Environment Diabetes

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Chapter 17 Environmental agents andtype 1 diabetesAmanda J. MacFarlane and Fraser W. Scott

Summary

Most of the evidence linking the environment (ornon-genetic factors) and type 1 diabetes in humans isindirect, based on epidemiological and animal studies.This includes the fact that the concordance rate fordiabetes in monozygotic twins is not 100% but about3050%, suggesting additional non-genetic influenceson causation. Type 1 diabetes incidence is increasing atabout 3% per year, a rate too high to be attributable tochanges in susceptibility genes. There is also a 350-foldvariation in the incidence of type 1 diabetes in differentcountries throughout the world; even in Europe, which isrelatively genetically homogeneous, there is a tenfoldrange in incidence. The environmental agents most often implicated

in type 1 diabetes are chemicals, viruses and foodcomponents. The N-nitroso compounds, streptozocin(streptozotocin) and alloxan, cause b-cell destructionin animals, and similar compounds in smoked meat andother foods and drinking water have been linked withtype 1 diabetes in humans, but there has been littledetailed investigation of this association. Viruses may act againstb cells by mechanisms thatinclude direct cytotoxicity and the triggering of anautoimmune process. Mumps, rubella, cytomegalovirus,enteroviruses such as coxsackie and retroviruses havebeen implicated. Some bacteria produce b-cell toxins(streptozocin and bafilomycin A1 from Streptomyces),and bacteria may also act as adjuvants for the immune

response to food antigens. Food components account for more than 50% ofdiabetes cases in rodents that spontaneously develop thedisease. The gut may play an important role in diabetes

pathogenesis. There is enhanced immune activation inthe gut of some type 1 diabetic subjects, with signs ofinflammation and increased gut permeability. Enhancedgut permeability to lumen antigens may lead to abreakdown in tolerance for dietary proteins. The most investigated dietary component associatedwith type 1 diabetes is cow-milk protein. Exposure tocow milk in early life (e.g. because of lack of breastfeeding) has been linked with type 1 diabetes inhumans and diabetes-prone BB rats. However, there isinconsistency in the studies, perhaps due to the variablecomposition of milk, with genetic variation in cowproteins. The findings are also consistent with theexistence of a subset of milk-sensitive diabetes-prone

individuals. Immune tolerance to insulin might also becompromised by early exposure to cow milk, whichcontains much less insulin than does human milk. Wheat gluten is a potent diabetogen in BioBreeding(BB) rats and NOD mice, animal models of type 1 diabetes.Between 5% and 10% of type 1 diabetic patients havegluten-sensitive enteropathy (coeliac disease) and manymore have antibodies to transglutaminase, a circulatingmarker of coeliac disease. Wheat may therefore beinvolved in the pathogenesis of type 1 diabetes, possiblyinducing subclinical gut inflammation. Other environmental factors linked with type 1diabetes include vitamin D, an immune modulator andsuppressant. This might explain the general northsouth

gradient of type 1 diabetes incidence in Europe, withlower mean sunshine hours in the north. Psychologicalstress has also been suggested as a trigger for diabetes,but data are sparse and inconsistent.

1

Type 1 diabetes is a multifactorial, immune-mediated

disease that occurs in individuals who have various

combinations of risk genes, the penetrance of which

is determined by exposure to environmental factors

(Fig. 17.1) [1]. The risk genes most strongly linked to

the development of diabetes (see Chapter 15) are the

class II major histocompatibility complex (MHC) geneslocated on chromosome 6p21.3HLA DQB1, DQA1,

HLA DRB1 (which includes HLA DR3 and DR4)

referred to collectively as IDDM1, which account for as

much as 50% of the risk, as well other non-MHC genes,

including insulin [1]. The environmental factors most

often considered are viruses (Chapter 16), diet, toxins

and stress, in order of suspected involvement [24].

Information about the risk genotype is incomplete, and

the inability of genome-wide scans to identify the full

complement of risk genes [1] indicates that there are

in fact several risk genotypes, interacting with a vari-

ety of environmental factors. It is becoming clear

that previous genetic association studies were under-powered, and new initiatives to develop an interna-

tional Type 1 Diabetes Genetics Consortium in order to

obtain sufficient numbers of subjects may address this

issue [5].

Direct evidence that environmental factors can cause

diabetes is rare. The case of individuals attempting to

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commit suicide by swallowing the rodenticide Vacor,

and surviving only to develop diabetes [6], speaks

more to the non-genetic effects of a massive dose of

a -cell toxin and does not reflect the natural course ofspontaneous autoimmune type 1 diabetes. Another

example is the children born with congenital rubella

syndrome in New York City in the 1960s; approxim-

ately 20% of these individuals developed type 1 dia-

betes [7]. Although this is strong evidence that rubella

virus can affect diabetes outcome, the fact that rubellahas essentially been wiped out by mumps/measles/

rubella (MMR) vaccine programmes in most developed

nations cannot explain the remarkable increase in dia-

betes over the past 40 years, from 13 per 100 000 to 42

per 100 000 in Finland, with other countries also

showing major increases [8].

Most of the evidence linking environment and

diabetes outcome is indirect and comes from epidemio-

logical and animal studies. More recently, several

prospective studies either in high-risk first-degree relat-

ives or individuals from the general population have

been initiated (the DIPP, DAISY, BabyDiab, TRIGR and

PANDA studies). It is not clear whether individuals

who are first-degree relatives of patients represent a

unique subset compared with the 90% of patients with

type 1 diabetes who do not have a first-degree relativewith the disease. It is now apparent that the genetics of

diabetes is far more complex than previously thought,

and there are probably several risk genotypes that react

in a unique manner to sequential or coincident envir-

onmental exposures. This chapter is not an exhaustive

review, but rather an attempt to alert the reader to

17.2 Chapter 17

Virus

Diet

Islet massMHC I

Toxins/stress

Repair/immune counter-regulation

Th1/Th2 cells

-cell death

Type 1 diabetes

Genes Environment

Fig. 17.1 Integrative biology of type 1 diabetes. There may be several diabetes genotypes, each of which may interact withone or more environmental factors. Evidence from animal studies suggests that some susceptible individuals may have lessislet mass compared with their non-diabetic counterparts. The exact event or combination of circumstances that initiatesthe immune attack on the b cells is not known. As the process wears on, the normal repair processes cannot counteract theloss ofb cells, until finally glucose homeostasis can no longer be maintained. Attempts to prevent or reverse the processhave focused mostly on various forms of immunosuppression, islet transplantation or avoidance of potential environmentaldiabetogens (e.g. the TRIGR trial, in which infants are exposed to a hydrolysed casein-based infant formula instead of amilk formula with intact protein). The complexity of the interactions may be better understood as newer technologies revealdiabetes-related patterns in the target tissue, effector cell populations and as the identity of various environmentaldiabetogens becomes known. MHC, major histocompatibility complex; Th, T helper (cell ).

