermore, some rats
without discernible IDD including normal glucose tolerance
tests were found to have PCA. However, observations of
matings between diabetic BB rats without PCA producing
litters with PCA-positive rats, while nondiabetic BB rats
with PCA produced offspring with IDD, do suggest that the
gene for IDD and PCA may be one and the same, possibly with
reduced penetrance. Alternatively, as hypothesized above,
the genes for IDD and PCA may be separate but interact to
increase the degree of penetrance of the other. Further
proof of these contentions should be provided by the F2
litters obtained from matings of male diabetic PCA-positive
BB rats with female Lewis or WF animals which have not yet
reached the age for susceptibility to IDD and PCA.
Other autoantibodies were sought and found in BB rats.
SMA occurred in high frequencies, but these autoantibodies
were not associated with either PCA or IDD. That a low
frequency of SMA was found in BB x WF FI animals and WF
control rats who never developed IDD, suggests that the
process leading to formation of SMA is independent of the
processes resulting in IDD and PCA. Thus, as is the case
with autoimmune endocrinopathies in man, the organ-specific


VHd %0I VHd %S'0 VHd %0
Z6
C PM x 10*


FIGURE 13. Responses (cpm) of splenic lymphocytes at 0.3 x
10^ cells/well from 2 nondiabetic BB rats (striped bars)
and 2 WF rats (open bars) to 1 yg/ml PWM and 4 yg/ml PWM.
Each value is mean of triplicate cultures + S.D.


Commonly Used Abbreviations
BB
BB/O
BB/W
BB x WF
BB x Lewis
BioBreeding
BB rats obtained from Ottawa, Ontario
BB rats obtained from Worcester, Mass.
Cross of male BB rat with female WF rat
Cross of male BB rat with female Lewis rat
C
Con A
cpm
Degrees Centigrade
Concanavalin A
Counts per minute
DR
HLA D region-associated antigen
FI
F2
FCS
First filial generation
Second filial generation
Fetal calf serum
HLA
Major histocompatibility complex of man
la
ICA
ICSA
IDD
ig
IgG
IgM
IL 2
Immune response-associated antigen
Islet cell antibodies (cytoplasmic)
Islet cell surface antibodies
Insulin-dependent diabetes
Immunoglobulin
Immunoglobulin Class G
Immunoglobulin Class M
Interleukin 2 or T cell growth factor
MLC
2-ME
MRC 0X6
Mixed lymphocyte culture
2-mercaptoethanol
Monoclonal antibody which defines rat la-
positive cells
MRC 0X8
Monoclonal antibody which defines rat
cytotoxic/suppressor T cell subset
n. d.
Not done
PBS
PCA
PHA
PWM
Phosphate-buffered saline
Gastric parietal cell autoantibodies
Purified phytohemagglutinin
Pokeweed motigen
RT.l
Major histocompatibility complex of the rat
S.D.
SMA
Standard deviation
Smooth muscle antibodies
vi


DISCUSSION
The BB Rat As A Model Of IDD And Organ-Specific
Autoimmunity ~
There are a number of similarities between the IDD
observed in humans and in BB rats. The age period that BB
rats were most susceptible to IDD occurred during late pub
erty and corresponds to the second peak of incidence of hu
man IDD, which is during adolescence (122). In both man and
rat, this time of onset of IDD coincides with an accelerated
growth period during which insulin requirements increase.
Several important findings suggest that the pancreatic
beta cell destruction observed in human IDD may be of
immunological origin. The degree of similarity of immuno
logical characteristics between diabetic BB rats and human
patients with IDD was therefore of great interest and an
important stimulus for these studies. Initial findings have
confirmed that pancreases from all BB rats with IDD in this
laboratory had lymphocytic infiltrations of the islets of
Langerhans as has been previously reported in other BB rats
and in man.
Studies of multiplex families with IDD have confirmed
that the disease is clearly inherited with HLA haplotypes,
while population studies have demonstrated that IDD is
149


25
were resuspended in RPMI 1640 without PCS or 2-ME when used
in microcytotoxicity assays.
Preparation of Pancreas and Peritoneal Cavity Suspensions.
Pancreases were removed aseptically, rinsed in PBS, minced
and pressed through nylon mesh screens. The suspension was
then centrifuged at 1600 rpm for 10 minutes at 20C and re
suspended in 1 ml of PBS. Peritoneal cells were obtained by
rinsing the open cavity with PBS and withdrawing the fluid
by pipette. This procedure was repeated several times until
the fluid removed from the peritoneal cavity was clear. The
suspension was then spun and resuspended in 1 ml of PBS.
Production of Con A Supernatants. Spleen cells from both
BB and WF rats at concentrations of 4-5 x 10^ cells/ml
were incubated with 5 yg/ml Con A in complete medium for
16-20 hours at 37C in 25 cm^ tissue culture flasks
(Corning, Medfield, MA). The contents of the flasks were
then centrifuged at 2000 rpm for 10 minutes, the cell-free
supernatants (Con A sups) removed and filtered through
0.45 ym filters (Sybron/Nalge, Rochester, NY) and stored at
-70 C for future use.
Detection of Organ-Specific Autoantibodies. BB and WF
rat sera were applied undiluted to slides of air-dried,
unfixed pancreatic or adrenal tissue and at 1:4 dilution and


FIGURE 15. Responses (cpm) of splenic lymphocytes at 2 x
1C>5 cells/well from 4 nondiabetic BB rats (striped bars)
and 4 WF rats (open bars) to 10 yg/ml PWM. Each value is
mean of triplicate cultures + S.D.


FIGURE 22. PHA responses (cpm) of PBL at 0.3 x 10^ cells/
well from 1 diabetic BB rat (first striped bar), 2 non
diabetic BB rats (remaining striped bars) and 3 WF rats
(open bars). Each value is mean of triplicate cultures +
S.D.


6
In contrast, ICSA are reported to be more common than ICA in
controls and seem to possibly occur independently of ICA in
patients with IDD (36).
ICSA in sera from patients with IDD have been shown to
be cytotoxic to mouse pancreatic islets in vitro (37), how
ever some nondiabetic sera without ICSA were also
cytotoxic to these cells. Sera from IDD patients have also
been shown to have complement-mediated cytotoxic effects on
hamster islets (38) and rat islets (39), correlating with
the detection of ICSA in the serum. In addition, ICSA-
positive sera in the presence of complement have been dem
onstrated to cause increased chromium release from labeled
rat islet cells (40,41) while sera containing ICA alone did
not have such effects (40). Difficulties with this study
include defects in the method of ICA detection, and findings
that 25% of ICSA-positive sera from nondiabetic first degree
relatives were also cytotoxic to islet cells.
There has been little convincing evidence to suggest
that ICSA react specifically with pancreatic beta cells.
Indeed in one study, cytotoxicity of sera with detectable
ICSA was shown to not be restricted to pancreatic beta
cells, but also affected a somatostatin-producing tumor line
(42). However, Dobersen and Scharff has recently demon
strated the preferential lysis of rat beta cells with
minimal killing of other types of islet cells by ICSA-
containing diabetic sera using double-label immunofluores
cence techniques (28). In addition, eleven of twenty-one


55


123
spleen cells to Con A sup. MLC responses by BB lymphocytes
to Lewis stimulators in the presence of Con A sup were only
comparable to WF MLC results without Con A sup. The pro
liferation was most likely due to the Con A sup alone as the
counts were not generally greater when Lewis stimulators
were added. Thus, the addition of Con A sup did not trigger
BB lymphoctes to specifically respond to allogeneic Lewis
cells.
Ability of Spleen Cells to Produce Lymphokines (IL 2). As
a possible cause of immunoincompetence, the ability of BB
spleen cells to produce IL 2 was determined. Supernatants
obtained from the Con A activation of BB spleen cells
contained significant amounts of IL 2 as measured by the
proliferation of IL 2-dependent cytotoxic T cells (Figure
28). However, the levels of IL 2 in BB Con A sups were
extremely variable, while all WF Con A sups had similar
amounts of IL 2. BB lymphocytes were thus able to produce
IL 2 but were probably deficient in their ability to respond
to this factor as indicated by the previous studies.
Ability of Thymocytes to Respond to Mitogens. In order to
determine if the T lymphocyte defect was only peripheral or
also included the thymus, mitogenic responses of BB and WF
thymocytes were compared. In sharp contrast to results
obtained with spleen cells and PBL, thymocytes from BB rats
proliferated as well to both Con A and Con A sup as did WF


MATERIALS AND METHODS
Animals. One hundred and fifty BB rats of both sexes and
of varying ages in addition to Wistar Furth (WF) rats
(Charles River Laboratories, Wilmington, MA) and Lewis rats
(Charles River) were used in these studies. Initially, 40
BB rats from 15 litters were obtained from Dr. Pierre
Thibert (Animal Resources Division, Health Protection
Branch, Ottawa, Ontario) and 28 BB rats from 3 litters were
obtained from Dr. Arthur Like (University of Massachusetts,
Worcester, MA). The animals obtained from Dr. Thibert and
Dr. Like are referred to as BB/O and BB/W rats
respectively. The remaining 82 BB rats were bred in our
laboratory from the original animals obtained above. Male
BB rats with IDD were also mated with inbred WF or Lewis
females. Male and female FI progeny were then interbred to
produce F2 rats.
The animals were given Purina Rodent Chow 5001 and
water ad libitum, with light regulation at 0730 and 1830
hours. Diabetic rats were maintained on PZI insulin given
between 1500 and 1700 hours daily by subcutaneous injections
into axillary skin folds at doses sufficient to sustain
weights, minimize polyuria and avoid clinical hypoglycemic
episodes.
21


