Functional Dyspepsia Microbiome Study

Recurrent abdominal pain has long been acknowledged to be one of the most common chronic
pain entities in children. In the US, ~13-17% of school-aged children and adolescents report
abdominal pain that occurs at least weekly and 21% of these report pain severe enough to
interfere with daily activities. (1) For most of these children (~90%), no specific organic
disease is found. However, these children experience decreases in quality of life that are
comparable to children with identifiable organic diseases such as inflammatory bowel disease
and gastro-esophageal reflux. (2) In addition, children with no identifiable organic disease
do not appear to get better on their own and often continue to have problems with abdominal
pain and associated symptoms into adulthood. (3,4)

Over 90% of children with chronic abdominal pain will fit the diagnostic criteria for a
functional gastrointestinal disorder (FGID). (5) As defined by Rome criteria, the FGIDs are
a set of diagnoses with specific symptom profiles in the absence of structural or
biochemical abnormalities to explain symptoms. FGIDs related to chronic or recurrent
abdominal pain include irritable bowel syndrome (IBS), functional dyspepsia (FD), functional
abdominal pain (FAP), and abdominal migraine. (6) FGIDs are probably best understood
utilizing a biopsychosocial model which states that abdominal pain occurs as a result of
varying contributions from, and interactions between, biological factors, psychological
factors, and social factors. The biological factors most implicated in the generation of
abdominal pain include motility disturbances, visceral hypersensitivity, and inflammation.
Stress/anxiety is the most implicated psychologic factor. Anxiety may interact with both
motility and inflammation. For example, the investigators have demonstrated an association
between anxiety and mucosal mast cells in children with post-prandial distress syndrome, a
subcategory of FD. (7) The scaffolding for the interaction between the various factors has
been termed the brain-gut axis consisting of both neural and humoral pathways.

There is increasing evidence that the investigators need to expand this paradigm to include
interaction of the biopsychosocial model with the intestinal flora, i.e. a
brain-gut-microbiota axis (B-G-M axis). It is becoming increasingly clear that host anxiety
and stress can effect the composition of the microbiome. (8) Likewise, the microbiome can
influence neural development, brain chemistry, and a wide range of behavioral phenomena
including emotional behavior, pain perception, and how the stress system responds. (9) Both
immune and neural pathways are involved intimately in visceral pain perception and
intestinal microbiota can modulate this communication. (8) The general scaffolding of the
brain-gut-microbiota axis includes the CNS, neuroendocrine and neuroimmune systems,
autonomic nervous system, and the enteric nervous system. (10) Microbiota communicate with
the brain-gut axis through different mechanisms including direct interaction with mucosal
cells, via immune cells, and via contact with neural endings. (11)

The B-G-M axis has been studied primarily under four conditions: 1. germ free intestines, 2.
infection or bacterial introduction, 3. antibiotic exposure, and 4. probiotic treatment.
Germ free mice exhibit anxiety-like behavior accompanied by decreased N-metyl-D-aspartate
receptor subunit mRNA expression in the amygdala and increased BDNF expression and decreased
serotonin receptor 1A expression in the dentate granule layer of the hippocampus. (12) The
brain is aware of the introduction of pathogenic microbes into the GI tract and this results
in brain stem nuclei becoming activated and in some instances, associated with the
development of anxiety-like behavior. (8) This occurs within hours after introduction of
pathogens at subclinical thresholds. (8) Even minute doses of microbes that do not trigger
an immune response are capable of influencing neurotransmission in the paraventricular
hypothalamus, the central nucleus of the amygdale, and the bed nucleus of the stria
terminalis, three regions involved in the processing of emotions related to anxiety and
mood. (13) Citerobacter rodentium (CR) is a bacteria that has been evaluated in a number of
studies involving mice. CR-infected mice demonstrated anxious-like behavior at 7-8 hours.
(14) CR has been demonstrated to induce anxiety-like behavior associated with no change in
plasma levels of IFN-γ, TNF-α, or IL-12 but with evidence of increased vagal transmission
without mucosal inflammation. (15) Additional work has also demonstrated that Helicobacter
pylori infected mice exhibit anxiety-like behavior corrected by treatment with a probiotic,
Bifidobacterium. Oral antibiotics have also been shown to induce anxiety-like behavior in
mice. (8) This behavior change is transient and behavior normalizes once normal flora has
been restored. This effect is not seen with systemic antibiotic administration. Changes in
behavior are accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the
hippocampus and amygdala. (8) Lastly, clinical evidence is mounting to support a role for
probiotics in reducing anxiety and the stress response as well as improving mood in patients
with IBS and those with chronic fatigue. (14) Probiotics have been shown to reduce anxiety
in rats and to have beneficial psychological effects with a decrease in serum cortisol in
humans. (14) Lactobacillus reuteri has been shown to decrease anxiety and the stress-induced
increase in corticosterone in mice. (A) In addition, there are a number of other studies
demonstrating the effects of probiotics on anxiety in humans. (8,9,13)

The DCX-domain family of proteins has been demonstrated to be involved in signal
transduction and cytoskeletal regulation. The investigators have demonstrated that DCDC3a
(DCLK1/DCAMKL1) is an intestinal epithelial stem cell marker expressed in the intestinal
mesenchymal stem cell niche which plays a critical role during intestinal tissue damage and
repair. DCDC2 is altered in the mouse model by Citrobacter rodentium infection and TNBS
induced colitis. DCDC2 knockout mice exhibit an increased anxiety phenotype. (16)