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Environmental agents and type 1 diabetes 1

(autoimmunity) compared with discordant dizygotic

twins and that discordant monozygotic twins have a

higher risk of disease development compared with

dizygotic twins [13,18]. The authors of this chapter

consider that these observations demonstrate a genetic

predisposition to an autoimmune process against ICAs,

but do not eliminate the environment as a major player

in the development of overt diabetes.

Several hypotheses have been put forward to

explain the discrepancy between genotype and pheno-

type. T-cell and -cell immune specificity occurs as aresult of V(D)J recombination of the antigen receptor

during cellular differentiation (reviewed in [20]). This

somatic recombination in lymphocytes may explain

differences in immune competence between identical

twins who are genetically identical at birth, but not

thereafter. Endogenous and exogenous influences will

differ among individuals, so that in essence each indi-

vidual is one experimental unit, with unique genetic

susceptibility, and an equally unique set of cumulative

environmental exposures. In effect, the disease process

waxes and wanes, producing a different natural course

(Fig. 17.2) for each individual, similar to the chaotic

model proposed by the late Kevin Lafferty to describe

how each individual non-obese diabetic (NOD) mouse

(an animal model of type 1 diabetes, see Chapter 19)

follows its own path to overt diabetes.

Secular changes in diabetes incidence

The overall incidence of type 1 diabetes in Europe

increased significantly at an annual rate of approxim-

ately 3% from 1989 to 1998 in children of 014 years

[21] (Chapter 5). This reflects the global annual

increase in diabetes incidence in this age group from

1960 to 1996, determined from 37 populations world-

wide, which was also reported as approximately 3%

[8]. Using these numbers, Onkamo et al. [8] estimated

that the incidence of type 1 diabetes in the year 2010

could be as high as 50 per 100 000 in Finland (a figure

that may in fact already have been reached) and 30 per

100 000 in many other countries. There was a remark-

able increase in disease incidence of almost 5% in

young European children from 0 to 4 years of age [21].

The incidence of type 1 diabetes appears to be increas-

ing in almost all populations worldwide at a rate that

is too high to attribute to changes in the frequency

of susceptibility genes [22]. Rather, it is likely thatchanges in the environment are playing a major role in

the changing incidence of diabetes.

Seasonality of first insulin injection is commonly

observed. There is an increase in patients who require

their first insulin injection in the winter months from

October to March, with a corresponding decrease in the

some of the important cornerstones and new findings in

this area. The focus is mainly on human type 1 diabetes,

with selected examples from studies using animal

models. The reader may refer to several reviews for

more details [24,912].

Evidence implicating environmentalfactors in type 1 diabetes

Twin studies

In order to study how the environment contributes to

the development of type 1 diabetes, some studies have

focused on disease concordance in mono- and dizy-

gotic twin pairs. If disease development is dependent

on genetic predisposition alone, then both individuals

from a genetically identical, monozygotic twin pair

would share the same risk of disease. Monozygotic

twins have an increased risk of progression to the dis-

ease state compared with dizygotic twins or siblings

[1315]. The increased pairwise concordance among

monozygotic twins is expected because of the strong

genetic component of the disease. However, the con-

cordance rate is not 100%, indicating that environment

and/or somatic genetic recombination may play a role

in disease induction and development. Concordance

rates in monozygotic twins vary between 23% and

70%, with a mean of approximately 3050%, depend-

ing on age at initial diagnosis [1317]. When the first

diabetic twin is diagnosed before 5 years of age, the

concordance rate is 6570% [14,15]. Concordance

rates in twin pairs in which the initial diagnosis is after

15 years of age range from 18% to 38% [13,16,17].

Although most monozygotic twin pairs are discord-

ant for diabetes, -cell autoimmunity can and doesoccur in the non-diabetic twin. -Cell autoimmunity(see Chapter 18) can be monitored by the presence of

autoantibody markers to glutamic acid decarboxylase

65 (GAD65), islet-cell antigen 2 (ICA2) and insulin

autoantibodies (IAA) (Table 17.1). These markers of

autoimmunity were observed more frequently than

overt diabetes in identical twins [17]. In monozygotic,

discordant twin pairs, 2066% of the twins that

remained diabetes-free had autoantibodies to ICA2,

insulin and/or GAD65, indicating the presence of an

autoimmune attack and damage to cells [17,18]. A

similar trend was observed in discordant dizygotictwins.

It has been argued, on the basis of a re-analysis of

twin studies, that diabetes outcome may be determined

mainly by genetics [19]. In spite of this, previous reports

demonstrated that discordant monozygotic twins have

an increased prevalence of islet-cell autoantibodies

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17.4 Chapter 17

Table

17.1

Autoantige

nsintype1diabetes(reviewedin[135

138]).

%ofrecent-onsetpatients

Autoantigen

Expressedin:

withautoantibodies

Antibodiescross-reactwith:

GAD65

Neuroendocrinepancreaticisletcells,b

rain

70

80

CoxsackievirusB4antigens

GAD67

Neuroendocrinepancreaticisletcells,b

rain

10

20

(Pro)-insulin

Pancreaticislet

b

cells

40

70

Retroviralproteinp73

ICA512(IA-2)

Receptor-typeproteintyrosinephosphatasein

50

60

pancreas,brain,pituitary,neuroendo

crinetissue

IA-2

b

(phogrin)

Pancreaticisletcells,brain,neuroendoc

rinetissue

30

50

38K/jun-B*

Nucleartranscriptionfactor

33

71

HumanCMVandherpesvirus

antigens

GLUT-2*

Glucosetransporter

Hsp60/Hsp65*

Ubiquitouslyproduced,maybeonsurfa

ceof

16

GAD65,coxsackievirusA9and

pancreaticislet

b

cells

coxsackievirusB4antigens

CarboxypeptidaseH*

Pancreaticisletsandneuroendocrinecells

Nodifferentfromcontrols

52kDa

Insulinomacellline

Rubellavirusantigens

p69*

Pancreaticisletcells,brain

20

30

ABBOSpeptidefrombovinese

rumalbumin

Glima38*

Mitochondrialproteinofbroaddistribu

tion

20

Aromatic

L-amino

Peripheralandcentralnervoussystems,liver,

0(foundinpatientswithau

toimmune

aciddecarboxylase

*

intestine,kidney,pancreatic

b

cells

polyendocrinesyndrometype1)

DNAtopoisomeraseII*

Nucleoprotein

48

Insulin,GAD65andHsp65

Imogen38

Broadtissuedistribution

n/a(T-cellreactive)

*Notroutinelyusedfordiagnosisandpredictionoftype1diabetes.CMV,cyto

megalovirus;GAD,glutamicaciddecarboxylase;GL

UT,glucosetransporter;ICA,islet-cellantigen.