156
there may have been an unknown requirement for the target
pancreatic beta cells to express IDD-specific or BB strain-
specific antigens. This important area is one in which
further studies are obviously required.
The BB Rat As A Model Of Immunodeficiency
Due to observations of the increased susceptibility of
BB rats to opportunistic infections especially of the
respiratory tract, the status of immunocompetence in the BB
rat was next studied. BB rats of all ages exhibited a pro
found lymphopenia which was independent of the presence of
IDD. This lymphopenia was due to the specific loss of T
cells as the absolute numbers of B lymphocytes observed in
BB rats were comparable to the numbers observed in WF
controls. PMNs were increased in BB rats, most likely as a
response to the various chronic respiratory infections
normally present in these animals. Monocytes and eosino
phils were found in similar proportions in both BB and WF
animals, albeit some BB rats had an obvious eosinophila. As
a possible explanation for the severely decreased numbers of
peripheral T cells, thymocytotoxic autoantibodies were
sought and found in sera from many BB rats with and without
IDD. The actual role, if any, of these antibodies in the
development of lymphopenia in the BB rat remains to be
explored, however, their contribution seems likely.
W3/25-positive cells are thought to be the prolifera
ting helper cells in MLCs, the T helper cell in the


Dedicated to the memory of
Mr. Mellow


164
TABLE 22
SUMMARY OF FINDINGS
Findings
IDD
Susceptibility to infections
Autoantibodies
PCA
SMA
Thyroid colloid antibodies
Thymocytotoxic antibodies
BB Rats
++
++
++
++
+
++
WF Rats
+
+
Immunological Parameters
PMNs t Na
Monocytes N N
Eosinophils N or + N
B lymphocytes N N
T lymphocytes 14- N
Helper T cells +4- N
Cytotoxic/suppressor T cells 4 N
Immunoglobulin levels N N
Immunological Functions
Peripheral lymphocytes
Allograft rejection 44 N
Mitogenic responses 44 N
MLC responses 44 N
IL 2 production N N
IL 2 responsiveness 44 N
Thymocytes
Mitogenic responses N N
Lymphoid Organ Histology
Thymus N N
Spleen
T cell areas 44 N
B cell areas N N
Lymph nodes
T cell areas 44 N
B cell areas N N
a
normal


TABLE 5
Ln
VO
LYMPHOCYTE SUBSETS IN BB, WF AND BB x WF FI RATS
Animals Studied
BB Rats With BB Rats Without
IDD (n=8) IDD (n=15) WF Rats (n=12) FI Rats (n=4)
Leukocytes/mn~
4837 + 2373
a,b
4921 + 1754
3
Lymphocytes /im
2986
+
99 lb
2847
+
943b
W3/13+ Cells/nm3
2060
+
817C
2069
+
818C
% W3/13+ Cells
70
+
14
71
+
10
W3/25+ Cells/mn3
1323
+
688C
1097
+
501C
% W3/25+ Cells
37
+
15C
38
+
13d
MRC OX8+ Cells/mn3
1650
+
729C
1258
+
505C
% MRC OX8+ Cells
46.5
+
9d
44
+
6e
MRC OX6+ Cells/mn3
1351
+
462
1377
+
460
% MRC 0X6+ Cells
43.5
+
15b
47
+
12b
10154 + 3858
8296 + 2714
6034 + 1421
74 + 9
4243 + 1035
50 + 9
3431 + 902
38 + 7
1592 + 649
18 + 4
9475 + 2306
7441 + 1840
5809 + 1051
79 + 8
3840 + 1343
47.5 + 2
3358 + 1327
41 + 2
1260 + 247
16.5 + 2


TABLE 8
I. EFFECT OF ADDITION OF IRRADIATED OR NONIRRADIATED ALLOGENEIC CELLS
ON MITOGENIC RESPONSES OF BB AND WF SPLEEN CELLS
Responder (cells/well)
Con A responses
(cpm)b
Concentrations of Con A (yg/ml)
0 0.5 1.0 2.0 4.0
A = BB
0.5 x 105
1455
+
242a
17481
+
1480
31858
+
611
30697
+
2631
29317
+
1506
B = WF
0.5 x 105
15459
+
485
202133
+
829
272274
+
17598
327044
+
7002
210640
+
97
A + BxC
5
0.5 x 10 each
6511
+
2591
16512
+
4095
20701
+
661
18479
+
1342
14751
+
2398
A + B 0.5 x 10^ each
21971
+
2106
180971
+
1114
266064
+
11759
294636
+
7358
246163
+
20079
B + Ax
5
0.5 x 10 each
19024
+
13958
144299
+
7819
254043
+
13185
306300
+
21026
302577
+
2361
Ax 0.5
x 105
4673
+
366
7232
+
17
7557
+
527
602
+
5
1492
+
133
Bx 0.5
x 105
1655
+
953
795
+
232
804
+
171
1340
+
1290
3726
+
266
Con A responses of BB and WF splenic lymphocytes with and without added cells were measured on day 3.
Counts per minute. Each value is mean of triplicate cultures + S.D.
x designates irradiated cells.
107


178
107. Jackson, R., Rassi, N., Crump, T., Haynes, B., and
Eisenbarth, G. S. 1981. The diabetic BB rat: Pro
found T-cell lymphocytopenia. Diabetes 30:887.
108. Wigzell, H., Sundqvist, K. G., and Yoshida, T. 0.
1972. Separation of cells according to surface anti
gens by the use of antibody-coated columns. Frac
tionation of cells carrying immunoglobulins and blood
group antigens. Scand. J. Immunol. 1:75.
109. Brideau, R. J., Carter, P. B., McMaster, W. R., Mason,
D. W., and Williams, A. F. 1980. Two subsets of rat
T lymphocytes defined with monoclonal antibodies.
Eur. J. Immunol. 10:609.
110. Amos, D. 1976-77. NIAID Manual of Tissue Typing
Techniques. Dept, of Health, Education and Welfare,
Public #78-545, p. 25.
111. Gunther, E., and Stark, 0. 1979. Overview. The
major histocompatibility system of the rat. Transplan
tation Proceedings 11:1550.
112. Lis, H., and Sharon, N. 1977. Lectins: Their chem
istry and application to immunology. In The Antigens.
Michael Sela (Ed.). Academic Press, New York.
p. 429.
113. Gilman, S. C., Rosenberg, J. S., and Feldman, J. D.
1982. T lymphocytes of young and aged rats. II.
Functional defects and role of Interleukin-2. J.
Immunol. 128:644.
114. Naot, Y., Merchav, S., and Ginsburg, H. 1979. Myco
plasma neurolyticum: A potent mitogen for rat B
lymphocytes. Eur. J. Immunol. 9:185.
115. Kasakura, S. 1970. A blastogenic factor derived from
x-irradiated leucocytes cultivated in vitro. In Pro
ceedings of the Fifth Leucocyte Culture Conference.
J. E. Harris (Ed.). Academic Press, New York, p. 619.
116. Ruiz, P., and Scornik, J. C. 1982. Role of monocytes,
alloantigens, and individual reactivity in the response
of human lymphocytes to mitogenic factor. J. Immunol.
128:1289.
117. Larsson, E-L, and Coutinho, A. 1980. Mechanism of T
cell activation. I. A screening of "step one" li
gands. Eur. J. Immunol. 10:93.


FIGURE 3. Section of pancreas from a BB rat at onset of
IDD. The islet in the center has disorganized architecture
and lymphocytic infiltration (black arrow). A normal beta
cell is indicated by a clear arrow.


INTRODUCTION
Autoimmune diseases result from loss of self toler
ance which leads to an immune response by the individual to
autologous antigens and subsequent cellular and tissue
destruction or other effects. Such diseases are separable
into two groups, i.e. organ specific disorders such as the
autoimmune endocrinopathies, and autoimmune diseases that
are systemic and not confined to any one organ such as
systemic lupus erythematosus.
A disease is generally considered to be an organ-
specific autoimmune disorder if there are mononuclear in
filtrations of the affected organ or tissue, organ-specific
autoantibodies, and a tendency for more than one of these
diseases to occur simultaneously in individual patients (1)
Such organ-specific autoimmune diseases include chronic
lymphocytic thyroiditis, Graves' disease, Addison's disease
acquired hypoparathyroidism and insulin-dependent diabetes
(IDD).
Most autoimmune endocrinopathies are associated with
disturbed frequencies of certain HLA antigens, especially
an increased frequency of the immune response gene HLA DR3
(1,2). HLA DR4 is also increased in IDD (1,2). For some
1


TABLE 20extended.
IDD+
+
PCA+
IDD PCA+
4
12
4
3
IDD
PCA
IDD PCA
5
9
7
2
IDD
PCA
IDD PCA
1
5
3
4
IDD
PCA
IDD PCA
2
12
2
5
IDD
PCA
IDD PCA
2
13
5
2
IDD
PCA
IDD PCA
1
1
0
0
a Only litters bom before 1/1/82 are
included because
the final
frequencies of IDD and
PCA in
younger litters have not yet been reached.
k The frequency of IDD was calculated independently of PCA (without regards to the presence or
absence of PCA).
c The frequency of PCA is only for 1 litter of 7 rats, as PCA were not sought in the other 2
litters.
^ The frequency of PCA was calculated independently of IDD.