BDNF has been found to promote neuronal survival and differentiation and to guide axon
extension both in vitro and in vivo. (17) Evidence suggests that BDNF may act as a
stress-responsive intercellular messenger modifying HPA axis activity. (17) Short-term acute
stress induces a significant increase in BDNF mRNA and protein in both younger and older
groups but changes in the younger group are substantially greater. (18) BDNF has
interactions with both inflammation and pain. BDNF knock out mice show a weaker visceral
response to colorectal distention. (19) A human study compared 40 adults with IBS to 21
healthy controls. (20) Biopsies from patients with IBS revealed significant upregulation of
BDNF as compared to controls. (20)

The transient receptor potential vanilloid type-1 (TRPV1) is expressed throughout the
gastrointestinal tract in myenteric ganglia, muscular layers, and mucosa and TRPV1
expression has been reported on mast cells. (21) TRPV1 is expressed by intestinal sensory
nerves and activated by capsaicin, heat, acid, and inflammation. (21) TRPV1 receptor has
been implicated as a mechanosensor involved in integrating painful stimuli and in the
generation of neurogenic inflammation and hyperalgesia. (22) In rodent models of
inflammation, TPRV1 is upregulated with subsequent visceral hyperalgesia to mechanical and
chemical stimuli which can be attenuated by TRPV1 antagonism. (22,23) In the rat model, it
appears that mast cells are required for upregulation TRPV1 under both infectious and stress
conditions. (24) In humans with IBS, there is a significant increase in rectosigmoid
TRPV1-immunoreactive fibers and TRPV1 and mast cells are related to abdominal pain scores.
(21) Likewise, IBS in quiescent inflammatory bowel disease is associated with an increase in
rectosigmoid colon TRPV1-immunoreactive fibers as compared to asymptomatic quiescent IBD or
healthy controls and again these fibers correlate with the abdominal pain score. (25) There
is evidence of a significant role for TRPV1 in visceral sensitivity and TRPV1 appears to
interact with inflammation in general, mast cells in particular, and to be influenced by
intestinal infection and stress.

The major initiator of the body's physiological stress response is the release of
corticotropin releasing hormone (CRH). CRH, produced within the hypothalamus (as well as by
immune cells, including human lymphocytes and mast cells), is the principal regulator of the
basal and stress-induced pituitary-adrenal axis which, in turn, activates glucocorticoid
(e.g. cortisol) and adrenal androgen secretion. Though results have been variable, the
majority of studies support an enhanced HPA axis responsiveness in at least a subset of
adults with IBS. (26-30) CRH receptors (CRH-R) are widely expressed within the
gastrointestinal tract and immune cells, where CRH-R activation has multiple effects which
may be relevant in the etiology of FGIDs. These effects include alteration of autonomic
balance, visceral sensitivity, motility disturbances, and inflammation. The most important
mechanism by which CRH may generate pain is through interaction with inflammatory cells,
directly and via epithelial cells and enteric nerves. CRH mediates visceral hypersensitivity
by activating mucosal mast cells with subsequent mediator sensitization of afferent sensory
enteric nerves. (31,32) Gastric mast cells have been shown to be increased in adults with
dyspepsia and may contain both pre-formed and newly synthesized cytokines (including IL-4,
IL-5, IL-6, and TNF-α, among others). (33-34) The investigators have demonstrated delayed
gastric emptying and increased gastric dysrhythmia in children/adolescents with FD with
elevated antral mast cell density. (35) The investigators have also demonstrated this
dysrhythmia to be associated with increased post-prandial pain. (36) Adult humans have
demonstrated selective luminal release of tryptase and histamine from jejunal mast cells
under cold stress; the magnitude of release was shown to be similar to that induced by
antigen exposure in food allergic patients. (37) This is a rapid response with peak
histamine and tryptase concentrations occurring between 15 and 30 minutes. Mast cells may
also recruit eosinophils as a secondary effector cell. Eosinophils are increased in the
duodenal mucosa of 71% of children with FD and they have also been found to be increased in
adults with FD. (38,39) These eosinophils are highly activated and the investigators have
demonstrated clinical improvement with treatment directed at mucosal eosinophils. (40,41)
The investigators have previously found a high correlation between anxiety scores and
mucosal eosinophil density, providing preliminary support for the role of CRH in downstream
eosinophil recruitment and activation. (42) Stress also has been shown to shift the relative
proportion and trafficking of T helper lymphocytes towards a Th2 or "allergic" phenotype.
This shift is driven by catecholamines and central CRH as well as CRH from peripheral nerves
and inflammatory cells. The Th2 phenotype is associated with release of IL 4,IL 10, and IL
13, which stimulate growth and activation of mast cells and eosinophils. (43) CRH also has
been associated with increased expression of TNF-α, MCP1, and IL 8, which have, in turn,
been associated with hyperalgesia. (44-46) As described above, CRH can initiate an
inflammatory cascade which may lead to pain directly or indirectly by causing dysmotility
and visceral hypersensitivity.

The purpose of the current study is to describe the microbiome in children with FD and to
explore relationships between the microbiome and postprandial distress syndrome, anxiety
scores, and mucosal biomarkers or anxiety.

SPECIFIC AIMS

1. To determine the frequencies and relative proportions for specific bacteria or bacteria
phyla in children with FD in both duodenal mucosal specimens and stool samples.

2. To determine if the frequencies or proportions of specific bacteria or bacteria phyla
differ between children with and without PDS.

3. To determine bi-variate correlations between bacteria/phyla frequency, bacteria/ phyla
proportion, anxiety scores, and mucosal biomarkers, respectively.

Inclusion Criteria:

- Diagnosis of FD as determined by the GI physician in accordance with Rome III
criteria

- Age 8-17 years inclusive

- Scheduled for upper endoscopy as part of routine care after failing to respond to
acid suppression therapy

Exclusion Criteria:

- Use of oral antibiotic or probiotic within 8 weeks prior to enrollment

- Use of systemic steroid or immunomodulating drug within 8 weeks of enrollment