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summer months [21]. This may reflect seasonal expos-

ure to viruses, food antigens or chemicals that induce

or modulate disease.

Geographical variation

There is a wide global variation in diabetes develop-

ment both between and within genetically similar

ethnic groups, indicating a role for factors in the envir-

onment (Fig. 17.3). The overall age-adjusted incidence

of type 1 diabetes ranges from 0.1/100 000 per year

in China and Venezuela to 36.8/100 000 per year in

Sardinia and 36.5/100 000 in Finland [22]. The differ-

ence represents more than a 350-fold variation in dis-

ease incidence among 100 populations worldwide,

unique among chronic diseases. Among the European

nations studied in the EURODIAB TIGER study group,there was a more than tenfold range in the disease

incidence [21]. The large variation cannot be explained

by genetic differences, as European populations are

relatively homogeneous in comparison with indigen-

ous populations from other continents. The incidence

rates are highest in northern and north-western Eur-

-Cellmass

Time

Geneticpredisposition

-Cellmass

Precipitatingenvironmental

event

Earlypre-diabetes Late

pre-diabetes Overt diabetes

Overt diabetes

Glucose intolerance

Time

C-peptide absence

C-peptide absence

Glucose intolerance

Progressive loss ofinsulin release

Islet-cell antibodyGADA, IAA

Overt immunologicalabnormalities

Modern model

Pre-diabetes

Variable insulitis-cell sensitivity

to injury

Interactionsbetween

genesimparting

susceptibilityand resistance

Immunedysregulation

Environmentaltriggers andregulators

IAA

GADA, ICA512A, ICA

Loss of first phaseinsulin response(IVGTT)

Traditional model

Fig. 17.2 Schematicrepresentation of the naturalcourse of diabetes development,showing a traditional model anda revised model in which thechanges in b-cell mass with ageare depicted. The modern modeldepicts the chronic interactionof various genes, immune

dysregulation and environmentaltriggers and regulators resulting innear-complete loss of functioningb cells, glucose intolerance andfinally overt diabetes. From [139],with permission from the Editor ofThe Lancet. GADA, glutamic aciddecarboxylase antibodies; IAA,insulin autoantibody; ICA, islet-cellantigen; IVGTT, intravenousglucose tolerance test.

ope, as opposed to those in central, southern and eastern

Europe, with Sardinia being an exception [21].

Candidate environmental factorsthat influence the outcome oftype 1 diabetes

Chemicals

N-nitroso compounds

Streptozocin (STZ), an N-nitroso compound, and

alloxan, a complex amine, are chemicals used to

induce diabetes in rodents [23]. N-nitroso compounds

that are structurally similar to STZ have been linked to

the development of type 1 diabetes in humans. Nitrite

and nitrate are common components in food and canreact with amines and amides to produce nitrosamines

and nitrosamides [24]. An increased incidence of type

1 diabetes in Icelandic boys born in October was

observed and linked to the consumption of smoked and

cured mutton that was eaten by the parents in the post-

Christmas season [25]. It was proposed that N-nitroso

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compounds present in smoked and cured meat were

mediating the increase in disease via the parental germ

cells [25]. Further studies focusing on the consumption

of N-nitroso compounds in either food or drinking

water found similar increased incidences of diabetes

[26]. It is thought that these compounds can directly

damage the cells, or may trigger the autoimmuneresponse against the islet cells [27], alone or in com-

bination with a viral infection. There have been few

follow-up studies of these findings.

Viruses (see Chapter 16)

Viruses are thought to act against cells by at leasttwo mechanisms. The first is by direct cytotoxicity to

the cells; the second is by triggering an autoimmune

process that targets the cells [28]. The following areexamples of viruses linked to the induction and devel-

opment of type 1 diabetes. Further details may be

found elsewhere [4,29,30] and in Chapter 16.

Mumps

A temporal relationship has been observed between

mumps and the onset of type 1 diabetes [31]. The

appearance of islet-cell antibodies after a mumps

infection suggests that the infection may play a signi-ficant role in the induction of an autoimmune process

and possibly type 1 diabetes [32].

Rubella virus

Some 1020% of patients diagnosed with congenital

rubella syndrome (CRS) developed autoimmune diabetes

17.6 Chapter 17

FinlandSwedenCanadaNorway

UKNew Zealand

KuwaitPuerto Rico

DenmarkUS

AustraliaItaly

PortugalVirgin Islands

The NetherlandsSpain

BelgiumLuxembourg

GermanyEstoniaGreeceAustria

HungarySlovakia

FranceBulgariaUruguay

BrazilSlovenia

LithuaniaTunisiaRussiaIsrael

ArgentinaLatvia

DominicaAlgeriaPoland

RomaniaSudan

ColombiaCuba

BarbadosJapanChile

MexicoMauritiusParaguay

ChinaPakistan

PeruVenezuela

0 5 10 15 20 25 30 35 40Incidence of type 1 diabetes (per 100 000/year)

Fig. 17.3 Geographicaldistribution of type 1 diabetes.Age-standardized incidence/100 000 per year of type 1diabetes in children 14 yearsof age in 100 populations. Data

for boys and girls have been pooled.From [22], with permission of theAmerican Diabetes Association.

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within 525 years after infection [33,34]. An auto-

immune process has been associated with CRS infec-

tion, as 5080% of diabetic patients with CRS have ICA

and/or IAA [7]. Molecular mimicry has been invoked

as a possible mechanism by which the autoimmune

process is triggered. For example, Karounas et al. [35]

demonstrated that monoclonal antibodies bound to

rubella virus capsid and envelope glycoproteins also

recognized a 52-kDa islet-cell protein.