162
deficient in their ability to respond to IL 2 or other
helper factors. This defective T cell responsiveness to
IL 2 could possibly be due to either the lack of or the
presence of suboptimal numbers of receptors for IL 2 or
other cytokines on BB cell membranes. The immunodeficiency
observed in the BB rat may thus possibly be more the result
of a primary defect in the T cell itself because of its
inabilty to respond with the generation of IL 2 receptors to
stimulation by mitogens or by allogeneic cells, rather than
being a consequence of disordered immunoregulatory
mechanisms. Obviously, these hypotheses remain to be
explored.
The T cell defect most likely occurs after T lymphocyte
maturation in the thymus, since responses by thymocytes from
weanling BB rats to Con A and Con A sup were comparable to
those by WF control thymocytes. Additionally, no abnormali
ties were seen histologically in BB thymuses in comparison
with thymuses from WF animals. In contrast, spleens and
lymph nodes from BB rats were observed to be severely
depleted of T lymphocytes. These findings and the previous
results suggest that T lymphocytes in BB rats have
peripherally acquired defects in immunoresponsiveness. It
is also possible that the T cell immunoincompetence is due
to stem cell defects that appear late in T lymphocyte
maturation which do not affect functions of immature T
cells. Such a hypothesis is supported by a recent study
demonstrating somewhat restored MLC responses to allogeneic


169
11. Junker, K., Egeberg, J., Kromann, H., and Nerup, J.
1977. An autopsy study of the islets of Langerhans in
acute-onset juvenile diabetes mellitus. Acta Path.
Microbiol. Scand. Sect. A 85:699.
12. Gepts, W. 1980. Islet cell morphology in Type I and
Type II diabetes. In Immunology of Diabetes.
W. J. Irvine (Ed.). Teviot Scientific Publications,
Edinburgh, p. 255.
13. Bottazzo, G. F., Florin-Christensen, A., and Doniach,
D. 1974. Islet-cell antibodies in diabetes mellitus
with autoimmune polyendocrine deficiencies. Lancet
2:1279.
14. MacCuish, A. C., Barnes, E. W., Irvine, W. J., and
Duncan, L. J. P. 1974. Antibodies to pancreatic
islet cells in insulin-dependent diabetes with co
existent autoimmune disease. Lancet 2:1529.
15. Neufeld, M., Maclaren, N., Riley, W., Lezotte, D.,
McLaughlin, J., Silverstein, J., and Rosenbloom, A.
1980. Islet cell and other organ-specific anti
bodies in . S. Caucasians and Blacks with insulin-
dependent diabetes mellitus. Diabetes 29:589.
16. Lendrum, R., Walker, J. G., and Gamble, D. R. 1975.
Islet-cell antibodies in juvenile diabetes mellitus of
recent onset. Lancet 1:880.
17. Bottazzo, G. F., Doniach, D., and Pouplard, A. 1976.
Humoral autoimmunity in diabetes mellitus. Acta
Endocr., Copenhagen. 83 (Suppl. 205) :55.
18. Bottazzo, G. F., and Lendrum, R. 1976. Separate
autoantibodies reacting with human pancreatic glucagon
and somatostatin cells. Lancet 2:873.
19. Maclaren, N. Autoimmunity and Diabetes. 1981.
S. J. Cooperstein and D. T. Watkins (Eds.). In Bio
chemistry, Physiology and Pathology of the Islets of
Langerhans. Academic Press, Inc., N. Y. p. 453.
20. Riley, W. J., Neufeld, M., and Maclaren, N. 1980.
Complement-fixing islet cell antibodies: A separate
species. Lancet 1:1133.
21. Lernmark, A., Freedman, Z., Hoffmann, C., Rubenstein,
A. H., Steiner, D. F., Jackson, R. L., Winter, R. J.,
and Traisman, H. S. 1978. Islet-cell-surface anti
bodies in juvenile diabetes mellitus. N. Engl. J. Med.
299:375.


177
97. Nakhooda, A. F., Wei, C., Like, A. A., and Marliss,
E. B. 1978. The spontaneously diabetic Wistar rat
(the "BB" rat). A study of rats with transient glyco
suria. Diabetes Metab. 4:255.
98. Seemayer, T. A., Oligny, L. L., Tannenbaum, G. S.,
Goldman, H., and Colie, E. 1980. Animal model:
Spontaneous diabetes mellitus in the BB Wistar rat.
Amer. J. Path. 101(2):485.
99. Patel, V. C., Wheatley, T., Malaisse-Lagae, F., and
Orci, L. 1980. Elevated portal and peripheral blood
concentration of immunoreactive somatostatin in spon
taneously diabetic (BBL) Wistar rats: Suppression
with insulin. Diabetes 29:757.
100. Rossini, A. A., Williams, R. M., Mordes, J. P.,
Appel, M. C., and Like, A. A. 1979. Spontaneous
diabetes in the gnotobiotic BB/W rat. Diabetes
28:1031.
101. Dyrberg, T., Nakhooda, A. F., Baekkeskov, S.,
Lernmark, A., Poussier, P., and Marliss, E. B. 1982.
Islet cell surface antibodies and lymphocyte anti
bodies in the spontaneously diabetic BB Wistar rat.
Diabetes 31:278.
102. Like, A., Rossini, A. A., Guberski, D. L., and
Appel, M. C. 1979. Spontaneous diabetes mellitus:
Reversal and prevention in the BB/W rat with anti
serum to rat lymphocytes. Science 206:1421.
103. Like, A. A., Williams, R. M., Kislauskis, E., and
Rossini, A. A. 1981. Neonatal thymectomy prevents
spontaneous diabetes in the Bio Breeding/Worcester
(BB/W) rat. Clin. Res. 29:542A.
104. Like, A. A., Kislauskis, E., Williams, R. M., and
Rossini, A. A. 1982. Neonatal thymectomy prevents
spontaneous diabetes mellitus in the BB/W rat.
Science 216:644.
105. Naji, A., Silvers, W. K., Bellgrau, D., and Barker,
C. F. 1981. Spontaneous diabetes in rats: Destruc
tion of islets is prevented by immunological toler
ance. Science 213:1390.
106. Colle, E., Gutmann, R. D., and Seemayer, T. 1981.
Spontaneous diabetes mellitus syndrome in the rat.
Association with the major histocompatibility complex
J. Exp. Med. 154:1237.


TABLE 12extended
Purifiedb WF
(0.5)
-
n.d.
5545 + 578
3931 + 1509
5899
4
1624
2179 + 231
1 ug/ml Oon A
n.d.
119938 + 5677
n.d.
146154
4
10244
128906 + 7243
+ Oon A sup
n.d.
n.d.
n.d.
216024
4
8342
155216 + 8752
Cbn A sup
n.d.
198630 + 9981
55768 + 5014
146502
4
18922
122799 + 1930
1 RIA
n.d.
n.d.
n.d.
82017
4
222
73759 +1158
Unseparated Lewis
(0.5)
-
539 + 49
n.d.
n.d.
23649

927
n.d.
1 ug/ml Con A
n.d.
n.d.
n.d.
346694

66
n.d.
" + Cbn A sup
n.d.
n.d.
n.d.
387475

3707
n.d.
Oon A sup
14307 + 3480
n.d.
n.d.
256265
4-
4559
n.d.
1 RIA
n.d.
n.d.
n.d.
180109
4
712
n.d.
Purified*5 Iiewis
(0.5)
-
n.d.
n.d.
n.d.
1505
4
71
n.d.
1 ug/ml Con A
n.d.
n.d.
n.d.
38767
4
176
n.d.
" + Con A sup
n.d.
n.d.
n.d.
97913
4
3225
n.d.
COn A sup
n.d.
n.d.
n.d.
46340
4
6814
n.d.
It RIA
n.d.
n.d.
n.d.
29554
4
1595
n.d.
a All responders were used at 0.5 x 10^ cells/well.
b Purified spleen cells were passed through rahbit Ig-antlrat Ig oolums.
c Each value is mean of triplicate cultures + S.D.
d 0.1 ml of WF Cbn A sup added to each well.
e Not (tone
115


27
was determined by gastric aspirates during the fasting state
and was quantitated by color changes of pH indicator paper.
Transfer Studies. Spleen, mesenteric lymph nodes and
peritoneal suspensions from BB rats 1 to 10 days after onset
of IDD were injected into the tail veins of anesthesized
recipient nondiabetic BB, WF or BB x WF FI rats using 25
gauge butterflys (Deseret, Sandy, UT). Pancreas suspensions
were given intraperitoneally. In some experiments, the
recipients were given 300-350 rads of irradiation 24 hours
before the transfer. Blood glucose levels were measured on
sera from recipient rats on day 0, day 3 and every week
thereafter for at least 90 days. Autoantibodies to the
cells of the pancreatic islets, the thyroid gland, and the
gastric mucosa were also sought monthly in sera recipient
rats. After approximately 90-120 days, the recipients were
sacrificed and the pancreases removed for histological
examination.
Determination of Leukocyte Populations and Rat T Cell
Subsets.
White blood cell counts were determined on whole blood after
1:200 dilution in acetic acid using a hemacytometer, and
differentials were made from slides of Wright-Giemsa stained
cells.
Monoclonal antisera to the various T cell subpopula
tions were kindly provided by Dr. Alan Williams (Medical


5
proven. A direct role for ICA in the pathogenesis of IDD in
fact seems unlikely, especially since these antibodies react
with a shared antigen found in the cytoplasm of all islet
cell types. Furthermore, autoantibody molecules are not
normally considered to be able to cross membranes of living
cells to effect damage. In addition, experimental evidence
to suggest that ICA do not cause IDD includes findings that
transfers of ICA from human patients with IDD to mice have
not resulted in IDD in these animals (19,27), nor has placen
tal passage of ICA resulted in the development of IDD in
newborn infants or even to affect neonatal insulin secretion
(19). Observations that ICA titers decrease with time after
clinical onset of IDD is probably related to the progressive
loss of the relevant antigen, whatever the disease
mechanism.
Islet Cell Surface Autoantibodies in IDD. Autoantibodies
reactive to antigens on pancreatic islet cell membranes
(ICSA) have also been demonstrated in sera of patients with
IDD (21,28-30). Using cell surface immunofluorescence tech
niques or 125i-iabeled protein A assays, ICSA have been
detected either by reaction with cultured human insulinoma
cells (29), beta cells isolated from dispersed rat or mouse
pancreatic islets (21,31,32) or human fetal pancreatic islet
cell cultures (33). Analagous to ICA, ICSA have been de
tected in about 67% of children at onset of IDD and decrease
in frequency within the first year after diagnosis (34,35).


