Leaky-Gut Autoimmunity - Crohns Dad

Leaky Gut and Autoimmune Diseases
Alessio Fasano
Clinical Reviews in Allergy &
Immunology
ISSN 1080-0549
Clinic Rev Allerg Immunol
DOI 10.1007/s12016-011-8291-x
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Clinic Rev Allerg Immunol
DOI 10.1007/s12016-011-8291-x
Leaky Gut and Autoimmune Diseases
Alessio Fasano
# Springer Science+Business Media, LLC 2011
Abstract Autoimmune diseases are characterized by tissue
damage and loss of function due to an immune response that is
directed against specific organs. This review is focused on the
role of impaired intestinal barrier function on autoimmune
pathogenesis. Together with the gut-associated lymphoid
tissue and the neuroendocrine network, the intestinal epithelial
barrier, with its intercellular tight junctions, controls the
equilibrium between tolerance and immunity to non-self
antigens. Zonulin is the only physiologic modulator of
intercellular tight junctions described so far that is involved
in trafficking of macromolecules and, therefore, in tolerance/
immune response balance. When the zonulin pathway is
deregulated in genetically susceptible individuals, autoimmune disorders can occur. This new paradigm subverts
traditional theories underlying the development of these
diseases and suggests that these processes can be arrested if
the interplay between genes and environmental triggers is
prevented by re-establishing the zonulin-dependent intestinal
barrier function. Both animal models and recent clinical
evidence support this new paradigm and provide the rationale
for innovative approaches to prevent and treat autoimmune
diseases.
Introduction
Keywords Antigens . Autoimmunity . Gut permeability .
Immune response . Tight junctions . Zonulin
Classical Theories on the Pathogenesis of Autoimmune
Diseases
A. Fasano (*)
Mucosal Biology Research Center,
University of Maryland School of Medicine,
20 Penn Street HSF II Building, Room S345,
Baltimore, MD 21201, USA
e-mail: afasano@mbrc.umaryland.edu
Soon after autoimmune diseases were first recognized more
than a century ago, it was believed that their development
was associated with viral and bacterial infections. The
connection between infection and autoimmune disease is
often explained by a mechanism known as “molecular
mimicry,” whereby microbial antigens are postulated to
resemble self-antigens [1]. The induction of an immune
response to the microbial antigens results in a cross-reaction
with the self-antigens and the induction of autoimmunity.
The intestinal epithelium is the largest mucosal surface
providing an interface between the external environment
and the mammalian host. Its exquisite anatomical and
functional arrangements and the finely-tuned coordination
of digestive, absorptive, motility, neuroendocrine, and
immunological functions are testimonial of the complexity
of the gastrointestinal (GI) system. Also pivotal is the
regulation of molecular trafficking between the intestinal
lumen and the submucosa via the paracellular space. The
dimensions of the paracellular space are estimated to be
between 10 and 15 Г…, suggesting that under physiological
circumstances, solutes with a molecular radius exceeding
15 Г… (~3.5 kDa) will be excluded from this uptake route.
Macromolecule trafficking is dictated mainly by intestinal
paracellular permeability, whose regulation depends on the
modulation of intercellular tight junctions (TJ). A fast
growing number of diseases, including autoimmune diseases, are recognized to involve alterations in intestinal
permeability related to changes in TJ competency.
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According to this theory, once the autoimmune process is
activated, it becomes independent of continuous exposure to
the environmental trigger and is therefore self-perpetuating
and irreversible. Epitope-specific cross-reactivity between
microbial antigens and self-antigens has been shown in some
animal models to initiate autoimmunity [2]. Conversely, in
most human autoimmune diseases, molecular mimicry seems
to be a factor in the progression of a pre-existing subclinical
autoimmune response, rather than in the initiation of
autoimmunity [2]. Another theory suggests that microorganisms expose self-antigens to the immune system by directly
damaging tissues during active infection, and that this leads
to the development of autoimmunity. This mechanism has
been referred to as the “bystander effect,” and it occurs only
when the new antigen is presented with the orally administered triggering antigen. Whether pathogens mimic selfantigens, release sequestered self-antigens, or both, however,
remains to be elucidated.
