Variables, Operacionalización Variables

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CD (CALM study), comparing a tight control arm using biomarkers vs. a conventional clinical management arm. Complete endoscopic remission rates were 18% (CDEIS=0) compared to 46% endoscopic remission (CDEIS <4) in the group with tight control. A 16% greater improvement in endoscopic remission rates in the tight control group (46% vs. 30% in Conventional arm) required a three times greater use of every week adalimumab (45% EW Adalimumab vs. 15% EW Adalimumab) (247).

Although not an intended outcome of our study, a treatment to target approach was followed with the EEN cohort. In children experiencing a first relapse after EEN therapy, despite therapeutic dosing of conventional immune-modulators, treatment was escalated to biologics either based on biomarkers alone or in combination with biomarkers+/- endoscopy.

We did not escalate treatment in asymptomatic children with abnormal biomarkers or endoscopy at the end of EEN induction therapy, all these relapsed over the following year leading to later treatment escalation. We observed a relatively high anti-TNF commencement rate in our EEN cohort reaching 71% within a median follow up of 4 years (41% combination therapy Anti-TNF’s+ IM’S, 30% Anti-TNF monotherapy; 29% immune-modulator monotherapy).

It’s time to embrace a treat to target approach but it remains unclear if therapeutic adjustments based on biomarkers are superior to repeated endoscopy examination in achieving mucosal healing. On one hand, FC and CRP are excellent non-invasive biomarkers but up to 20% patients with a normal CRP<5mg/L and FC<250 μg/ gm of stools at 48 weeks failed to achieve MH (CDEIS<4) in the CALM study (248). The other important issue is that escalation of therapies based on biomarker alone may result in overtreatment of substantial proportion of patients.

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but emerging data suggest that this approach may be feasible and is associated with superior outcomes if escalation of therapy is performed in those failing to achieve MH (249). Recently published expert paediatric guidelines suggest that repeat endoscopic examination for MH is helpful but it does carry a risk associated with multiple general anaesthetics and health care burden. Future trials need to evaluate whether treating to an endoscopic endpoint is better than a biomarker based approach (250).

Chapter 6 reports longitudinal mucosal associated microbiome changes (MAM) at diagnosis and after completion of EEN (Median 7.9 weeks, 95% CI=7.5-8.2 weeks) from 8 treatment naïve children diagnosed with CD. Employing 16S rRNA gene sequencing, we confirmed an overall reduction in MAM diversity following EEN. Previous studies indicated reduction in stool microbial diversity post EEN to be a crucial step associated with clinical remission but our study is the first to document significant reduction in mucosal microbial diversity to be associated with complete mucosal healing (p=0.003). A significant increase in the relative abundance of Lachnospiraceae family, particularly Dorea genus and reduction in the relative abundance of Faecalibacterium community were observed after EEN therapy. These findings are interesting as a recent paediatric study exploring longitudinal stool microbiome changes in CD demonstrated lower levels of Lachnospiraceae to be associated with absence of mucosal healing (non-responders) to Immunomodulators and biologics. Whether increased abundance of Dorea in patients following EEN is a reflection of its sustained growth during period of nutritional starvation or a consequence of reducing inflammation requires further studies.

Faecalibacterium spp. are now widely recognized for their “anti-inflammatory” properties and their persistence in the gut is associated with improved health outcomes in adult CD patients, our results

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suggest significant reduction in Faecalibacterium spp. following EEN presumably indicating that their abundance may not be required for mucosal healing as part of the mechanisms driven by EEN induction therapy.

There are several limitations of our study, including small size and lack of functional metabolomics analysis. Alterations of the gut microbial metabolic functional activity on EEN have been reported in recent studies. Reductions have been observed in faecal butyric acid, genetic expression of biotin and thiamine biosynthetic pathways. In contrast an increase in stool pH, stool sulphides and spermidine/putrescine biosynthesis are documented (14,15). Dunn et al recently reported an increase in the functional metagenomics pathways involved in the degradation of environmental pollutants and xenobiotics and reduction in NOD-like receptor signalling in those with a poor clinical response to EEN (134)

There are only limited data on metabolomic changes during dietary interventions. In treatment naïve children with CD, stool metabolic alterations similar to those seen in children on EEN were observed on a CD-TREAT diet, an ordinary diet with a nutritional composition identical to EEN (Svolos V et al. ECCO, March 2016). As we look to using more normal, less restrictive diets than EEN in CD, these observations are encouraging but preliminary. However, a more objective endpoint of response such as mucosal or endoscopic healing are required to establish therapeutic efficacy of dietary interventions. To better characterise underlying functional metabolomics mechanisms associated with mucosal remission, endoscopic assessment and biopsies should be obtained before and after dietary interventions and on normal diet.