160
T lymphocytes from BB rats obtained after passage
through rabbit Ig-antirat Ig columns had enhanced but still
severely decreased mitogenic and MLC responses in comparison
with WF cells. Because the passage of spleen cells through
these columns is thought to greatly augment lymphocyte
responsiveness by removing suppressor cells (121), these
results suggest that suppressor cells may in part contribute
to the T lymphocyte unresponsiveness seen in BB rats.
The data concerning the effects of the addition of
either irradiated or nonirradiated BB cells on mitogenic and
MLC responses by WF lymphocytes were equivocal. In several
experiments, some inhibition of proliferation by WF lympho
cytes was noted in the presence of BB spleen cells, yet in
other studies no suppressor activity by BB cells was
observed. Imbalances between helper and suppressor T cells
may indeed play a role in the T lymphocyte immunoincompe-
tence in the BB rat. However, because the suppression of WF
responses by added BB cells was modest and inconsistent and
because BB spleen cells were good stimulators in MLCs, other
factors besides increased suppressor activity must
contribute to the lack of significant T cell-mediated immune
responses by BB rats.
One aspect of the immune system of the BB rat that was
considered was the ability of BB lymphocytes to respond and
to produce IL 2. IL 2 is a lymphokine produced by helper T
cells which has a broad spectrum of biological activities.
One of the most important actions is that IL 2 appears to


TABLE 9
II. EFFECT OF ADDITION OF IRRADIATED OR NONIRRADIATED ALLOGENEIC CELTS
O MITOGENIC RESPONSES OF BB AND WF SPLEEN CELLS
Counts Per Minute3
Responder (oells/well)
PWM Concentration (yg/ml)
PHA Concentration (%)
0 1.0 5.0 0.5 1.0
A = BB
0.5 x
105
1455
+
242
7977
+
118
6897
+
392
6842
+
470
4329
+
489
B = WF
0.5 x
io5
15459
+
485
81736
+
641
100007
+
9988
106333
+
1712
128479
+
10372
A + Bxb
0.5 x
105
each
6511
+
2591
7129
+
1342
6435
+
664
4881
+
111
3615
+
808
B + Ax
0.5 x
io5
each
19024
+
13958
82276
+
9143
106082
+
3437
88380
+
1290
90076
+
5484
Ax 0.5
x 105
4673
+
3661
2819
+
944
2475
+
1846
1211
+
318
1428
+
0
Bx 0.5
x 105
1655
+
953
1121
+
107
1102
+
79
533
+
134
2046
+
441
Each value is expressed as mean of triplicate cultures + 1 S.D.
x designates irradiated cells.


H uo3 [w/rt s y uoD Iw/Brt 0
ZOT
CPM (xl0


134


145
observations that matings between diabetic BB rats resulted
in litters with frequencies of IDD of only 50%, while
matings between 2 nondiabetic BB rats produced a few
diabetic offspring (Table 20). Interestingly, matings of
nondiabetic BB rats with normal glucose tolerance tests who
were both PCA-positive, produced offspring with IDD.
Crosses of diabetic BB rats without PCA resulted in
offspring positive for PCA. These results raise questions
as to whether IDD and PCA are due to the same or separate
genes.
As was the case with BB x WF FI rats, BB x Lewis FI
animals did not develop either IDD or PCA (Table 21). None
of the rats in F2 litters from matings of either BB x WF FI
rats or BB x Lewis FI animals were positive for either IDD
or PCA. However, many of these F2 rats, especially from the
Lewis crosses, were not yet old enough at the time of this
writing to develop these autoimmunities.


Responder
RB
TABLE 17
MITOGENIC RESPONSES OF BB AND WF THYMOCYTES
(Cells/Well)
0.5 x 105
1.0 x 105
Counts Per Minute3
Mitogen Added
Expt. 1
Expt. 2
Expt.
3
-
, c
n.d.
4724 + 1275
1075
+
363
0.1 yg/ml
Con
A
n.d.
n.d.
1300
+
250
0.1 yg/ml
Con
A
+
b
sup
n.d.
n.d.
52296
+
6632
1.0 yg/ml
Con
A
n.d.
112208 + 1000
12353
+
2753
1.0 yg/ml
Con
A
+
sup
n.d.
320578 + 3440
67367
+
2886
sup
n.d.
n.d.
53851
+
6993
-
950 + 6
455 + 313
2153
+
287
0.1 yg/ml
Con
A
n.d.
n.d.
1203
+
212
0.1 yg/ml
Con
A
+
sup
n.d.
n.d.
91726
+
8295
1.0 yg/ml
Con
A
3812 + 37
176397 + 49111
28292
+
1001
1.0 yg/ml
Con
A
+
sup
23225 + 2685
323411 + 7001
133644
+
18797
sup
32606 + 689
n.d.
117650
+
1716
127


79
0 Mg/ml PWM
10 yg/ml PWM


45
The intensity of immunofluorescence of the PCA-positive sera
tended to increase with duration of PCA. Except for an
equivocal result from the serum of one WF rat, none of the
WF or BB x WF FI animals had demonstrable PCA in their
serum. As was the case with IDD, PCA did not seem to have a
dominant mode of inheritance. Thyroid colloid autoanti
bodies were of low immunofluorescence intensity and were
less frequent than PCA, being found in only 5% (1/20) and
18% (5/28) of the sera from BB/0 rats and BB/W rats,
respectively. None of the control animals had autoanti
bodies to thyroid colloid. SMA were demonstrated in 55%
(11/20) of BB/0 rats, in 61% (17/28) of BB/W rats, and in 9%
(7/80) of WF and BB x WF FI animals. The presence of SMA
was unrelated to the presence of IDD or PCA in the BB rats
(Table 2).
Some 71% (5/7) of the BB/0 rats with PCA and 79%
(15/19) of the BB/W rats with PCA had IDD (Table 2).
However, two PCA-positive BB animals (one BB/O and one BB/W)
without clinical evidence of IDD had abnormal glucose
tolerance tests as previously mentioned. Thus, only 15%
(4/26) of the BB rats with detectable PCA had no discernible
IDD confirmed by normal glucose tolerance tests.
The appearance of PCA in the serum usually shortly
preceded the onset of IDD in BB rats that developed the
disease. The frequency of PCA in the BB rats was seen to
increase at an age coincident with the development of IDD
and did not further rise after the critical age span for


98


136


FIGURE 5. Positive indirect immunofluorescence staining of
smooth muscle in rat gastric fundus.


96
0 pg/ml Con fl
1 yg/ml Con fl
5 yg/ml Con fl


FIGURE 33. Section of lymph node from a nondiabetic BB rat
stained with hematoxylin and eosin. B cell follicles (black
arrow) are relatively normal, but parafollicular area is
severely depleted of T lymphocytes (clear arrow).


158
in vivo and in vitro T cell-mediated immune responses were
noted in all BB rats studied, irrespective of age or the
presence of IDD. Defective graft rejection across both
major and minor histocompatibility barriers was observed in
these animals. The inability of BB rats to reject allo
grafts normally probably was related to the severe T
lymphopenia present in these animals.
Con A, PHA and PWM are considered to be mainly T
lymphocyte mitogens in the rat, but B cells may also be
stimulated to divide (112,114,129). Even though these
mitogens are extremely potent nonspecific stimulators of T
cell proliferation, lymphocytes from BB rats were unable to
mount proliferative responses comparable to those by lympho
cytes from WF rats to any concentration of PWM, PHA, Con A
or Con A sup. The lack of mitogenic responses by BB spleen
cells or PBL probably reflects to a large degree the fewer T
lymphocytes present in BB spleen cells or PBL compared to
the number of responding T cells in the same concentrations
of WF spleen cells or PBL. Comparable mitogenic responses
by BB and WF lymphocytes were only observed when at least
ten times fewer WF spleen cells or PBL than BB cells were
used. However, the absolute numbers of peripheral blood T
lymphocytes in BB rats were only two-fold to three-fold less
than the absolute numbers of T cells seen in WF animals,
suggesting that decreased numbers of T lymphocytes were not
the only factor contributing to the lack of mitogenic
responses by BB rats in vitro.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
/t
Paul A. Klein, Ph.D.
Associate Professor of Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Edward K.
Assistant
Wakeland,
Professor
Ph.D.
of Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
L
AJ
hi
Christopner M. West, Ph.
Assistant Professor of Anatomy
L
hi5T