New Proposed Hypothesis: the Leaky Gut as Third
Element in Autoimmune Pathogenesis
A common denominator in autoimmune diseases is the
presence of several pre-existing conditions that lead to an
autoimmune process [3]. The first of these conditions is the
genetic susceptibility of the host immune system to
recognize, and potentially misinterpret, an environmental
antigen presented within the gastrointestinal tract. The
second is that the host must be exposed to the antigen.
Finally, the antigen must be presented to the gastrointestinal
mucosal immune system following its paracellular passage
from the intestinal lumen to the gut submucosa; this process
is normally prevented by competent TJ [3–5]. In many cases,
increased intestinal permeability seems to precede disease
and causes an abnormality in antigen delivery that triggers
the multiorgan process leading to the autoimmune response
[3–5]. Taking the above information into consideration, we
propose that the pathogenesis of autoimmune diseases can be
described by three key points [6]:
1. Autoimmune diseases involve a miscommunication
between innate and adaptive immunity;
2. Molecular mimicry or bystander effects alone might not
explain entirely the complex events involved in the
pathogenesis of autoimmune diseases. Rather, the continuous stimulation by nonself-antigens (environmental
triggers) seems to be necessary to perpetuate the process.
Contrary to general belief, this concept implies that the
autoimmune response can theoretically be stopped and
perhaps reversed if the interplay between genes predisposing individuals to the development of autoimmunity
and environmental triggers is prevented or eliminated;
3. In addition to genetic predisposition and exposure to
triggering nonself-antigens, the loss of the protective
function of mucosal barriers that interact with the
environment (mainly the gastrointestinal and lung
mucosa) is necessary for autoimmunity to develop.
Evidence Supporting This New Theory
Celiac disease (CD) is the best testimonial of the validity to
the accuracy of the new paradigm for the pathogenesis of
autoimmunity proposed above. Celiac disease is an autoimmune condition triggered by the ingestion of glutencontaining grains in genetically susceptible individuals (for
more details, see CD section below).
Given the undisputable role of gluten in causing inflammation and immune-mediated tissue damage, CD is a unique
model of autoimmunity in which, in contrast to most other
autoimmune diseases, a close genetic association with HLA
genes, a highly specific humoral autoimmune response
against tissue transglutaminase auto-antigen, and, most
importantly, the triggering environmental factor (gliadin), are
all known. It is the interplay between genes (both HLA and
non-HLA associated) and environment (i.e., gluten) that leads
to the intestinal damage typical of the disease [7]. Under
physiological circumstances, this interplay is prevented by
competent intercellular TJ. Early in CD, TJs are opened [8–
12]. Combined, this information provides the rationale for
the treatment of the disease based on complete avoidance of
gluten-containing grains from the patients’ diet. Following
gluten withdrawal, the symptoms resolve, the biomarkers of
the autoimmune process return within normal limits, and the
intestinal autoimmune insult heals. These outcomes support
the notion that the autoimmune process can be reverted
provided that the interplay between genes and environmental
trigger(s) can be prevented.
Besides celiac disease, several other autoimmune diseases, including type 1 diabetes [13, 14], multiple sclerosis
[15, 16], and rheumatoid arthritis [17], are characterized by
increased intestinal permeability secondary to noncompetent TJs that allow the passage of antigens from the
intestinal flora, challenging the immune system to produce
an immune response that can target any organ or tissue in
genetically predisposed individuals [18–21].
Intestinal Barrier Function and Its Regulation
A century ago, TJs were conceptualized as a secreted
extracellular cement forming an absolute and unregulated
barrier within the paracellular space [22]. Biological studies
of the past several decades have shown that TJs are
dynamic structures subjected to structural changes that
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The discovery of zonula occludens toxin (Zot), an
enterotoxin elaborated by Vibrio cholerae that reversibly
opens TJ [23], increased our understanding of the intricate
mechanisms that regulate the intestinal epithelial paracellular pathway and led to the discovery of its eukaryotic
counterpart zonulin [24, 25]. The physiological role(s) of
the zonulin system remains to be established. This pathway
appears to be involved in several functions, including TJ
regulation responsible for the movement of fluid, macromolecules, and leukocytes between the bloodstream and the
intestinal lumen and vice versa. Another possible physiological role of intestinal zonulin is the protection against
microorganism colonization of the proximal intestine
(innate immunity) [26].