Cytomegalovirus

Like CRS, cytomegalovirus (CMV) can be transmitted

from the mother to the fetus during pregnancy, either

transplacentally or at conception from an infected par-

ent whose genome carries the CMV genome [2]. The

CMV genome has been observed at an increased pre-

valence in lymphocytes from patients with newly dia-

gnosed type 1 diabetes [36]. The virus can also be

transferred prenatally or postnatally by close contact,

or through breast milk. However, no difference in CMV

antibodies in early pregnancy has been observed

among mothers whose offspring develop type 1 dia-

betes and those whose offspring do not [37]. Nicoletti

et al. [38] reported an association between islet-cell

and CMV-IgG antibodies, but no relationship was

observed between CMV antibodies and HLA-DR alleles

in unaffected siblings of patients with type 1 diabetes.

This suggests that a CMV infection may induce an

autoimmune process and the production of ICA, but

other factors must be required for the development of

overt type 1 diabetes [2]. However, another study did

not reveal any association between ICA and CMV anti-

bodies [37]. These contrasting observations do not

support the hypothesis that primary CMV infections

in utero or early childhood promote the development

of type 1 diabetes, or indicate it may be involved in a

small subset of individuals.

Enteroviruses

Enteroviruses (EVs) belong to the picornavirus family,

and typically infect the stomach and intestine [39].

EVs are a group of RNA viruses with four subfamilies

including the polioviruses, coxsackievirus A and B

(CAV and CBV) and echoviruses. EVs were initially

linked to type 1 diabetes through a temporal associ-

ation between CBV infection and disease incidence.

There appears to be a delay of 26 months between

CBV infection and disease onset [40] and a temporalrelationship has been reported in several other case

control studies linking EV infection and diabetes. As

many as 4069% of newly diagnosed type 1 diabetes

patients have elevated levels of antibodies to CBV, spe-

cifically CBV4, in comparison to 04% in controls

[41,42]. CBV-induced diabetes is associated with an

induction of autoantibodies against GAD65 [43], and

there is amino-acid homology between a CBV4 protein

and GAD65, which has led to the hypothesis that CBV4

can induce diabetes via molecular mimicry. It may be

that various strains of CBV4 that are present in the

general population are able to induce -cell damagein susceptible individuals [44]. It is thought that EVs

in general may trigger or potentiate existing -cellautoimmunity, either by direct cytotoxicity or by indir-

ect triggering of an autoimmune response [2].

Retroviruses

Insulin autoantibodies from type 1 diabetic patients

and their unaffected first-degree relatives have been

demonstrated to cross-react with the retroviral antigen

p73 [45]. Sixty-three per cent of IAA-positive diabetic

patients had antibodies that bound p73. When the sera

were preabsorbed with insulin, p73 was no longer

bound and vice versa, indicating the presence of cross-

reactive antibodies. It has been suggested that retro-

viral infections can lead to -cell autoimmunity viamolecular mimicry.

Bacteria

Streptomyces species are found ubiquitously in soil,

and can affect tuberous vegetables such as potatoes

and beets. STZ and bafilomycin A1 are macrolide anti-

biotics produced byStreptomyces species. Bafilomycin

A1 produced by these bacteria can cause glucose intol-

erance, decreased proinsulin and insulin release, and

decreased pancreatic islet size [46]. Thus, bafilomycin

A1 could be a source of-cell toxins in the human diet.Other bacteria may also act as adjuvants for the

immune response to food antigens. It has been

observed that heat-killed bacteria or microbial prod-

uctsincluding bacterial lipopolysaccharide, pertussis

toxin, cholera toxin and bacterial DNA oligonu-

cleotides with CpG motifscan act as adjuvants. The

administration of certain microbes or their metabolic

products can result in an altered cytokine pattern,

and they can enhance the immune response to orally

administered soluble antigens. Dysregulation of the

handling of dietary antigens by the immune system

could result in the activation of a destructive auto-

immune response to the cells [12,47].

Vaccination

In infants, immunization after 2 months of age has

been associated with an increased risk for type 1 dia-

betes [48]. However, other studies have not demon-

strated any link between the Haemophilus influenzae

type b vaccine, the bacille CalmetteGurin (BCG)

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vaccine, the diphtheria, tetanus and pertussis vaccine,

and the MMR vaccine, and type 1 diabetes [29,49]. It

has also been demonstrated that timing of vaccination

with the H. influenzaetype b vaccine is not linked to

the development of disease [50].

Perinatal factors

The triggers that initiate the autoimmune process in

young-onset type 1 diabetes patients may be present at

the fetal or perinatal stage of life. Maternal rubella

virus infection is linked temporally to diabetes incid-

ence, as is fetal exposure to EVs [34,5153]. Maternal

child blood group incompatibility is also strongly

linked to type 1 diabetes [54]. Other perinatal factors

include maternal age, pre-eclampsia, caesarean sec-

tion delivery, birth weight, gestational age and birth

order [9].

Food components

Dietary factors, mainly in cereal-based diets, account

for 50% or more of diabetes cases in rodents that

develop spontaneous diabetes [2,12]. Diet influences

insulitis and diabetes incidence and may modify cyto-

kine production by enterocytes or immunocytes [55].

Preliminary reports of the only prospective nutritional

intervention trial in humans, TRIGR, show that Finnish

infants at high risk fed a hydrolysed casein (HC)-based

infant formula, Nutramigen, were less likely to develop

islet-cell autoantibodies (GAD65, IA-2, insulin, ICA

[56]). The link between diet and type 1 diabetes has

generated interest in a possible role of the gut immune

system in diabetes pathogenesis. Our understanding

of the role of the gut in diabetes pathogenesis and its

modification by diet is rudimentary, particularly in

humans, in whom studies of the gastrointestinal tract

are difficult and are rarely conducted [57,58]. Because

diet has such major effects on diabetes development, it

is important to understand how an intraluminal stimu-

lus alters disease expression.

Savilahti and colleagues [58] found that jejunal

biopsies from patients with type 1 diabetes showed

increased MHC class II expression (HLA DR, DQ and

DP), a sign of enhanced immune activation, that had

expanded in most of the villi and crypts in addition to

the normal expression seen only on the upper villi.

Patients had more cells in the lamina propria that werepositive for47 integrin, indicating the potential tohome to the gut. Another study compared peripheral

blood mononuclear cells of young type 1 diabetic pa-

tients and healthy children [59]. Peripheral blood

mononuclear cells were sorted into high and low 47+

cells; interferon- (IFN-) secretion was higher and

transforming growth factor- (TGF-) was lower in47-high cells compared with controls [59]. The twomajor inducers of MHC class II expression in the in-

flamed gut are IFN- and tumour necrosis factor-(TNF-

). Enhanced T-helper type 1 (Th1) cytokine

responses in the gut can cause gut inflammation and

damage [60], and they are associated with increased

gut permeability in coeliac patients, Crohns disease

and other chronic gut inflammatory conditions. En-

hanced permeability is seen in several animal models

of gut inflammation [61,62]. Increased gut permeab-

ility to mannitol was recently reported in type 1

diabetic patients [63] and in BioBreeding (BB) rats [64].