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171
32. Lernmark, A., Kanatsuna, T., and Patzelt, C. 1980.
Antibodies directed against the pancreatic islet-cell
plasma membrane: Detection and specificity. Dia-
betologia 19:445.
33. Pujol-Borrell, R., Bottazzo, G. F., Khoury, E. L.,
Doniach, D. 1980. Detection of autoantibodies direc
ted against plasma membranes of human fetal islet
cells in cultures in Type I diabetes. Diabetologia
19:308.
34. Cahill, G. F., and McDevitt, H. 0. 1981. Insulin-
dependent diabetes mellitus: The initial lesion.
N. Engl. J. Med. 304:1454.
35. Nerup, J., and Lernmark, A. 1981. Autoimmunity in
insulin-dependent diabetes mellitus. Amer. J. Med.
70:135.
36. Freedman, Z. R., Irvine, W. J., Lernmark, A., Hiren,
H. J., Steiner, D. F., and Rubenstein, A. H. 1980.
Cell surface and cytoplasmic islet-cell antibodies in
insulin-dependent diabe:tes. In Immunology of Dia
betes (Ed.) W. J. Irvine. Teviot Scientific Publica
tions. Edinburgh. p. 169.
37. Lernmark, A., Kanatusna, T., Rubenstein, A. M., and
Steiner, D. F. 1979. Treatment of early diabetes:
Detection and possible functional influence of anti
bodies directed against the pancreatic islet cell
surface. R. A. Camerini-Davalos and B. Hanover (Eds.),
Plenum Publishing Corp. p. 157.
38. Rittenhouse, H., Oxender, D., Pek, S., and Ar, D.
1980. Complement-mediated cytotoxic effects on pan
creatic islets with sera from diabetic patients.
Diabetes 29:317.
39. Soderstrom, W. K., Freedman, Z. R., Lernmark, A. 1980.
Complement-dependent cytotoxic islet-cell surface
antibodies in insulin dependent diabetes. Diabetes
29:317.
40. Dobersen, M. J., Scharff, J. E., Ginsberg-Fellner, F.,
Notkins, A. L. 1980. Cytotoxic autoantibodies to beta
cells in the serum of patients with insulin-dependent
diabetes mellitus. N. Engl. J. Med. 303:1493.
41. Kanatsuna, T., Lernmark, A., Rubenstein, A. H., and
Steiner, D. F. 1980. Block in insulin release from
column-perifused pancreatic beta-cells induced by
islet cell surface antibodies and complement.
Diabetes 30:231.


40
control WF animals or BB x WF FI rats, which were followed
for at least 180 days after birth, developed IDD or
pancreatic insulitis (Table 1), indicating that IDD in the
BB rat is not due to a single dominant gene.
Presence of Organ-Specific Autoantibodies. ICA and other
organ-specific autoantibodies were sought in BB/O and BB/W
rats because these antibodies are characteristically found
in human IDD, are evidence for an autoimmune etiology for
IDD in the BB rat, and can be used as markers for animals
with autoimmune tendencies. ICA were never detected in any
BB rats, regardless of age or duration of IDD. To rule out
the possibility that ICA were present but were directed
against diabetic antigens, ICA were also sought on pancre
atic sections from both nondiabetic BB rats and BB rats at
onset of IDD. However, no such autoantibodies were identi
fied. In addition, ICA were not demonstrated in BB rat sera
when using fluorescein conjugated antirat Ig (all classes)
instead of antirat IgG in case ICA of the IgM class were
present. Thyroid microsomal autoantibodies and adrenal
autoantibodies were never found in any BB animals.
However, autoantibodies to the parietal cells of the
gastric mucosa (PCA) (Figure 4), thyroid colloid antigens
and smooth muscle (SMA) (Figure 5) were demonstrated in the
sera of a considerable number of BB/O and BB/W rats (Table
1). PCA were detected in the sera of 35% (7/20) of the BB/O
rats and in the sera of of 68% (19/28) of the BB/W animals.


TABLE 15extended
Ax
(0.5) 4
c*
(0.5)
50742 4
22)6
52419

50)8
358)3 4 3231
n.d.
-

-
(1.0)
56669 4
14589
9)079

4)40
36087 4 8130
161905 4 10950



(2.0)
91666 4
1816
126262

14651
60852 4 2886
65956 4 3371
A
(0.5) 4
Cx
(0.5)
76911 4
)207
62198

66
41008 4 1229
n.d.
-


(1.0)
84B14 4
2497
94954

7)47
45451 4 1078
125788 4 8754

*
(2.0)
11790 ) 4
6885
110192

162)6
54014 4 1864
105592 4 7670
Ctii
A sup
12)625 4
1669
1076)2

6091
84132 4 4909
151792 4 20574

*
Cx
(0.5)
n.d
112544

5182
n.d.
n.d.
-


(1.0)
n.d
10)62)

)4I6
n.d.
132912 4 1609
t
(2.0)
n.d
924)6

4)20
n.d.
12)526 4 1)14
liewis (0.5)
-
15704 4
1009
12177

1289
n.d.
21663 4 7698
Ax
(0.5)
295)0 4
4681
25)09

2510
n.d.
n.d.

(1.0)
50)4) 4
10262
)7655

8828
n.d.
59077 4 4865

(2.0)
91072 4
741)
n.d.
n.d.
82321 4 1981
Bx
(0.5)
)5452 4
6126
17128

12)0
n.d.
n.d.
-
(1.0)
45)51 4
)22)
37830

)955
n.d.
51747 4 6677
-
(2.0)
54059 4
1768
68045

10402
n.d.
55670 4 20746
Cbn
A sup
n.d
.
1
n.d
1.
n.d.
11258) 4 24165
a 5
Rali)it Ig antlrat Ig treated spleen cells were used as ail responders at 0.5 x 10 cells/well.
b Useparated spleen cells vere used as st imitators at 0.5-2.0 x 10^ cells/well.
C Each value is expressed as mean of triplicate cultures + 8.D.
^ 0.1 ml of WF Odn A sup was added to each well.
e Mot (hne
120


42


181
139. Naji, A., Silvers, W. K., Bellgrau, D., and Barker,
C. F. 1982. Restoration of T cell dependent immune
response defect in BB rats. Diabetes 31 (Suppl. 2):
188.
140. Kindred, B. 1978. The nude mouse in studying T cell
differentation. In The Nude Mouse in Experimental
and Clinical Research. J. Fogh and G. C. Giovanella
(Eds.). Academic Press, New York. p. 111.
141. Kirchner, H., Fenkl, H., Zawatzky, R., Engler, H., and
Becker, H. 1980. Dissociation between interferon
production induced by phytohemogglutin and Concanav-
alin A in spleen cell cultures of nude mice. Eur. J.
Immunol. 10:224.
142. Brooks, C. G., Webb, P. J., Robins, R. A., Robinson,
G., Baldwin, R. W., and Festing, M. F. W. 1980. Stu
dies on the immunobiology of rnu/rnu "nude" rats with
congenital aplasia of the thymus. Eur. J. Immunol.
10:58.
143. Svendsen, V. G., Koch, C., Werdelin, 0., and Chris
tiansen, P. 1981. Studies on the thymus-dependent
immunity in the rat mutant "nude." Acta Path.
Microbiol. Scand. C. 89:293.
144. Vos, J. G., Kreeftenberg, J. G., Kruijt, B. C.,
Kruizinga, W., and Steerenberg, P. 1980. The athymic
nude rat. II. Immunological characteristics. Clin.
Immunol, and Immunopatho. 15:229.
145. Talal, N. 1977. Autoimmunity and lymphoid malignancy
Manifestations of immunoregulatory disequilibrium. In
Autoimmunity (Ed.) N. Talal. Academic Press, New
York. p. 184.
146. Talal, N. 1974. Autoimmunity and lymphoid malignancy
in New Zealand black mice. Prog. Clin. Immunol.
2:101.
147. Michalski, J. P., McCombs, C. C., and Talal, N. 1979.
Suppressor cells and immunodeficiency in (NZB x NZW)
FI hybrid mice. Eur. J. Immunol. 9:440.
148. Ammann, A. J. 1977. Immunodeficiency disorders and
autoimmunity. In Autoimmunity (Ed.) N. Talal.
Academic Press, New York. p. 479.


142
results suggested that the functions as well as the numbers
of B lymphocytes may possibly be unimpaired in these BB
rats.
Transfer Experiments. Attempts were made to transfer IDD
with pancreas extracts and cells from the spleen, the mesen
teric lymph nodes, and the peritoneal cavity of diabetic BB
rats to nondiabetic BB, WF and BB x WF FI hybrid animals.
These studies were performed because positive results would
provide definitive proof for an autoimmune etiology for IDD
in the BB rat.
As shown in Table 19, no combination of cell types,
cell numbers or recipients resulted in the development of
hyperglycemia, as defined by blood glucose levels greater
than 250 mg/dl, or pancreatic insulitis in any rats. PCA
were also not detected in the sera of any recipients,
although they were sought in the sera for more than 90
days. SMA were demonstrated in the sera of four WF
recipients, but as shown previously in Table 1, SMA were
normally found in some WF rats.
Genetics of IDD. Studies of the mode of inheritance of
IDD and PCA were carried out in order to see genetic simi
larities between the BB rat and humans with IDD. In
addition to the findings that BB x WF FI rats do not develop
IDD (Table 1), further indications that IDD in the BB rat
probably does not have a dominant inheritance include


