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

Review: Ontogeny of oral drug absorption processes in children

A large proportion of prescribed drugs to children are administered orally. Age-related change in factors affecting oral absorption can have consequences for drug dosing. Areas covered: For each process affecting oral drug absorption, a systematic search has been performed using Medline to identify relevant articles (from inception till February 2012) in humans. This review presents the findings on age-related changes of the following processes affecting oral drug absorption: gastric pH, gastrointestinal motility, bile salts, pancreatic function, intestinal pH, intestinal drug-metabolizing enzymes and transporter proteins. Expert opinion: Clinicians should bear in mind the ontogeny of oral drug absorption processes when prescribing oral drugs to children. The authors’ review shows large information gaps on almost all drug absorption processes. It is important that more knowledge is acquired on intestinal transit time, intestinal pH and the ontogeny of intestinal drug-metabolizing enzymes and drug transporter proteins. Furthermore, the ultimate goal in this field should be to predict more precisely the oral disposition of drugs in children across the entire pediatric age range

The gut microbiome in patients with intestinal failure: Current evidence and implications for clinical practice

Intestinal failure (IF) is the reduction of gut function or mass below a minimum needed to absorb nutrients and fluids, such that patients are dependent on parenteral nutrition (PN). Patients with IF have an altered gut microbiome. Our aim was to review and evaluate the current evidence on gut microbiome and its metabolic activity as well as its association with disease characteristics in adults and children with IF. We performed a PubMed literature search for articles published after 2000 using the following terms: intestinal failure, microbiome, microbiota, short-chain fatty acids, short bowel syndrome and parenteral nutrition. Literature search was restricted to human studies only. The gut microbiome diversity is remarkably reduced and community structure is altered with a noticeable over-abundance of Proteobacteria, especially the Enterobacteriaceae family. A substantial increase in Lactobacillus level is often reported in patients with IF. Gut microbiome characteristics have been associated with poor growth, liver disease, D-lactic acidosis and duration of intestinal adaptation. Differences in microbiome characteristics have been found between patients on PN and those whose guts have adapted and have been weaned off PN. Future research with prospective sample collection should explore the value of the gut microbiome as a biomarker to guide clinical practice and as a modifiable therapeutic target to optimize outcomes of patients with IF

Standardized and Individualized Parenteral Nutrition Mixtures in a Pediatric Home Parenteral Nutrition Population

OBJECTIVES: Studies evaluating efficacy or safety of standardized parenteral nutrition (PN) versus individualized PN are lacking. We aimed to assess effects on growth and safety of standardized PN compared with individualized PN in our Home PN group. METHODS: Descriptive cohort study in Dutch children on Home PN, in which standardized PN was compared with individualized PN. Both groups received similar micronutrient-supplementation. Primary outcome was growth over 2 years, secondary outcomes were electrolyte disturbances and biochemical abnormalities. Additionally, patients were matched for age to control for potential confounding characteristics. RESULTS: Fifty patients (50% girls, median age 6.5 years) were included, 16 (32%) received standardized PN mixtures. Age (11 vs 5 years), gestational age (39.2 vs 36.2 weeks) and PN duration (97 vs 39 months) were significantly higher in the group receiving standardized PN (P: ≤0.001; 0.027; 0.013 respectively). The standardized PN group showed an increase in weight-for-age (WFA), compared with a decrease in the individualized PN group (+0.38 SD vs -0.55 SD, P: 0.003). Electrolyte disturbances and biochemical abnormalities did not differ. After matching for age, resulting in comparable groups, no significant differences were demonstrated in WFA, height-for-age, or weight-for-height SD change. CONCLUSIONS: In children with chronic IF, over 2,5 years of age, standardized PN mixtures show a comparable effect on weight, height, and weight for height when compared with individualized PN mixtures. Also, standardized PN mixtures (with added micronutrients) seem noninferior to individualized PN mixtures in terms of electrolyte disturbances and basic biochemical abnormalities. Larger studies are needed to confirm these conclusions. TRIAL REGISTRATION: Academical Medical Center medical ethics committee number W18_079 #18.103

Accuracy of Predicted Genomic Breeding Values in Purebred and Crossbred Pigs

Genomic selection has been widely implemented in dairy cattle breeding when the aim is to improve performance of purebred animals. In pigs, however, the final product is a crossbred animal. This may affect the efficiency of methods that are currently implemented for dairy cattle. Therefore, the objective of this study was to determine the accuracy of predicted breeding values in crossbred pigs using purebred genomic and phenotypic data. A second objective was to compare the predictive ability of SNPs when training is done in either single or multiple populations for four traits: age at first insemination (AFI); total number of piglets born (TNB); litter birth weight (LBW); and litter variation (LVR). We performed marker-based and pedigree-based predictions. Within-population predictions for the four traits ranged from 0.21 to 0.72. Multi-population prediction yielded accuracies ranging from 0.18 to 0.67. Predictions across purebred populations as well as predicting genetic merit of crossbreds from their purebred parental lines for AFI performed poorly (not significantly different from zero). In contrast, accuracies of across-population predictions and accuracies of purebred to crossbred predictions for LBW and LVR ranged from 0.08 to 0.31 and 0.11 to 0.31, respectively. Accuracy for TNB was zero for across-population prediction, whereas for purebred to crossbred prediction it ranged from 0.08 to 0.22. In general, marker-based outperformed pedigree-based prediction across populations and traits. However, in some cases pedigree-based prediction performed similarly or outperformed marker-based prediction. There was predictive ability when purebred populations were used to predict crossbred genetic merit using an additive model in the populations studied. AFI was the only exception, indicating that predictive ability depends largely on the genetic correlation between PB and CB performance, which was 0.31 for AFI. Multi-population prediction was no better than within-population prediction for the purebred validation set. Accuracy of prediction was very trait-dependent

