Part 1 focuses primarily on the pathophysiology of mucositis, in order
to gain more insight different experimental mouse models were used.
Chapter 2 describes mucositis induced by high dose doxorubicin (DOX)-
treatment. DOX is a frequently used cytostatic drug in childhood
cancer, often causing severe mucositis. DOX-induced mucositis closely
resembles the characteristics of previously studied methotrexate (MTX)-
induced mucositis. Both drugs induce severe damage to the epithelial
morphology, characterized by severe villus atrophy, changes in
epithelial proliferation and loss of epithelial differentiation. We
did not expect these similarities in morphological damage as DOX
attacks epithelial cells much closer to the stem cell than MTX does.
DOX was suspected to have a more severe influence on intestinal
homeostasis in comparison to MTX. The resemblance suggests a general
mechanism in intestinal damage and repair. The time-line however, in
which both drugs induced their damage to the intestine was different.
DOX-treatment leads to immediate hyper-proliferation (day 1 and 2)
with subsequent inhibition of proliferati!
on during severe morphological damage (day 3). MTX causes
proliferation inhibition within one day, followed by a period of hyper-
proliferation during severe intestinal damage. Furthermore, we studied
changes in epithelial-mesenchymal cross talk during DOX-induced
mucositis. The expression of the intestinal morphogene and TCF4, the
main Wnt pathway transcription factor in the intestinal epithelium
were followed by immunohistochemistry during the different stages of
DOX-induced mucositis. BMP4- and TCF4 expression appeared to be
linked, shown by the fact that BMP signaling seem to suppress Wnt
signaling and visa versa during mucositis development and
regeneration. This suggests a balance between epithelial proliferation
and subsequent intestinal differentiation.
Chapter 3 The objective of this study was to investigate the
expression of the small intestinal transcription factors HNF-1a, Cdx2,
GATA-4 in an experimental model of MTX-induced intestinal damage, and
to correlate these alterations with histological damage, epithelial
proliferation and differentiation. HNF-1a, Cdx2 and GATA-4 are
critical transcription factors in epithelial differentiation, and in
combination they act as promoting factors of the sucrase-isomaltase
(SI) gene, an enterocyte-specific differentiation marker which is
distinctly down regulated after MTX-treatment. Intestinal damage was
most severe at day 3 and was associated with decreased expression of
the transcriptional factors HNF-1a, Cdx2 and GATA-4, which correlated
well with decreased expression of SI, and seemed inversely correlated
with enhanced proliferation of epithelial crypt cells. During severe
damage, the epithelium was preferentially concerned with proliferation
rather than differentiation, most l!
ikely in order to restore the small intestinal barrier function rather
than maintaining its absorptive function.
In Chapter 4 we show that there were no major differences found in
intestinal pathology or protein expression during MTX-induced
mucositis in Muc2+/+ mice in comparison to MTX-induced mucositis in
Muc2-/- mice. Mucositis regeneration however, could not be assessed in
the absence of Muc2 as almost all mice died spontaneously 1 day prior
to sacrifice for evaluation. Surprisingly, however, the intestine of
the Muc2 deficient mice evaluated just a few days after MTX-treatment
showed already increased regeneration compared to the wild type mice.
In addressing this question it became clear that the cytokine
production by the mucosal immune system of Muc2 deficient mice was
different compared to wild type littermates. Both the pro-inflammatory
cytokine TNF-a as the anti-inflammatory cytokine Il-10 was increased
in naïve Muc2 deficient mice, indicating that Muc2 deficiency leads
to induction of an inflammatory response. This suggests that MTX
induced damage in the Muc2-/- mice may !
be tempered by triggering the immune system to release IL-10, an anti-
inflammatory cytokine, prior to MTX-treatment.
Chapter 5 MTX is associated with severe damage of the intestinal
epithelium. As a result, the mucosal immune cells become increasingly
exposed to a vast amount of microbial stimuli. In this study we aimed
at determining if and to what extent these cells are still functional
during MTX treatment. Furthermore, we assessed whether activation of
the mucosal immune system would play a role in the pathogenesis of
mucositis.