UNIVERSITY OF FLORIDA
| III III Hill -m ct A-r
3 1262 08554 7817


TABLE 16
III. MLC RESPONSES OF PURIFIED SPTEFN CETJE FROM BB, WF AND LEWIS RATS
c
Counts Per Minute
Responder
(Cells/Well)a
Stimulator ,
(Cells/Well;
Expt. 1
Expt.
2
A = BB (1.0)
-
2199 + 250
4797
+
1016
Bx
(1.0)
2103 + 801
3226
+
571
II
(2.0)
2728 + 172
3329
+
254
Cx
(1.0)
5346 + 1876
3679
+
64
II
(2.0)
2844 + 62
3164
+
62
Con
A sup^
, e
n.d.
87825
+
2619
II
+ Cx
(1.0)
n.d.
29633
+
5397
II
+ "
(2.0)
n.d.
12742
+
1597
B = WF (1.0)
-
74155 + 8028
39977
+
17259
Ax
(1.0)
128158 + 558
71425
+
12699
II
(2.0)
101730 + 7025
74309
+
5049
Cx
(1.0)
104270 + 2589
69389
+
11321
II
(2.0)
178659 + 175
119614
+
17349
121


Table 5extended.
Ig+ Cells/mn3
1158
+
446
1384
+
559
1324
+
509
1370
+
899
% Ig+ Cells
36.5
+
iob
47
+
14b
16
+
4
17
+
8
a Each value is stated as mean + S.D.
p <0.0005 by Student's t test when compared to WF rats.
c
p <0.005 by Student's t test when ocmpared to WF rats.
^ p <0.025 by Student's t test when compared to WF rats.
0
p <0.01 by Student's t test when ocmpared to WF rats.
CT*
O


71
PHR Concentration (/)


58
numbers of both total peripheral blood leukocytes and
lymphocytes, when compared to 28 control WF rats and 8 FI
hybrid animals (p <0.0005). The lymphopenia was more
striking than the leukopenia, reflecting significant
complementary decreases in the percentage of lymphocytes
seen in BB rats from that observed in WF and BB x WF FI rats
(p <0.0005). Significant differences were also seen in both
the percentages and absolute numbers of lymphocytes in
diabetic BB rats when compared to BB rats without IDD (p
<0.01).
Due to the severe lymphopenia observed in all BB rats,
analyses of lymphocyte subpopulations were next performed.
Irrespective of age or the presence of IDD, increased
percentages (p <0.0005) but similar absolute numbers of la-
positive cells (MRC 0X6 monoclonal antibody reactive) and Ig-
positive cells were found in BB rats in comparison with WF
rats and BB x WF FI rats (Table 5). In contrast, the
absolute numbers of peripheral T lymphocytes (W3/13 mono
clonal antibody reactive) were significantly lower in all BB
rats when compared to WF and FI animals (p <0.001). Corre
spondingly significant depressions of both absolute numbers
(p <0.005) and percentages (p <0.025) of W3/25-positive
cells (helper T cells) were observed in all BB rats,
independent of age or IDD, in comparison with WF and FI
animals. Increased percentages (p <0.005) but decreased
absolute numbers (p <0.005) of cytotoxic/ suppressor T
lymphocytes (MRC 0X8 monoclonal antibody reactive) were also


113
enhanced BB proliferative responses to PHA, Con A and Con A
sup in comparison to the responses seen with unseparated BB
spleen cells (Tables 12 and 13). However, these augmented
responses were still considerably lower than WF mitogenic
responses by either unseparated or purified spleen cells.
These results are further evidence that suppressor T lympho
cytes are not the major cause of unresponsiveness of BB
lymphocytes because removal of at least a subpopulation of
suppressor T cells did not restore normal proliferative
capacity to BB spleen cells when stimulated with mitogens.
Purified BB spleen cells proliferated slightly more in
MLCs than unseparated cells (Tables 14-16). This was
especially true when 0.5 x 10^ spleen cells were used as
responders per well. But again, these responses were
minimal in comparison with responses by purified WF splenic
lymphocytes. The levels of proliferation seen by purified
BB cells to Lewis or WF stimulators were, at best, compara
ble to responses by WF unseparated spleen cells to BB stimu
lating cells. The addition of irradiated or nonirradiated
unseparated BB spleen cells did not strongly inhibit WF MLC
responses, but these results were variable. However, the
presence of irradiated WF cells did slightly increase MLC
responses by purified BB lymphocytes to Lewis stimulators,
but even these improved responses were still much less than
those seen with WF lymphocytes. Stimulation of BB respond
ers was noted to Con A sup, but again, the proliferation
seen was 2-fold to 4-fold less than that observed by WF


46
TABLE 2
RELATIONSHIPS BETWEEN IDD, PCA AND SMA
IN BB/O, BB/W AND WF RATS
Rats Studied
Total
PCA %
SMA %
I. Total BB rats with IDD
37
54
57
Total BB rats without IDD
11
54
64
BB/O rats with IDD
16
31
50
BB/O rats without IDD
4
50
75
BB/W rats with IDD
21
71
62
BB/W rats without IDD
7
57
57
Rats Studied
Total
IDD %
SMA %
II. Total BB rats with PCA
26
77
46
Total BB rats without PCA
22
77
73
BB/O rats with PCA
7
71
57
BB/O rats without PCA
13
85
54
BB/W rats with PCA
19
79
42
BB/W rats without PCA
9
67
100


26
at 1:10 dilution to thyroid and stomach sections respect
ively. Optimal results (lowest background and highest sen
sitivity) were obtained when these specified dilutions were
used on the various tissues. After incubation with rat sera
for 30 minutes in the dark, the slides were washed three
times in PBS. Rabbit antirat IgG-fluorescein isothiocya
nate conjugate (Cappel Laboratories, Cochranville, PA) was
then added at 1:60 final dilution to the slides and
incubated for 60 minutes in the dark. After washing in PBS,
the slides were dried, covered with glycerol and glass slips
and read under a Leitz Dialux 20 ultraviolet glass micro
scope fitted with an HB-100 mercury lamp and KP490, TK510
and K515 filters (E. Leitz, Inc., Rockleigh, NJ). All
results were read double blind with control negative and
antibody-positive sera in each batch. Sere were considered
to have autoantibodies if positive immunofluoresence of the
tissue was observed. Sera were tested for the presence of
autoantibodies at least monthly after the rats were 40 days
of age.
Measurement of Gastric Acidity and Serum Iron and Vitamin
B12 Levels.
Serum vitamin B12 levels were measured by radioimmunoassay
(Diagnostic Products, Los Angeles, CA). Serum iron levels
and iron binding capacities were measured by the ACA-III
autoanalyzer (Dupont Inc., Wilmington, DL). Gastric acidity
after pentagastrin administration (0.06 mg/kg body weight)


14
influences have been implicated as at least secondary
factors in the development of IDD (86). Support from animal
models for a viral role in the pathogenesis of IDD includes
evidence that encephalomyocarditis virus causes a diabetes
like syndrome in susceptible SJL mice, associated with beta
cell loss and pancreatic insulitis (89).
There is limited direct evidence for such viral partic
ipation in human IDD. Coxsackie B4 virus was isolated from
the pancreas of a child with fatal diabetic ketoacidosis
which proved capable of inducing insulitis and hyperglycemia
in some strains of mice (90). An epidemiological relation
ship between annual cycles of infection with Coxsackie B4
virus in people and the seasonal incidence of IDD has also
been found (91), and a high frequency of IDD in children who
have suffered severe congenital rubella or mumps infection
has been reported (45,92). One study has suggested that the
frequency of IDD in patients after infections with these
viruses is positively associated with HLA B8 and thus by
linkage disequilibrium with HLA DR3 (93). If confirmed,
such findings would suggest a relationship between the
immune response of an individual to a virus and the
development of IDD. Several investigators suggest that
these viruses do not usually cause IDD, but instead may
trigger IDD by a stress effect in the susceptible predia
betic individual with preexisting insulinopenia. To this
effect, it is notable that no epidemics of IDD have been
reported in several large registry studies in the United
States, London and Denmark.


CPM
125


81
10
8 -
0 jug/m 1 PWM
1 Mg/ml PWM
5 jug/ml PWM


4
been found to precede development of the clinical disease
(13,15,21,22). Neufeld et al. determined the frequency of
ICA to be approximately 74% in Caucasian children tested
within three months of onset of IDD, whereas ICA were
detected in less than 50% of patients three years after
diagnosis (15). However, 10-15% of IDD patients have been
noted to have persistent ICA for many years and have been
shown to have higher frequencies of associated thyroid,
gastric and adrenal autoimmune diseases as well as higher
frequencies of HLA B8-bearing haplotypes than patients with
IDD who become ICA-negative over similar periods (19,23).
In contrast, black insulin-dependent diabetics have only
about half the frequency of ICA in relation to duration of
IDD as do Caucasian patients with IDD, suggesting that much
of the IDD among black patients may be different from IDD
seen in Caucasian populations (15,24). In addition,
children who are ICA-negative at onset of IDD rarely become
positive for these antibodies later (2,3). ICA have also
been demonstrated in approximately 3-5% of nondiabetic
relatives of IDD probands (15,23) and in about 0.5% of
healthy controls (15,23).
Observations that ICA-positive family members of
patients with IDD and noninsulin-dependent diabetics with
ICA tend to become insulin-requiring with time (22,25,26)
suggest that these antibodies may be of clinical value in
predicting the subsequent development of IDD (22). However,
a causal relationship between ICA and IDD remains to be


FIGURE 25. Proliferative responses (cpm) of PBL at 1 x
105 cells/well from 1 diabetic BB rat (first striped bar),
3 nondiabetic rats (remaining striped bars) and 5 WF rats
(open bars) to 2 Con A concentrations. Each value is mean
of triplicate cultures + S.D.