Since zonulin is overexpressed in tissues and sera of
subjects affected by autoimmune diseases, we elected to use
sera from zonulin-positive and zonulin negative type 1
diabetes (T1D) and CD subjects to characterize further the
molecular nature of zonulin. Through proteomic analysis of
human sera, we have recently identified zonulin as prehaptoglobin (HP)2 [11], a molecule that, to date, has only
been regarded as the inactive precursor for HP2, one of the
two genetic variants (together with HP1) of human HPs.
Mature human HPs are heterodimeric plasma glycoproteins
Fig. 1 Composition of intercellular tight junctions. The structural
components of intercellular tight junctions can be classified in integral
membrane proteins (occludin, claudins, and JAM), junctional complex
proteins (ZO-1, ZO-2, p130 or ZO-3, 7H6, symplekin, cingulin), and
the cell cytoskeleton structures (microtubules, intermediate filaments,
and microfilaments)
dictate their functional status under a variety of developmental
scenarios. To meet the many diverse physiological challenges
to which the epithelial and endothelial barriers are subjected,
TJs must be capable of rapid and coordinated responses. This
requires the presence of a complex regulatory system that
orchestrates the state of assembly of the TJ multiprotein
network (Fig. 1). While our knowledge on TJ ultrastructure
and intracellular signaling events have significantly progressed during the past decade, relatively little is known
about their pathophysiological regulation secondary to
extracellular stimuli. Therefore, the intimate pathogenic
mechanisms of diseases in which TJs are affected have
remained unexplored owing to limited understanding of the
extracellular signaling involved in TJ regulation.
The Zonulin Pathway
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Fig. 2 Proposed zonulin intracellular signaling leading to the opening of
intestinal TJ. Zonulin interacts with a specific surface receptor [1] whose
distribution within the intestine varies. The protein then activates
phospholipase C [2] that hydrolyzes phosphatidyl inositol [3] to release
inositol 1,4,5-tris phosphate (PPI-3) and diacylglycerol (DAG) [4].
PKCО± is then activated [5], either directly (via DAG) [4] or through the
release of intracellular Ca2+ (via PPI-3) (4a). Membrane-associated,
activated PKCО± [6] catalyzes the phosphorylation of target protein(s),
with subsequent polymerization of soluble G-actin in F-actin [7]. This
polymerization causes the rearrangement of the filaments of actin and
the subsequent displacement of proteins (including ZO-1) from the
junctional complex [8]. As a result, intestinal TJ becomes looser (see
freeze fracture electron microscopy). Once the zonulin signaling is over,
the TJs resume their baseline steady state
composed of О±- and ОІ-polypeptide chains. While the ОІ
chain (36 kDa) is constant, the О± chain exists in two forms,
i.e., О±1 (~9 kDa) and О±2 (~18 kDa). The presence of one or
both of the О± chains results in the three human HP
phenotypes, i.e., HP1-1 homozygote, HP2-1 heterozygote,
and HP2-2 homozygote. The zonulin pathway modulating
TJ permeability is described in Fig. 2.
peptides involved in the disease pathogenesis is greater than
appreciated previously, with at least 50 toxic epitopes in
gluten peptides exerting cytotoxic, immunomodulatory, and
gut permeating activities. These activities have been partially
mapped to specific domains in О±-gliadin: the cytotoxic
peptide 31–43, the immunomodulatory peptide 57–89 (33mer), the CXCR3 binding, zonulin releasing (gut permeating)
peptides 111–130 and 151–170, and the IL8-releasing peptide
261–277 [21]. The effect of the permeating gliadin peptides
in vivo was confirmed by the analysis of intestinal tissues
from patients with active CD and non-CD controls probed
for zonulin expression [8]. Quantitative immunoblotting of
intestinal tissue lysates from active CD patients confirmed
the increase in zonulin protein compared to control tissues
[8]. Zonulin upregulation during the acute phase of CD was
confirmed by measuring zonulin concentration in sera of 189
CD patients using a sandwich ELISA. Compared to healthy
controls, CD subjects had higher zonulin serum concentrations (p<0.000001) during the acute phase of the disease
that decreased following a gluten-free diet [25].