Thus, the gut of patients with type 1 diabetes shows a

similar Th1 cytokine profile as the inflamed, diabetes-

susceptible pancreas, cells of the gut appear to be activ-

ated, and there is gut damage, as indicated by passage

of mannitol across the gut barrier.

Milk proteins

By far the most investigated diet component is cow

milk protein and its effect on autoimmunity or diabetes

outcome; several reviews are available [2,6568].

Exposure to cow milk early in life has been linked to

development of diabetes in humans [69,70], diabetes-

prone BB rats [71,72] and NOD mice [73,74]. A measure

of the intense interest in this relationship is the fact

that more than 25 casecontrol studies have been car-

ried out to examine the link between early exposure to

cow milk, lack of breast-feeding and later development

of type 1 diabetes. The results of these studies have

been controversial, with some showing a relationship

while others did not [75]. Two meta-analyses showed

an odds ratio of approximately 1.6 in favour of an

increased risk of developing diabetes in those exposed

early to cow milk [70,76]. Some, but not all, studies

in genetically susceptible animals have shown a rela-

tionship between milk-based diets and diabetes. We

conclude that milk powder-based diets can be diabeto-

genic in some susceptible individuals, but the diabetes-

inducing potential varies, possibly because of the

genetic variation in cow proteins. This might account

for much of the variability seen in the epidemiological

data and in animal studies, and might explain in part

why all infants at genetic risk do not develop diabetes

when exposed to cow milk proteins. Because milk is

pooled from several sources, studies in the past may

have been confounded. Thus, the controversy surround-ing milk most likely relates to variable milk composi-

tion, and there may be a subset of milk-sensitive at-risk

individuals distributed unevenly in the total at-risk

population. At present, there is no way to identify them.

Elliott et al. reported that milk protein induced dia-

betes in NOD mice, and it was proposed that this effect

17.8 Chapter 17

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may be attributable to the A1 -casein variant ratherthan the A2variant [77]. The consumption of-casein

A1 and B was later reported to be associated with the

incidence of diabetes in children 014 years of age

[78]. A group from Iceland recently reported that-

casein fractions may influence type 1 diabetes incidence

[79]. A recent international trial in which standardized

diets with purified milk -casein variants, A1 or A2,were fed to NOD mice and BB rats, showed that in most

instances diets based on either variant protected the

animals from developing diabetes [80].

Vaarala and colleagues first reported that immune

tolerance to insulin may be compromised by early

exposure of Finnish infants to insulin in cow milk

[81,82]. They found that cow milk-based products con-

tained low levels of insulin that were associated with

increased immunoglobulin G (IgG) antibodies in the

first 9 months of life. Peripheral blood T-cell responses

to bovine insulin were increased compared with the

response to human insulin in some subjects. Human

milk contains approximately four times as much

insulin as cow milk. This has prompted the suggestion

that human insulin should be added to infant formulas

to increase the oral immune tolerance to insulin and

possibly prevent diabetes [83]. There are also animal

data suggesting that early oral exposure to insulin can

prevent diabetes [84]. However, this could also pose

risks, as the effect of oral antigens is dose-dependent,

and other attempts at oral tolerizing in fact induced

autoimmunity [85].

Others have reported no relation with the duration

of breast-feeding or the introduction of cow milk

products [86], and one study showed no relationship

between cellular and humoral immunity to -casein,-casein, -lactoglobulin or bovine serum albumin(BSA) [87]. With respect to effects of the BSA-derived

ABBOS peptide (and other milk proteins) on cytokine

production by peripheral blood lymphocytes from

patients and controls, BSA caused a weak Th2 res-

ponse, and in general, milk proteins did not show an

immune deviation in patients that was different from

controls [88].

Wheat proteins

Wheat contains a complex mixture of proteins. Pro-

lamins is the general term for cereal storage proteins

from the plant endosperm; those from wheat make up

80% of the total protein and are known as wheatgluten (WG). Gluten is made up of gliadin and glutenin

proteins that remain after water extraction of wheat

dough. Wheat proteins can cause inappropriate immune

stimulation in susceptible individualsfor example,

the immune-mediated damage to the gut caused by

coeliac toxic (gliadin) peptides and the development of

immunoglobulin E (IgE)-mediated Bakers asthma from

exposure to water/salt-soluble wheat allergens.

WG is the most potent individual food diabetogen in

BB rats [12,89]. Wheat also induces diabetes in NOD

mice [90,91], and as many as 510% of patients with

type 1 diabetes have gluten-sensitive enteropathy

(coeliac disease). Even more patients have antibodies

against the coeliac autoantigen, tissue transglutam-

inase [92,93]. These data are consistent with the

involvement of dietary wheat proteins in diabetes

pathogenesis [12,89,94,95]. The identity of the dia-

betogenic agents in wheat and the mechanisms by

which they participate in diabetogenesis are unclear.

The majority of cereal-containing diets fed to laborat-

ory rodents are based on wheat and other plants. For

example, the new NTP-2000 diet contains 37.3%

wheat, is milk-free and produces a diabetes frequency

of 65% in BBdp rats. Similarly, the ProLab RMH 1000

diet fed to NOD/LtJmice by Karges et al. resulted in

62% of animals becoming diabetic, and was also a

milk-free diet containing 80% wheat [74]. We char-

acterized the diabetes-inducing potential of all the

major ingredients of the NIH-07 diet [12,96] which is

composed of 83.5% plant materials, and found that

wheat gluten was the major diabetogenic component

[12,89].

Coeliac diseasesimilarities and lessons

There are some parallels between the proposed effects

of wheat in diabetes and another wheat-induced

immune-mediated condition, coeliac disease. Coeliac

disease is caused by exposure of susceptible indi-

viduals to storage proteins from wheat, barley and

rye. Coeliac disease is the prototype food-induced

immune disorder [97], involving IFN--dependentimmune responses to gluten that produce intestinal

damage [98]. There is an unusually high frequency

of type 1 diabetes patients with overt coeliac disease

(510%), which is 1733 times that in the general popu-

lation, and many type 1 diabetes patients have high

levels of antibodies for tissue transglutaminase, a

recently identified coeliac autoantigen [99]. In a group

of 68 type 1 diabetic patients who were DQ2 homozy-

gotes, 33% had immunoglobulin A (IgA) antibodies to

transglutaminase [92]. Another report indicates that

10% of patients have transglutaminase C antibody

levels similar to those of coeliac patients, while another

30% have low-level transglutaminase C binding [93].The two diseases have in common certain genetic risks

[100,101], in particular the HLA DQB1*0201 allele.

