Chemotherapy induced intestinal mucositis; from bench to bed

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.

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