Current data suggest that altered processing by intraluminal enzymes, changes in intestinal permeability, and
activation of innate immunity mechanisms seem to precede
the activation of the adaptive immune response [28]. Based
on these data and on the gliadin epitope mapping described
above, it is conceivable to hypothesize the following
sequence of events: after oral ingestion, gliadin interacts
Zonulin-Dependent Impaired Intestinal Barrier
Function and Autoimmune Diseases
Celiac Disease
CD is an immune-mediated chronic enteropathy with a wide
range of presenting manifestations of variable severity. It is
triggered by the ingestion of gliadin fraction of wheat gluten
and similar alcohol-soluble proteins (prolamines) of barley
and rye in genetically susceptible subjects with subsequent
immune reaction leading to small bowel inflammation and
normalization of the villous architecture in response to a
gluten-free diet [27]. CD not only affects the gut, but it is a
systemic disease that may cause injury to any organ. It is a
complex genetic disorder, and HLA status appears to be the
strongest genetic determinant of risk for celiac autoimmunity.
Gluten is a complex molecule made of gliadin and
glutenins, both toxic for CD patients. The repertoire of gluten
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with the small intestinal mucosa causing interleukin (IL)8 release from enterocytes (peptide 261–277), so leading to
immediate recruitment of neutrophils in the lamina propria.
At the same time, gliadin permeating peptides 111–130 and
151–170 initiate intestinal permeability through a MyD88dependent release of zonulin (as we have recently confirmed by identifying CXCR3 as the receptor that releases
zonulin in a MyD88-dependent manner, see ref. [29]) that
enables paracellular translocation of gliadin and its subsequent interaction with macrophages (through 33-mer and
other immunomodulatory peptides) within the intestinal
submucosa [30]. This interaction initiates signalling
through a MyD88-dependent, but TLR4 and TLR2independent pathway, resulting in the establishment of a
proinflammatory (Th1-type) cytokine milieu [30] that
results in mononuclear cell infiltration into the submucosa.
The persistent presence of inflammatory mediators such as
tumor necrosis factor (TNF)-О± and interferon (IFN)-Оі
causes further increase in permeability across the endothelial and epithelial layers [31], suggesting that the initial
breach of the intestinal barrier function caused by zonulin
can be perpetuated by the inflammatory process after the
access of gliadin to the submucosa. In genetically predisposed individuals this, in turn, may permit the interaction of
T cells with antigen presenting cells, including macrophages, leading ultimately to the antigen-specific adaptive
immune response causing the autoimmune insult of the
intestinal mucosa seen in patients with CD [32].
Once gluten is removed from the diet, serum zonulin
levels decrease, the intestine resumes its baseline barrier
function, the autoantibody titers are normalized, the
autoimmune process shuts off and, consequently, the
intestinal damage (that represents the biological outcome
of the autoimmune process) heals completely.
Type 1 Diabetes
Type 1 diabetes (T1D) is an autoimmune condition,
sometimes associated to diseases that are characterized by
marked immunologic features, such as CD and thyroiditis
[33, 34]. GI symptoms in diabetes mellitus have been
generally ascribed to altered intestinal motility secondary to
autonomic neuropathy [35].However, other studies suggest
that an increased permeability of intestinal TJ is responsible
for both the onset of the disease and the GI symptoms that
these patients often experience [36]. This hypothesis is
supported by a study performed on a spontaneously
diabetic animal model [37]. The authors of this study
showed an increased permeability of the small intestine of
Bio Breeding Diabetes Prone (BBDP)/Wor diabetic-prone
rats that precedes at least a month the onset of diabetes.
Further, histological evidence of pancreatic islet destruction
was absent at the time of increased permeability but clearly
present at a later time [37]. Therefore, the authors presented
evidence that increased permeability occurred before either
histological or overt manifestation of diabetes in this animal
model. We confirmed these data by reporting in the same
rat model that zonulin-dependent increase in intestinal
permeability precedes the onset of T1D by 2–3 weeks
[38]. Oral administration of the zonulin inhibitor, AT1001
(now called Larazotide acetate), to BBDP rats blocked
autoantibody formation and zonulin-induced increases in
intestinal permeability, so reducing the incidence of
diabetes [38]. These studies suggest that the zonulindependent loss of intestinal barrier function is one of the
initial steps in the pathogenesis of T1D in the BBDP animal
model of the disease. The involvement of zonulin in T1D
pathogenesis was corroborated by our studies in humans
showing that ~50% of T1D patients has elevated serum
zonulin levels that correlated with increased intestinal
permeability [39]. We also provided preliminary evidence
suggesting that, as in the BBDP rat model of the disease,
zonulin upregulation precedes the onset of diabetes in T1D
patients [39]. Interestingly, a smaller percentage (~25%) of
unaffected family members of probands with T1D have
also been found to have increased serum zonulin levels and
increased gut permeability [39], suggesting that loss of
intestinal barrier function is necessary but not sufficient for
the onset of the autoimmune process.