Patients with type 1 diabetes also display increased

T-cell stimulation in response to WG [102]. Diabetes

in BB rats [89] and NOD mice [90,91] can be induced

by feeding WG diets. In very young infants newly

TODC17 8/22/02 4:17 PM Page 9


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diagnosed with diabetes, antigliadin antibodies have

been reported [103,104]. These similarities between

coeliac disease in humans and diabetes in BB rats, NOD

mice and type 1 diabetic patients are consistent with the

idea that wheat is involved in diabetes pathogenesis,

possibly by inducing a subclinical, gut inflammation in

individuals that develop diabetes.

Vitamin D

Vitamin D has been shown to have immunomodulat-

ory, specifically immunosuppressive, characteristics.

It may down-regulate the autoimmune process that

leads to overt diabetes. The biologically active form of

vitamin D is 1,25-dihydroxyvitamin D3 [1,25(OH)2D3][105]. Peripheral lymphocytes, macrophages and thy-

mus and pancreatic islet tissues carry the nuclear-

localized 1,25(OH)2D3 receptor, with activated T

lymphocytes having significant quantities (reviewed

in [105]). It has been shown that T-cell-mediated

responses can be blunted by 1,25(OH)2D3, either dir-

ectly by inducing a Th1Th2 switch in lymphocytes, or

indirectly by its action on antigen-presenting cells.

The northsouth gradient in the disease incidence

of type 1 diabetes may be correlated with lower mean

monthly sunshine hours, which in turn may correlate

with decreased biologically active vitamin D3 [106].

In a Finnish study, Hypponen et al. [107] observed a

lower rate of type 1 diabetes incidence in children whose

diets were supplemented with vitamin D, regardless of

dose. Similar trends were observed in seven centres

across Europe [106]. Long-term treatment of NOD mice

with 1,25(OH)2D3 also resulted in a decrease in the

incidence of insulitis, without impairment of cellular

immunity [108]. Cod-liver oil, a source of vitamin D,

when taken during pregnancy was also associated

with a lower risk for type 1 diabetes in the offspring

[109]. This may be a reflection of the effect of vitamin

D, or of eicosapentaenoic acid and docosahexaenoic

acid which are also present in cod liver oil, or a com-

bination of both. In-vitro studies using human pan-

creatic islets demonstrated that the addition of

1,25(OH)2D3 decreased nitrite, interleukin-6 (IL-6) and

MHC class I expression [110]. These characteristics

indicate a decrease in oxidative stress and inflam-

mation. The authors suggested that 1,25(OH)2D3 may

reduce the vulnerability of pancreatic islets to cyto-

toxic T cells and cytotoxic challenge. Caution must be

exercised when the diet is supplemented with vitaminD, as it has the potential to cause systemic toxicity

after a single overdose. In addition, the hypercalcaemic

effect of vitamin D3 or 1,25(OH)2D3 in test subjects

may be cause for concern if it were used for pro-

phylaxis in individuals at risk. For this reason, non-

hypercalcaemic analogues of 1,25(OH)2D3 have been

17.10 Chapter 17

investigated for their effect on disease incidence in

NOD mice [111].

There also may be a genetic link between vitamin D

and susceptibility to type 1 diabetes. It has been shown

that a polymorphism within the vitamin D receptor ini-

tiation codon or within the vitamin D receptor gene is

linked to increased risk for disease [112,113]. If uptake

of vitamin D is decreased by a defective receptor, dys-

regulation of T-cell-mediated activity could occur.

Stress

Psychosocial stress has been indicated as a precipitat-

ing factor in the development of type 1 diabetes. Studies

in the BB rat demonstrated a relationship between

exposure to multiple, concurrent and unpredictable

environmental stressors and a significant decrease in

age of onset, in comparison to control groups [114].

Lehman et al. [115] demonstrated that chronic moder-

ate stress resulted in a significantly increased incidence

of diabetes over the control subjects. However, treated

male rats developed disease at approximately the same

time as control rats, and treated female rats showed

delayed onset of disease [115]contrasting with the

study by Carteret al. [114].

In humans, it has been demonstrated that severe

life events prior to diagnosis may be a risk factor for

disease. Although the frequency of stressful life events

was not different from reference controls, life events

reported by diabetic children tended to be more severe

in nature [116]. Children aged 59 years who had suf-

fered, or were threatened with, a loss within the family

were significantly more likely to be diabetic. Events in

the first 2 years of life in which difficult adaptation,

child behaviour problems or family dysfunction

occurred were more common in diabetic children than

in controls [117]. In contrast, recent-onset diabetic

patients between the ages of 15 and 34 years appeared

to have had no major stress factors within the year

before diagnosis [118]. It may be that stress early in life

is a risk factor for diabetes development, but not in

young adults.

Suggested mechanisms by whichenvironmental factors may trigger

autoimmunityBreakdown of tolerance

Breakdown of tolerance to -cell antigens can occurif the cells are damaged or destroyed, resulting in the

release of sequestered antigensfor example during

a viral infection or exposure to chemical toxins.

TODC17 8/22/02 4:17 PM Page 10


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Antigen-presenting cells can pick up and present -cell antigens to autoreactive T cells, activating them

and possibly targeting an immune response against the

cells. The end result may be the selective destructionof the insulin-producing cells (Fig. 17.4).

Molecular mimicry

Several mechanisms have been proposed to explain

how environmental antigens trigger a -cell-specificautoimmune response. Molecular mimicry is thought

to occur when a single lymphocyte recognizes a for-

eign antigen that shares conformational or amino-acid

sequence homology with an endogenous antigen [119].

Therefore, a lymphocyte activated by the presentation

of a foreign antigen may be able to recognize and target

self protein molecules or self peptide MHC complexes.

CBV is an example in which molecular mimicry may

contribute to the autoimmune process. Both the 65 and

67 kDa isoforms of GAD are autoantigens proposed

to be early targets in the destruction of cells. The2C protein (P2C) of CBV4 shares a six amino-acid

sequence, PEVKEK, with the GAD proteins, and anti-

body and T-cell cross-reactivity among these peptides

has been demonstrated [120122]. Synthetic peptides

consisting of the homologous region have been pro-duced and are reported to stimulate human peripheral

blood lymphocytes at a higher frequency in type 1 dia-

betes patients than in controls [123]. These findings led

to the hypothesis that the homologous region acted as

a molecular mimic between the foreign CBV4 protein

and the endogenous GAD proteins.