The fact that the adaptive immune system contributes to mucositis was
established by showing that lamina propria lymphocytes that were
derived from MTX-treated mice responded by an enhanced production of
various cytokines to ex vivo polyclonal (anti-CD3e and anti-CD28
mAb)stimulation. Next, in vitro experiments revealed that macrophages,
either a cell-line or cells isolated from the murine peritoneal
cavity, were not affected by MTX in the capacity to produce TNF-α and
IL-10 upon lipopolysaccharide (LPS) exposure. Moreover, in vivo
experiments showed that peritoneal macrophages isolated from MTX
treated mice produced more IL-10 and TNF-α upon LPS stimulation,
compared to cells derived from control mice. These data indicate
persistence of both innate and adaptive immune responses in this
model. The clinical relevance of these findings was further
established by the fact that LPS exposure prior to MTX treatment
aggravated the course of mucositis. Furthermore, LPS responsive !
mice recovered more slowly compared to LPS unresponsive mice during
MTX induced intestinal damage. Finally, we found an increase in weight
loss and intestinal damage upon MTX treatment in IL-10 deficient mice
in comparison to wild type (WT) controls, which suggests a protective
role for IL-10 in mucositis.
Part 2 focuses on intestinal metabolism during mucositis and mucositis
prophylaxis in childhood cancer patients.
In Chapter 6 we validate a new method for collecting breath samples
that simplifies the collection of breath samples in young children in
order to use this method in studies described in chapter 7. Stable
isotope tracers are used in clinical studies to measure (intestinal)
metabolism of various substrates. Nowadays, the oxidation of [13C]
labeled substrates to 13CO2 and the measurement of the appearance of
excess 13CO2 in expiratory air is a common method. The collection of
respiratory CO2, occurs via trapping of CO2 in sodium hydroxide
(trapping method) sometimes in conjunction with indirect calorimetry.
The aim of the present study was to determine the accuracy of direct
nasal-pharyngeal sampling method for the collection of breath samples
in preterm infants compared with the currently used trapping method.
Seven pre-term infants were studied while receiving full enteral
feeding. A primed constant 3-h intragastric infusion of [13C]
bicarbonate was given and breath samples !
were collected by means of direct nasal-pharyngeal sampling and by a
sodium hydroxide trap simultaneously. Breath CO2 isotopic enrichments
rose rapidly to reach a plateau by 120 min with < 5% variation of
plateau in both methods. 13CO2 breath isotopic enrichments obtained by
the direct nasal-pharyngeal sampling method correlated highly with the
trapping method, showing that direct nasal-pharyngeal sampling for the
collection of breath samples is as accurate as the trapping method.
Chapter 7 The aim of this study was to evaluate systemic availability
of dietary amino acids (leucine) during chemotherapy-induced
mucositis. We studied eight childhood cancer patients (age 1.5 to 16
years) on two days, i.e. the day before chemotherapy and 3-5 days
after. Chemotherapy-induced oral mucositis and diarrhea were scored on
a WHO toxicity scale. Stable isotope tracers were used to measure
first-pass splanchnic leucine uptake and whole-body leucine kinetics.
Patients showed increased mucositis and/or diarrhea toxicity scores
after chemotherapy. Systemic availability of enterally administered
leucine was not significantly affected by chemotherapy. Interestingly
however was that most of the children were already catabolic prior to
start of a new cycle of chemotherapy.
Therefore, all efforts should be directed at initiating enteral
feeding even before start of chemotherapy in order to reduce catabolic
state. Our data imply that this might be accomplished best by
hydrolyzed formula.
In Chapter 8 the efficacy and feasibility of a TGF-b2-enriched feeding
for preventing oral and gastro-intestinal mucositis in childhood
cancer patients were studied. The study was designed as a 2-period
crossover, randomized, double-blinded, placebo controlled trial.
Patients who had a high risk for developing mucositis and who would
receive two comparable cycles of chemotherapy were eligible to the
study. During one cycle of chemotherapy TGF-b2-enriched feeding was
administered; during the other a ‘placebo’ (not enriched) feeding
was used. WHO toxicity scales of diarrhea, oral mucositis, fever, anal
lesions and nausea/vomiting were scored daily. In addition, the
incidence of occurrence of blood cultures, antibiotic therapy and
interventions or diagnostics related to mucositis were measured. The
feasibility of the study was good: 83% of the patients completed two
cycles and 86% of the study feeding was consumed. Administration of
TGF-b2 was safe, as serum TGF-b2 did not !
increase and renal and liver function were not affected. The degree of
toxicity, scored during the whole observation period and the number of
days with WHO 3/4 toxicity did not significantly differ between cycles
with TGF-b2 enriched and normal feeding. These studies do not provide
evidence that TGF- decreases the incidence or degree of mucositis
induced by combination therapy in childhood cancer-patients.
In Part 3 all studies presented in this thesis are summarized, and new
insights for future studies are discussed