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Conclusion:

Exclusive Enteral Nutrition (EEN) in children with Crohn’s disease (CD) is associated with early complete mucosal healing (MH) in up to one-third of children. Completeness of mucosal healing following EEN induction is strongly associated with improved long-term outcomes. In the two thirds of children failing to achieve complete MH the majority will suffer moderate to severe clinical relapse within 2 years, requiring anti-TNF, surgery or both despite dose optimised Immuno- modulators.

This thesis highlights an urgent need to revise current treatment paradigms for escalating therapies targeting mucosal remission.

Our data reinforces the finding of poor correlation of endoscopic measured inflammation with symptom based scores (PCDAI); serum CRP and stool calprotectin are more reliable. A composite score of normalised PCDAI, CRP and stool calprotectin cut-off <500 µgram was the most reliable for predicting endoscopic remission during EEN induction therapy. a practical prediction tool for endoscopic response, however further validation in large multicentre studies is required to establish theirutility. Using combination of biomarkers targets to make treatment adjustments can lead to better outcomes as demonstrated by the recent CALM study. Aiming for CRP<5 mg/L and FC <250 micogram/gm strongly correlated with endoscopic remission (246).

Conflicting results from the changes in stool microbiota vs. mucosal microbiota during EEN, and lack of knowledge of microbiome signatures associated with endoscopic mucosal healing were examined in a limited cohort of children with CD. Our results indicate that reduction in bacterial diversity following EEN is strongly associated with mucosal healing. Whether this is cause or consequence of mucosal healing needs further evaluation.

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Faecalibacterium spp. are now widely recognized for their “anti-inflammatory” properties and their persistence and recovery in the gut is associated with improved health outcomes in adult CD patients via prolonged remission. Our results are in agreement with two previous stool microbiome studies reporting reduction in F. prausnitzii subgroups studies on EEN (Jia et al. FEMS Microbiol.2010, Gerasimidis et al. Inflamm Bowel. Dis 2014) suggesting Faecalibacterium spp. presence, while beneficial, may not be required for mucosal healing as part of the mechanisms driven by EEN induction therapy.

We observed significant increases in the relative abundance of Lachnospiraceae family, particularly Dorea genus. While enhanced growth of Dorea during periods of nutritional starvation could be related to its inherent ability to thrive on host derived nutrients (mucins and proteoglycans) but these findings may be relevant, as two recent paediatric studies exploring longitudinal stool microbiome changes in CD also identify of Lachnospiraceae Spp. as key bacterium associated with prolonged remission (Gevers et al. Cell Host Microbiome 2014, Dunn et al. Inflamm Bowel. Dis 2016).

Nonetheless, we did not examine the impact of EEN on functional activity of gut microbiome and future studies are warranted to better elucidate both structure-function activity of the microbiota in those achieving MH vs. active endoscopic disease. Lack of resources precluded our planned comprehensive analysis of cytokines, mucosal CD25+FoxP3+ expression in predicting therapeutic response to EEN.

Future studies should also perform longitudinal analysis of MAM signatures while on normal diet associated with sustained remission vs. those relapsing. Critical information from MAM signatures and metabolic pathways of endoscopic responders vs. non-responders will enhance our understanding of “pro-inflammatory vs. anti-Inflammatory microbiota” providing grounds to

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improve dietary interventions by promoting the growth of anti-Inflammatory gut microbiota.

It is time to also harness newer technology, using human intestinal organoid (HIO) for studying interaction between dietary components including EEN and other elimination diets with intestinal epithelium, to device best dietary strategies to maintain remission in CD. (218, 251)

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