Phase

Targettissue

Draininglymph node

Combinationof conditionsgenerating

autoimmunedisease

APC influxinduced by:

1 Aspecific necrosisof target cells(virus, toxins)

2 Altered metabolismof growth of targetcells

Aberrant regulation ofimmune response:

1 Defects in intrathymicgeneration of T cells

2 Defects in T-celldeletion (AICD)

3 Th1:Th2 imbalance

4 Defects in Tr circuitsdue to altered APCfunction

Pathological reactionof target cells:

1 Excessive accumulationof lymphoid cells andtheir products. Effects of:(a) receptor antibodies;(b) ADCC;(c) blocking/toxic effects

of cytokines andmacrophage-derivedradicals;

(d) CD8 cytotoxicity.

2 Excessive susceptibilityof target cells

Tr

TGF-

IFN

IFN-

IL-4IL-4

P

enen

Aabs

APC APCAPC

Cy E E E E E E

Ag

+

RadicalsCy

Cy+

+

+

ReceptorAabsADCC

Effector phaseCentral phaseAfferent phase

M

Time

Th1 Th2

APC

CD8

Th1

Th2

Th1Th1

Th2

Cy

CD8

momo

Fig. 17.4 Breakdown of immunetolerance in autoimmunity. In theafferent phase, antigen-presentingcells (APCs) accumulate in thetarget tissue. The influx of APCs

can be induced by a specificinflammatory stimulus, such asnecrosis of target cells by viralinfection or chemical toxins. TheAPCs can take up autoantigens,some of which may normally besequestered, leave the tissue andtraffic to the local draining lymphnode. In the central phase, theAPCs will prime autoreactive T cellswithin the lymph node, followedby an aberrant immune response.Tolerance to the target tissue isovercome, and an autoimmunereaction occurs. Listed are anumber of abnormalities foundin animal models underlying thedysregulation of the immuneresponse. In the effector phase,autoreactive T cells, b cells andantibodies damage and destroy thetarget tissue. Aabs, autoantibodies;ADCC, antibody-dependent cell-mediated cytotoxicity; Ag, antigen;AICD, activation-induced celldeath; b, b cell; Cy, cytokines; E,endocrine cell; en, endothelial cell;IFN, interferon; IL, interleukin; mo,monocyte; Mf, macrophage; P,plasma cell; Th1, T helper -1 cell;Th2, T helper-2 cell; Tr, T-

regulatory cell; +, stimulation;, suppression; , traffic. From[140], with permission from theNature Publishing Group.

TODC17 8/22/02 4:17 PM Page 11


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However, recent findings do not support this hypo-

thesis. Schloot et al. [30] demonstrated that none out of

four GAD65 peptide-reactive T-cell lines derived from

type 1 diabetes patients cross-reacted with the homo-

logous P2C peptide. Two T-cell lines cross-reacted with

GAD65 and a P2C peptide, but it was found that the

recognition was occurring through different restriction

elements and not by cross-reaction to the homologous

peptide [30]. It is becoming clear that bystander activa-

tion and cell death (see below) may play a larger role in

CBV4-induced autoimmune diabetes than molecular

mimicry. It is important to remember that although it is

an appealing concept and the basis of several hypo-

theses, molecular mimicry has not been demonstrated.

Bystander activation and death

Bystander death of cells is a process in whichinflammation occurs in the vicinity of or within the

islets of Langerhans. During an inflammatory process,

immune mediators such as IFN-, TNF- or productionof nitric oxide can damage or destroy cells (reviewedin [124]). Bystander activation is the activation of a

lymphocyte without direct cell-to-cell contact [125].

For example, during a viral infection, an immune

response is activated that provides the appropriate

cytokine environment, which can lead to the recogni-

tion of-cell self antigens by indirect activation of-cell-specific lymphocytes [126]. Horwitz et al. [127]demonstrated that CBV4 infection could induce dia-

betes, not by activating T cells with a molecular mimic,

but by causing local inflammation and tissue damage,

releasing sequestered islet antigen, which re-stimulated

resting autoreactive T cells. It has also been shown that

disease induction may require the presence of a signi-

ficant population of pre-existing autoreactive T cells

and insulitis within the pancreas before CBV4 infection

[128]. It is proposed that the viral infection of cells

within the islets of Langerhans activates an indirect

antiviral defence resulting in the presentation of-cellantigen to the autoreactive T-cell population, leading

to disease induction [128,129]. Bystander activation

of autoreactive lymphocytes and bystander death ofcells can result in the breakdown of tolerance to self.

Diet, gut dysfunction and diabetes

The gut of susceptible individuals may be the site ofa mild, subclinical, inflammatory process [63] charac-

terized by increased expression of pro-inflammatory

mediators [55]. Inflammation can damage the gut bar-

rier, increasing permeability to lumen antigens, making

it difficult to induce normal oral tolerance [130]. In

diabetes-prone animals and possibly in humans, there

17.12 Chapter 17

may be a breakdown in the tolerogenic mechanisms

that normally prevent Th1 responses to dietary pro-

teins; this could be at the level of immunoregulation

or barrier function/permeability. Among the T-cell

repertoire of diabetes-prone individuals, there are-

cell-reactive T cells that could be activated either

non-specifically at a site of chronic inflammation or

following exposure to antigenic structures or immune

mediators from the gut lumen [57]. Our data [12,131]

suggest that dietary modulation has effects at two (or

more) levels:

1 At the target cells before classic insulitis, changingthe growth pattern of insulin-producing cells, enhan-

cing islet mass [131,132] and changing metabolism

and insulin reserves [133].

2 Dampening an ongoing inflammatory condition in

the gut [134].

We have therefore proposed that the gut may play

an essential role in the pathogenesis of diet-induced

cases of diabetes, possibly as a source of pancreatic

islet-directed inflammatory cells and stimulatory anti-

gens, and also as a major contributor of information

that controls the mass, function and metabolism of

insulin-containing cells in the pancreas [132].