179
118. Smith, K. A., and Ruscetti, F. W. 1981. T-cell
growth factor and the culture of cloned functional T
cells. Advances in Immunol. 31:137.
119. Watson, J., Mochizuki, D., and Gillis, S. 1980.
T-cell growth factors: Interleukin 2. Immunol.
Today, December:113.
120. Watson, J. D. 1981. Lymphokines and the induction
of immune responses. Transplantation 31:313.
121. Peck, A. B., Wigzell, H., Janeway, C., and Andersson,
L. C. 1977. Environmental and genetic control of
T cell activation in vitro: A study using isolated
alloantigen-activated T cell clones. Immunological
Reviews 35:146.
122. Bloom, A., Hayes, T. M., Gamble, D. R. 1975. Regis
ter of newly diagnosed diabetic children. Br. Med.
J. 3:580.
123. Colle, E., Guttmann, R. D., Michel, F., and Seemayer,
T. A. 1982. Genetics of T lymphopenia in the syndrome
of spontaneous diabetes mellitus in the rat. Dia
betes 31 (Suppl. 2): 84.
124. Riley, W., Maclaren, N., Rand, K., and Bejar, R. 1980.
Inherited autoimmunity versus Coxsackie B4 in insulin
dependent diabetes. Diabetes 29 (Suppl. 2):53A.
125. King, C. E., Leibach, J., and Toskes, P. P. 1979.
Clinically significant vitamin B12 deficiency secon
dary to malabsorption of protein-bound vitamin B12.
Digestive Dis. Sci. 24:397.
126. Shearman, D. J. C., Delamore, J. W., and Gardner,
D. L. 1966. Gastric function and structure in iron
deficiency anemia. Lancet 1:845.
127. Riley, W. J., Maclaren, N. K., Winer, A., and Gold
stein, D. Coincident onset of thyro-gastric auto
immunities and diabetes in insulin dependent diabetes.
Submitted for publication.
128. White, R. A. H., Mason, D. W., Williams, A. F.,
Galfre, G., and Milstein, C. 1978. T-lymphocyte
heterogeneity in the rat: Separation of functional
subpopulations using a monoclonal antibody. J. Exp.
Med. 148:664.


THE SPONTANEOUSLY DIABETIC BB RAT: A MODEL OF
AUTOIMMUNITY AND IMMUNODEFICIENCY
By
MELISSA ELLEN ELDER
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1982


155
autoimmunities do not seem to be genetically associated with
systemic autoimmunity like SMA in BB rats. Because thyroid
colloid antibodies were found in only a few BB rats, these
observations are insufficient to relate the presence of
thyroid colloid antibodies to the other autoimmune processes
resulting in IDD and PCA.
In summary, in respect to frequencies of autoantibodies
and associated diseases, there are both similarities and
differences between BB rats and human patients with IDD.
The number of autoantibodies present in BB rats suggests
that the strain has an underlying autoimmune diathesis, of
which IDD may be only one possible result. The BB rat
appears to have a general genetic predisposition for
autoimmunity, with IDD and PCA being the result of either
the same or separate genetic influences and SMA occurring
independently of either PCA or IDD.
Although the presence of autoantibodies provides
further evidence that the IDD seen in the BB rat may result
from an autoimmune process, transfers of pancreas
suspensions, lymphocytes and peritoneal macrophages from
diabetic BB rats did not result in the development of IDD or
pancreatic insulitis in any recipient animals. However,
serum was not transferred from BB rats with IDD neither
alone or in combination with lymphocytes and pancreas, which
may have contributed to the negative results obtained if
specific humoral immunity or antibody-dependent
cell-mediated cytotoxicity were involved. Furthermore,


31
0.25-1 x 10^ unseparated or purified responder spleen
cells and 0.5-3 x 10^ irradiated (3000 rads) unseparated
spleen cells as stimulators. In addition, third-party
irradiated or nonirradiated unseparated spleen cells at a
concentration of either 0.25 x 10^ or 0.5 x 10^ cells
were added to some wells with a final volume in each well
always of 0.2 ml of complete medium. The plates were incu-
batd at 37C in 5% CO2 for 5 days, pulsed with 1.0 yCi/
well of 3H-thymidine as previously described, and
harvested 18 hours later. Also, 0.1 ml of WF Con A sup was
also added in some cases to wells containing either
responders alone or both responder and stimulator cells.
Interleukin 2 (IL 2) Assay. Levels of IL 2 in Con A sups
from both BB and WF rats were determined by the stimulatory
activity of these samples on a murine IL 2-dependent cyto
toxic T cell line (anti-EL4, generously provided by Dr.
Shiro Shimuzu, University of Florida). Some 20 x 103 cyto
toxic T cells in 0.1 ml of complete medium were incubated at
37C for 22 hours with 0.1 ml of various Con A sups and 6
dilutions (50%-1.5%) of a reference murine Con A sup. The
cultures were then pulsed with 0.5 yCi/well of 3H-thymi-
dine and harvested 6 hours later.
Measurement of Gamma Globulin Levels. Gamma globulin
levels were determined on 10 yl serum samples from BB rats
with and without IDD, and WF rats using the Beckman micro
zone serum protein electrophoresis system.


47
onset of IDD in these rats had passed (Figures 1 and 2).
Although BB/W rats were susceptible to IDD for a longer age
range than BB/O animals, the marked increase in the
frequency of PCA in BB/W rats between 70 and 110 days of age
mirrored the increase in PCA frequency observed in BB/O rats
between 90 and 113 days of age. The frequency of SMA in BB
rats increased with age and, unlike PCA, did not closely
parallel the development of IDD in BB/O rats. The frequency
of SMA, however, increased in parallel with PCA in the BB/W
animals.
Functional abnormalities of the gastric parietal cells
were sought in BB rats with PCA as evidence that gastric
autoimmunity may result in clinical disease. Achlorhydria
was not demonstrated in the gastric aspirates of BB rats
with PCA, the pH values of which ranged from 2 to 3.
Vitamin B12 levels were measured in sera from 14 BB rats
with PCA, 14 PCA-negative BB rats, and 7 control WF rats.
No statistically significant differences were seen in serum
vitamin B12 levels between the three groups as evaluated by
Student's t-test (Table 3). Comparison of serum iron levels
and total iron binding capacities between 14 BB rats with
PCA, 11 BB rats without PCA and 7 control WF animals also
revealed no significant differences (Table 3).
Histological examinations of stomach sections from 12
BB rats with PCA were performed in order to see if lympho
cytic infiltration suggestive of autoimmunity were present.
All sections revealed mild to moderate lymphocytic


FIGURE 9. A plot of the ratio of the W3/25-positive subset
(helper T lymphocytes) to the MRC 0X8-positive subset
(cytotoxic/ suppressor T cells) versus age of the BB rats.
N designates nondiabetic BB rats and D designates BB rats
with IDD.


22
Detection of IDP and Sera Collection. All BB rats had
blood and urinary glucose levels determined weekly. Blood
samples were drawn from the periorbital venous sinus using
heparinized capillary tubes while the rats were under light
ether anesthesia. After clotting, serum glucose levels were
run in duplicate on a Beckman II glucose analyzer (Beckman
Instruments, Inc., Fullerton, California). Sera were
collected weekly and then stored at -20C until testings for
autoantibodies were made. Rats were considered to have IDD
if the animals had glycosuria or serum glucose levels above
250 mg/dl. For some studies, intraperitoneal glucose toler
ance tests (1.75 g/kg) were performed after an overnight
fast on BB rats aged 6-9 months who had not developed overt
IDD.
Handling and Preservation of Tissues for Autoantibody
Detection.
Normal WF rat tissues were used for detection of organ-
specific autoantibodies. After removal, the tissues (thy
roid, pancreas, adrenal and stomach) were cubed and immedi
ately snap frozen in isopentane cooled in a mixture of dry
ice and acetone and then stored at -80C. Four micrometer
tissue sections were cut out on a SLEE HR Mark II cryostat
(Slee Medical Equipment Ltd., London, England) at -20C,
placed on slides and air-dried. The slides were either used
immediately or stored for no more than one month at -80C.


180
129. Rozing, J., and Valssen, L. M. B. 1979. Mitogen
Responsiveness in rats. Transplantation Proceedings
11:1657.
130. Sporer, R., Manson, L. A., and Gotze, D. 1979. A
biochemical analysis of the membrane-associated gene
products of major histocompatibility complex of the
rat. J. Immunol. 122:2162.
131. Natori, T., Kanda, M., Ohhashi, T., Iwabuchi, K.,
Komura, K., and Aizawa, M. 1979. An estimate of the
gene sequence in the major histocompatibility complex
of the rat: RT1. Transplantation Proceedings
11:1568.
132. Cramer, D. V., Shonnard, J. W. and Gill, T. J. 1974.
Genetic studies in inbred rats. II. Relationship
between the major histocompatibility complex and mixed
lymphocyte reactivity. J. Immunogenetics 2:421.
133. Kunz, H. W., and Gill, T. J. 1982. Major histocom
patibility complex of the rat. Survey Immunol.
Res. 1:5.
134. Gunther, E., and Stark, 0. 1978. At least two loci
of the major histocompatibility complex can determine
mixed lymphocyte stimulation in the rat. Tissue
Antigens 11:465.
135. Gillis, S., Smith, K. A., and Watson, J. 1980. Bio
chemical characterization of lymphocyte regulatory
molecules. II. Purification of a class of rat and
human lymphokines. J. Immunol. 124:1954.
136. Watson, J., and Mochizuki, D. 1980. Interleukin 2:
A class of T cell growth factors. Immunological
Reviews 51:257.
137. Andersson, J., Gronvik, K., Larsson, E-L., and
Coutinho, A. 1979. Studies on T lymphocyte activa
tion. I. Requirements for the mitogen-dependent
production of T cell growth factors. Eur. J. Immunol
9:581.
138. Talal, N., Dauphinee, M., Wofsy, D., Kipper, S.,
Roths, J., and Murphy, E. 1981. Defective inter
leukin- 2 activity in C57BL/6 mice bearing the gene
lpr for autoimmunity and lymphoproliferation. Fed.
Proceedings 40:4705A.