Several reports have linked gliadin (the environmental
trigger of CD autoimmunity that also causes zonulin release
from the gut, see refs. [8] and [26]) to T1D autoimmunity
both in animal models and in human studies [40–42]. More
recently, we reported a direct link between antibodies to
Glo-3a (a wheat-related protein), zonulin upregulation, and
islet autoimmunity in children at increased risk for T1D
[43]. Glo-3A antibody levels were inversely associated with
breast-feeding duration and directly associated with current
intake of foods containing gluten in islet autoimmunity
cases but not in controls [43]. Further, zonulin was directly
associated with Glo-3A antibody levels in cases but not in
controls, suggesting that the presence of Glo-3A antibodies
and zonulin upregulation in islet autoimmunity cases are
related to an underlying difference in mucosal immune
response as compared to controls.
Asthma
Asthma is a complex clinical syndrome characterized by
airflow obstruction, airway hyperresponsiveness, and inflammation. The mechanisms by which airway inflammation and alterations in airway function are maintained are
incompletely understood. Because wheezing can also be
triggered by food challenges in some asthmatic children,
increased intestinal permeability of asthmatics [44] may
play a role in susceptibility to environmental allergens. We
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have generated preliminary data suggesting that serum
zonulin levels are high in a subset of subjects affected by
asthma and that approximately 40% of asthmatic patients
have an increased intestinal permeability [21]. This preliminary observation suggests that, besides inhalation, an
alternative route for the presentation of specific antigens
or irritants may occur through the GI mucosal immune
system following their paracellular passage (normally
prevented by the intercellular TJ).
Multiple Sclerosis
Besides an increase in blood–brain barrier permeability,
multiple sclerosis (MS) patients may also experience an
increased permeability of intestinal TJ. Yacyshyn and
coworkers have demonstrated that 25% of MS patients
studied had an increased intestinal permeability [45]. The
fact that patients with MS [45] and Crohn’s disease [46]
both present an increased number of peripheral B cells
exhibiting CD45RO, a marker of antigen exposure, further
support the concept of preexisting, genetically determined
small intestinal permeability abnormalities with subsequent
altered antigen exposure as a pathogenic factor common to
these diseases.
To challenge this hypothesis, we measured serum levels
of zonulin in MS patients with different subtypes—
relapsing–remitting [RRMS] vs. secondary–progressive
[SPMS]—and activities to ascertain whether expression of
zonulin into peripheral circulation can differentiate these
two groups. Approximately 29% of patients with either
RRMS or SPMS had elevated serum zonulin levels (a
percentage similar to increased intestinal permeability in
MS patients reported by Yacyshyn et al., see ref. [45]), with
overall average serum levels ~2.0-fold higher than in
controls. Interestingly, patients with RRMS in remission
showed serum zonulin levels comparable to controls [21].
Inflammatory Bowel Diseases
Crohn’s disease and ulcerative colitis are inflammatory
diseases involving the GI tract in which abnormal paracellular permeability defects precede the development of
both syndromes and, therefore, appear to play an important
role in disease pathogenesis [46, 47]. The pathogenesis of
inflammatory bowel disease (IBD) remains unknown,
although in recent years there is convincing evidence to
implicate genetic, immunological, and environmental factors in initiating the autoimmune process. Several lines of
evidence, however, suggest that an increased intestinal
permeability plays a central role in the pathogenesis of
IBD. In clinically asymptomatic Crohn’s disease patients,
increased intestinal epithelial permeability precedes clinical
relapse by as much as 1 year, suggesting that a permeability
defect is an early event in disease exacerbation [48]. The
hypothesis that abnormal intestinal barrier function is a
genetic trait involved in the pathogenesis of IBD is further
supported by the observation that clinically asymptomatic
first-degree relatives of Crohn’s disease patients may have
increased intestinal permeability [48]. We have recently
generated evidence suggesting that zonulin upregulation is
detectable in the acute phase of IBD and that its serum
levels decrease (but still are higher than normal) once the
inflammatory process subsides following specific treatment
[21]. While a primary defect of the intestinal barrier
function (possibly secondary to activation of the zonulin
pathway) may be involved in the early steps of the
pathogenesis of IBD, the production of cytokines, including
IFN-Оі and TNF О± secondary to the inflammatory process
serve to perpetuate the increased intestinal permeability by
reorganizing TJ proteins ZO-1, junctional adhesion molecule 1, occludin, claudin-1, and claudin-4 [49]. In this
manner, a vicious cycle is created in which barrier
dysfunction allows further leakage of luminal contents,
thereby triggering an immune response that in turn
promotes further leakiness.