Clinical evaluation of an evidence-based method based on food characteristics to adjust pancreatic enzyme supplements dose in cystic fibrosis

[EN] Background: Patients with cystic fibrosis (CF) and pancreatic insufficiency need pancreatic enzyme replacement therapy (PERT) for dietary lipids digestion. There is limited evidence for recommending the adequate PERT dose for every meal, and controlling steatorrhea remains a challenge. This study aimed to evaluate a new PERT dosing method supported by a self-management mobile-app. Methods: Children with CF recruited from 6 European centres were instructed to use the app, including an algorithm for optimal PERT dosing based on in vitro digestion studies for every type of food. At base-line, a 24h self-selected diet was registered in the app, and usual PERT doses were taken by the patient. After 1 month, the same diet was followed, but PERT doses were indicated by the app. Change in faecal fat and coefficient of fat absorption (CFA) were determined. Results: 58 patients (median age 8.1 years) participated. Baseline fat absorption was high: median CFA 96.9%, median 2.4g faecal fat). After intervention CFA did not significantly change, but range of PERT doses was reduced: interquartile ranges narrowing from 1447-3070 at baseline to 1783-2495 LU/g fat when using the app. Patients with a low baseline fat absorption (CFA<90%, n= 12) experienced significant improvement in CFA after adhering to the recommended PERT dose (from 86.3 to 94.0%, p=0.031). Conclusion: the use of a novel evidence-based PERT dosing method, based on in vitro fat digestion studies incorporating food characteristics, was effective in increasing CFA in patients with poor baseline fat absorption and could safely be implemented in clinical practice.We acknowledge the support of the MyCyFAPP Project consortium. We especially thank the participation and the effort of the patients involved in the study and their families. This work was fully funded by the European Union and the Horizon 2020 Research and Innovation Framework Programme (PHC-26-2014 call Self management of health and disease: citizen engagement and mHealth) under grant number 643806.Calvo-Lerma, J.; Boon, M.; Colombo, C.; De Koning, B.; Asseiceira, I.; Garriga, M.; Roca, M.... (2021). Clinical evaluation of an evidence-based method based on food characteristics to adjust pancreatic enzyme supplements dose in cystic fibrosis. Journal of Cystic Fibrosis. 20(5):e33-e39. https://doi.org/10.1016/j.jcf.2020.11.016Se33e3920

Gastrointestinal biomarkers and their association with feeding in the first five days of pediatric critical illness

OBJECTIVES: Predicting the patients' tolerance to enteral nutrition (EN) would help clinicians optimize individual nutritional intake. This study investigated the course of several gastrointestinal (GI) biomarkers and their association with EN advancement (ENA) longitudinally during pediatric intensive care unit (PICU) admission.METHODS: This is a secondary analysis of the PEPaNIC RCT. EN was started early and increased gradually. The cholecystokinin (CCK), leptin, glucagon, intestinal fatty acid-binding protein 2 (I-FABP2), and citrulline plasma concentrations were measured upon PICU admission, day three and day five. ENA was defined as kcal EN provided as % of predicted resting energy expenditure (pREE). The course of the biomarkers and ENA was examined in patients with samples on all time points using Friedman and Wilcoxon signed-rank tests. The association of ENA with the biomarkers was examined using a two-part mixed-effects model with data of the complete population, adjusted for possible confounders.RESULTS: For 172 patients, median age 8.6 years (first quartile (Q1); third quartile (Q3): 4.2; 13.4), samples were available, of which 55 had samples on all time points. The median ENA was 0 (0; 0) on admission, 14.5 (0.0; 43.8) on day 3 and 28.0 (7.6; 94.8) on day 5. During PICU stay, CCK and I-FABP2 concentrations decreased significantly, whereas glucagon concentrations increased significantly, and leptin and citrulline remained stable. None of the biomarkers was longitudinally associated with ENA.CONCLUSIONS: Based on the current evidence, CCK, leptin, glucagon, I-FABP2, and citrulline appear to have no added value in predicting ENA in the first five days of pediatric critical illness.</p

Exome sequencing in patient-parent trios suggests new candidate genes for early-onset primary sclerosing cholangitis

BACKGROUND & AIMS Primary sclerosing cholangitis (PSC) is a rare bile duct disease strongly associated with inflammatory bowel disease (IBD). Whole-exome sequencing (WES) has contributed to understanding the molecular basis of very early-onset IBD, but rare protein-altering genetic variants have not been identified for early-onset PSC. We performed WES in patients diagnosed with PSC METHODS In this multicentre study, WES was performed on 87 DNA samples from 29 patient-parent trios with early-onset PSC. We selected rare (minor allele frequency <2%) coding and splice-site variants that matched recessive (homozygous and compound heterozygous variants) and dominant (de novo) inheritance in the index patients. Variant pathogenicity was predicted by an in-house developed algorithm (GAVIN), and PSC-relevant variants were selected using gene expression data and gene function. RESULTS In 22 of 29 trios we identified at least 1 possibly pathogenic variant. We prioritized 36 genes, harbouring a total of 54 variants with predicted pathogenic effects. In 18 genes, we identified 36 compound heterozygous variants, whereas in the other 18 genes we identified 18 de novo variants. Twelve of 36 candidate risk genes are known to play a role in transmembrane transport, adaptive and innate immunity, and epithelial barrier function. CONCLUSIONS The 36 candidate genes for early-onset PSC need further verification in other patient cohorts and evaluation of gene function before a causal role can be attributed to its variants.Peer reviewe