Conclusions

Classic type 1 diabetes mellitus is a common, polygenic

chronic disease affecting one in 300 individuals, most

of whom are of Caucasian descent. Those affected are

mainly children and young adults, with a sizeable

affected group above 30 years of age. As many as 14%

of patients with type 2 diabetes may have a slow form

of autoimmune, type 1 diabetes, known as latent

autoimmune diabetes in adults (LADA). The expression

of overt diabetes is affected by exposure to factors in

the environmentthose most often cited are infectious

agents, mainly viruses, and certain chemicals, includ-

ing dietary proteins such as milk and wheat. Current

evidence suggests there are more than 20 diabetes

genes in humans, which in various combinations rep-

resent different risk genotypes. People with these

genotypes may react differently to one or several com-

binations of environmental factors. Epidemiological

data show variations in disease incidence by region,

with certain hot spots (Finland, Sardinia), and there are

changes in the disease incidence in migrants (YemeniteJews in Israel). If external factors that influence dia-

betes are common in the environment, the value of

ecological associations is questionable, and a dose

response relationship may be absent. It will be difficult

or even impossible to identify environmental factors

using traditional epidemiological approaches.

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7 Ginsberg-Fellner F, Witt ME, Yagihashi S etal. Congenitalrubella syndrome as a model for type 1 (insulin-dependent)diabetes mellitus: increased prevalence of islet cell surfaceantibodies. Diabetologia1984; 27 (Suppl.): 879.

8 Onkamo P, Vaananen S, Karvonen M, Tuomilehto J. Worldwide

increase in incidence of type 1 diabetes: the analysis of thedata on published incidence trends. Diabetologia 1999; 42:1395403.

9 Dahlquist GG, Patterson C, Soltesz G. Perinatal risk factors forchildhood type 1 diabetes in Europe. The EURODIAB Substudy2 Study Group. Diabetes Care1999; 22: 1698702.

10 Leiter EHIC, Gerling JC, Flynn. Spontaneous insulin-dependentdiabetes mellitus (IDDM) in nonobese diabetic (NOD) mice:comparisons with experimentally induced IDDM. In: McNeill J,ed. Experimental Models of Diabetes. Boca Raton, FL: CRCPress, 1999: 25794.

11 Mordes JP, Bortell R, Groen H etal. Autoimmune diabetes mel-litus in the BB rat. In: Sima A , Shafrir E, eds. Frontiers in AnimalDiabetes Research: Primer on Animal Models of Diabetes.Reading, UK: Harwood Academic, 2001: 141.

12 Scott FW. Food-induced type 1 diabetes in the BB rat. DiabetesMetab Rev1996; 12: 34159.

13 Redondo MJ, Rewers M, Yu L etal. Genetic determination ofislet cell autoimmunity in monozygotic twin, dizygotic twin,and non-twin siblings of patients with type 1 diabetes: pro-spective twin study. BMJ1999; 318: 698702.

14 Kyvik KO, Green A, Beck-Nielsen H. Concordance rates ofinsulin dependent diabetes mellitus: a population based studyof young Danish twins. BMJ1995; 311: 91317.

15 Kumar D, Gemayel NS, Deapen D etal. North-American twinswith IDDM: genetic, etiological, and clinical significance ofdisease concordance according to age, zygosity, and the inter-val after diagnosis in first twin. Diabetes1993; 42: 135163.

16 Kaprio J, Tuomilehto J, Koskenvuo M etal. Concordance for type1 (insulin-dependent) and type 2 (non-insulin-dependent)diabetes mellitus in a population-based cohort of twins inFinland. Diabetologia1992; 35: 10607.

17 Verge CF, Gianani R, Yu L etal. Late progression to diabetesand evidence for chronic beta-cell autoimmunity in identical

twins of patients with type I diabetes. Diabetes 1995; 44:11769.

18 Petersen JS, Kyvik KO, Bingley PJ etal. Population based studyof prevalence of islet cell autoantibodies in monozygotic anddizygotic Danish twin pairs with insulin dependent diabetesmellitus. BMJ1997; 314: 15759.

19 Gale EA, Bingley PJ, Eisenbarth GS etal. Reanalysis of twinstudies suggests that diabetes is mainly genetic. BMJ 2001;323: 9978.

20 Nemazee D. Receptor selection in B and T lymphocytes. AnnuRev Immunol2000; 18: 1951.

21 Green A, Patterson CC. Trends in the incidence of childhood-onset diabetes in Europe 198998. Diabetologia 2001; 44(Suppl. 3): B38.

22 Karvonen M, Viik-Kajander M, Moltchanova E etal. Incidenceof childhood type 1 diabetes worldwide. Diabetes Mondiale(Diamond) Project Group. Diabetes Care2000; 23: 151626.

23 Ganda OP, Rossini AA, Like AA. Studies on streptozotocin dia-betes. Diabetes1976; 25: 595603.

24 World Health Organization. Nitrates, Nitrites and N-NitrosoCompounds. Geneva: World Health Organization, 1978.

25 Helgason T, Jonasson MR. Evidence for a food additive as acause of ketosis-prone diabetes. Lancet1981; ii: 71620.

26 Kostraba JN, Gay EC, Rewers M, Hamman RF. Nitrate levels incommunity drinking waters and risk of IDDM: an ecologicalanalysis. Diabetes Care1992; 15: 15058.

Genomic and proteomic analyses may reveal more

fruitful predictors of risk and progression to -celldestruction, particularly in the form of altered disease-

specific patterns of gene and protein expression. Data

from the two most-studied animal models of spon-

taneous type 1 diabetes, the NOD mouse and BB rat,

are consistent with this interpretation, as numerous

changes in the environment can alter diabetes outcome

in these highly inbred animals. Thus, although the risk

of developing diabetes varies with ones complement

of risk and protective genes, the process that destroys

insulin-producing cells appears to be rather plastic.As the details of the pathogenesis and how it is affected

by external factors become clearer, the hope is that

primary prevention or deviation of the autoimmune

process and beneficial modification of islet homeos-

tasis will prevent those at risk from developing overt

diabetes. One approach to achieving this is to under-

stand and modify the environmental factors that

induce disease or equip those at risk with better means

of avoiding or handling these agents. In order to better

understand how diabetes occurs, we need to consider

the integrative biology, which involves complex gene

and protein expression patterns of target tissues, effec-

tor cells, and how these are altered by environmental

components.

Acknowledgements. The authors would like to thank

the following agencies for supporting the research pro-

gram in Fraser Scotts laboratory: Juvenile Diabetes

Research Foundation (JDF), Canadian Institutes of Health

Research (CIHR), Ontario Research and Development

Challenge Fund, Canada Foundation for Innovation,

Health Canada. Amanda MacFarlane is the recipient of

a scholarship from the Ontario Graduate Scholarship

Program and the University of Ottawa.

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