Ankylosing Spondylitis
Ankylosing spondylitis (AS) is a common and highly
familial rheumatic disorder that typically affects young and
middle-aged adults and is characterized by stiffness and
pain in the back. The link between increased intestinal
permeability and AS has been clearly established [50].
Using different markers of TJ permeability, two independent studies [51, 52] found an increased intestinal permeability in both AS patients and their relatives. These
changes precede the clinical manifestations of the disease,
suggesting a pathogenic role of TJ dysfunction in AS.
Using a proteomic approach, Liu et al. have identified
HP as an AS biomarker [53]. The authors investigated the
serum protein profiles of AS patients and healthy controls
from a large Chinese AS family using two-dimensional
electrophoresis analysis. A group of four highly expressed
protein spots was observed in all ankylosing spondylitis
patients’ profiles and subsequently identified as isoforms of
HP by ESI-Q-TOF MS/MS [53].
Proof of Pathogenic Role of Zonulin-Mediated Intestinal
Barrier Defect in Autoimmunity: the Celiac Disease
and Type 1 Diabetes Paradigms
CD and type 1 diabetes autoimmune models suggest that,
when the finely tuned trafficking of macromolecules is
deregulated due to a leaky gut, autoimmune disorders can
occur in genetically susceptible individuals [21]. This
theory implies that removing any of the three key elements
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(genes, environmental trigger(s), or impaired barrier function) should block the autoimmune process. To challenge
this hypothesis, zonulin inhibitor Larazotide acetate was
used with encouraging results in the BBDP rat model of
autoimmunity [38]. Besides preventing the loss of intestinal
barrier function, the appearance of autoantibodies, and the
onset of disease, pretreatment with Larazotide acetate
protected against the insult of pancreatic islets and,
therefore, of the insulitis responsible for the onset of type
1 diabetes [21].
This proof-of-concept in an animal model of autoimmunity
provided the rationale to design human clinical trials in which
Larazotide acetate was initially tested in an inpatient, doubleblind, randomized placebo controlled trial to determine its
safety, tolerability, and preliminary efficacy [54]. No increase
in adverse events was recorded among patients exposed to
Larazotide as compared to placebo. Following acute gluten
exposure, a 70% increase in intestinal permeability was
detected in the placebo group, while no changes were seen in
the Larazotide acetate group [54]. Gastrointestinal symptoms
were significantly more frequent among patients of the
placebo group as compared to the Larazotide acetate group
[54]. Larazotide acetate has now been tested in approximately
500 subjects with excellent safety profile and promising
efficacy as concern protection against symptoms caused by
gluten exposure in CD patients [55].
Conclusions
The classical paradigm of autoimmune pathogenesis involving specific gene makeup and exposure to environmental
triggers has been recently challenged by the addition of a third
element, the loss of intestinal barrier function. Genetic
predisposition, miscommunication between innate and adaptive immunity, exposure to environmental triggers, and loss of
the intestinal barrier function secondary to dysfunction of
intercellular TJ seem to be all key ingredients involved in the
pathogenesis of autoimmune diseases. Both in CD and T1D
gliadin may play a role in causing loss of intestinal barrier
function and/or inducing the autoimmune response in genetically predisposed individuals. This new theory implies that
once the autoimmune process is activated, it is not autoperpetuating, rather can be modulated or even reversed by
preventing the continuous interplay between genes and
environment. Since TJ dysfunction allows this interaction,
new therapeutic strategies aimed at reestablishing the intestinal barrier function offer innovative, unexplored approaches
for the treatment of these devastating diseases.
Funding Work presented in this review was supported in parts by
grants from the National Institutes of Health Grants DK-48373 and
DK-078699 to AF.
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