30
splenic lymphocytes or PBL from nondiabetic or well con
trolled diabetic BB and WF rats at various cell concentra
tions ranging from 0.3-2 x 10^ cells/well were cultured in
round-bottom microtiter plates (Costar, Cambridge, MA) with
several mitogen concentrations. Pokeweed mitogen (PWM;
1-25 yg/ml) (Sigma, St. Louis, MO), PHA (0.025-1%) (Difco,
Detroit, MI), Con A (0.5-10 yg/ml) (Miles-Yeda, Rehovoth,
Israel) and WF Con A sup (0.l/ml/well) were used for a total
volume of 0.2 ml of complete medium in each well. After 48
hours incubation at 37C in 5% CO2, the cultures were
pulsed with 1.0 yCi/well of 3H-thymidine (Schwarz/Mann,
Spring Valley, NY, specific activity of 6 Ci/mM) and
harvested 18 hours later onto filter paper with a 24-line
cell harvester (Otto Hiller, Madison, WI). The filters were
then air-dried, placed into vials containing scintillation
fluid, and counted in a LKB Model 8100 liquid scintillation
counter (LKB Instruments, Rockville, MD). In several
experiments, 0.5 x 10^ spleen cells irradiated with 3000
rads from a 137Cs source (Gammator Model M) were also
added to some wells. Some 0.5 x 10^ or 1 x 10^ BB and
WF thymocytes/well were also incubated with 0.1 yg/ml and 1
yg/ml Con A and 0.1 ml/well WF Con A sup for 48 hours at 37
C, pulsed with 1.0 yCi/well of 3H-thymidine and harvested
18 hours later.
Mixed Leukocyte Cultures (MLCs). MLCs were performed in
round-bottom microtiter plates, with each well containing


CPM
100


66
TABLE 7
ALLOGRAFT REJECTION BY
BB AND WF RATS
Recipients
Donor
Graft Survival (Days)
8 BB
rats without
IDD
Lewis
30
+ 4
6 WF
rats
Lewis
12
+ 2
6 BB
rats without
IDD
WF
>
90
6 WF
rats
BB
17
+ 3
The period of graft survival was measured from day of
graft placement to day of complete graft rejection. Each
value is expressed as mean + S.D.


ACKNOWLEDGEMENTS
First and foremost, I wish to thank my parents,
Wayne and Nora Elder, for their lifelong love and support.
I would like to also especially acknowledge my sister,
Melanie Elder, for her love, understanding, and friendship.
I wish to likewise thank Phillip Ruiz for his affection and
help during my graduate education.
I wish to express my deep appreciation to Dr. Noel
Maclaren for his expert guidance and assistance during the
course of this research. The members of my committee,
Dr. Juan Scornik, Dr. Ammon Peck, Dr. Edward Wakeland,
Dr. Paul Klein and Dr. Christopher West, are also thanked
for their interaction, assistance and interest in this
project. Other members of the Pathology faculty to whom I
express special thanks for their help are Dr. William Riley,
Dr. Raul Braylan and Dr. Arthur Kimura. I am grateful to
the Department of Pathology for the financial support I
received during my graduate career.
I would like to express my gratitude to Lee Glancey,
Tom McConnell and Edith Rosenbloom for their invaluable,
technical assistance and support in the completion of these
studies. Their excellent work aided in the development and
completion of this project. My thanks also go to everyone
else associated with the Lab. The help of Crystal Grimes
in


FIGURE 7. Hematoxylin and eosin stained section of gastric
fundus from a BB rat with PCA showing lymphocytic
infiltration of the mucosa (black arrow).


FIGURE 29. Hematoxylin and eosin stained section of a
thymus from a nondiabetic BB rat at 21 days of age.


CPM
83


TABLE 16extended.
Con
A sup
168991 + 5435
125904 + 962
II
+
Cx (1.0)
n.d.
n.d.
II
+
" (2.0)
n.d.
n.d.
C = Lewis (1.0)
-
n.d.
35052 + 6513
Ax
(1.0)
n.d.
55566 + 3239
II
(2.0)
n.d.
98563 + 18159
Bx
(1.0)
n.d.
42363 + 10671
II
(2.0)
n.d.
45317 + 10961
a Rabbit Ig antirat
Ig treated spleen
cells were
used as responders at
1.0 x 105
cells/well.
b 5
Unseparated spleen cells were used as stimultors at 1.0-2.0 x 10 cells/well.
c .
Each value is expressed as mean of triplicate cultures + S.D.
d 0.1 ml of WF Con A sup added to each well.
6 Not done
122


TABLE 11
MLC RESPONSES OF UNSEPARATED SPLEEN CELLS FROM BB, WF AND LEWIS RATS
Counts Per Mimitec
Responder Stimulator
(cells/wel l)
(cel 1s/well)
Expt. 1
Expt.
2
Expt. 3
A = DB (0.5)
-
3342 + 545
515 +
230
412 + 94
Bx
(0.5)
2769 345
1503 +
436
n.d.

(1.0)
. d
n.d.
530 +
148
309 + 152
-
(2.0)
n.d
796 +
220
n.d.
Cx
(1.0)
3561 + 92
505
in
1288 + 255

(2.0)
3763 + 2562
612 +
153
n.d.
Dx
(1.0) *
n.d.
n.d
305 + 117
Bx
(0.5) + Cx
(1.0)
2372 + 150
1023 +
25
780 + 99

+ "
(2.0)
2475 + 422
1837 +
121
n.d.
Con
. e
A sup
n.d.
1401 l
92
1551 + 49

Cx
(1.0)
n.d.
n.d.
1123 + 480

"
(2.0)
n.d.
1072
340
n.d.
M
Bx
(1.0)
n.d.
n.d.
883 250
R = WF (0.5)
-
7916 1277
705 +
66
446 + 11
Ax
(0.5)
100441 2877
6260 +
899
3900 + 020
M
(1.0)
n.d.
2744 +
169
2702 + 1047
H
(2.0)
n.d.
4656 +
724
664 + 125
A
(0.5)
110574 + 1650
5473 +
1470
2572 + 42
Cx
(1.0)
130844 + 404
17709 +
2567
4679 + 465

(2.0)
9B728 + 2823
15846 +
2066
3622 1142
Dx
(1.0)
n.d.
n.d.
1970 + 710
M
(2.0)
n.d.
n.d.
879 + 650
Ax
(0.5) + Cx
(1.0)
136771 + 3335
12005 +
1030
3226 + 40
-
-t
(2.0)
131057 + 13639
10333 +
2844
n.d.
110


126
thymocytes (Table 17). The variability in levels of
proliferation between the three experiments was due to
differences in ages of the thymuses used, however, the BB
and WF thymuses were age matched for each experiment. The
ages of the thymuses used were 15 days, 26 days and 21 days
for experiments 1, 2 and 3 respectively. From these
findings, the defect in T lymphocyte immunocompetence would
seem to be expressed only in peripheral T cells.
Lymphoid Tissue Histology. Because the above findings
suggested that thymocytes from BB rats were functionally
normal, while splenic lymphocytes and PBL were obviously
immunoincompetent, histological examinations of BB thymuses,
spleens and lymph nodes were performed for comparison with
WF lymphoid tissues. No significant differences were seen
at the light microscopic level between thymuses from BB rats
(Figure 29) and WF rats (Figure 30). In sharp contrast, T
cell but not B cell-dependent areas of spleens (Figure 31)
and lymph nodes (Figure 33) from BB rats were severely
depleted of lymphocytes in comparison with spleens (Figure
32) and lymph nodes (Figure 34) from WF rats.
Gamma Globulin Levels. Serum gamma globulin levels were
measured in BB rats as a gross indication of B lymphocyte
function. The levels of immunoglobulins in sera from BB
rats with and without IDD were not significantly different
from those measured in WF controls (Table 18). These


112
responses in vitro. Unlike WF lymphocytes, BB spleen cells
were also unable to respond to Con A sup alone in the six
day MLC assay. The ability of WF cells to respond to Con A
sup may possibly be due to the presence of submitogenic
levels of Con A that in the presence of IL 2 were sufficient
for stimulation of proliferation.
No significant MLC responses were exhibited by BB
spleen cells to 0.5 x 10^ irradiated WF cells or to
1 x 10^ irradiated cells from another BB rat. In
contrast, lymphocytes from WF rats were able to proliferate
to 0.5-2 x 10^ irradiated BB cells in MLCs, confirming the
skin grafts results. The levels of proliferation by WF
lymphocytes to BB stimulators were generally lower than that
seen to irradiated Lewis spleen cells. The results were
variable, but no major inhibition of WF responses to Lewis
stimulator cells was observed in the presence of either
irradiated or nonirradiated BB spleen cells. These
findings, in addition to the mitogen assays, suggest the
lack of proliferative responses by BB lymphocytes in vitro
was not due primarily to increased suppressor activity.
Mitogenic and MLC Responses Using Rabbit Ig Antirat Ig
Purified T Cells.
To obtain highly purified populations of T lymphocytes
strongly reactive in alloresponses, BB spleen cells were
passed through rabbit Ig antirat Ig columns. The removal
of Fc receptor-bearing spleen cells (which include many B
cells, monocytes and suppressor T lymphocytes) (121)


69
PWM Concentration (pg/tnl)