Cytokine and Microbiome Changes in Adolescents with Anorexia Nervosa at Admission, Discharge, and One-Year Follow-Up.

Anorexia nervosa (AN) is a severe eating disorder that predominantly affects females and typically manifests during adolescence. There is increasing evidence that serum cytokine levels are altered in individuals with AN. Previous research has largely focused on adult patients, assuming a low-grade pro-inflammatory state. The serum levels of the cytokine tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, IL-6 and IL-15, which are pro-inflammatory, were examined in 63 female adolescents with AN and 41 age-matched healthy controls (HC). We included three time points (admission, discharge, and 1-year follow-up) and investigated the clinical data to assess whether the gut microbiota was associated with cytokine alterations. Relative to the HC group, serum levels of IL-1ß and IL-6 were significantly lower during the acute phase (admission) of AN. IL-1ß expression was normalised to control levels after weight recovery. TNF-α levels were not significantly different between the AN and HC groups. IL-15 levels were significantly elevated in patients with AN at all time points. We found associations between cytokines and bodyweight, illness duration, depressive symptoms, and the microbiome. In contrast to most findings for adults, we observed lower levels of the pro-inflammatory cytokines IL-1ß and IL-6 in adolescent patients, whereas the level of IL-15 was consistently increased. Thus, the presence of inflammatory dysregulation suggests a varied rather than uniform pro-inflammatory state.

Improvement in male pelvis magnetic resonance image contouring following radiologist-delivered training.

INTRODUCTION: The magnetic resonance linear accelerator (MRL) combines both magnetic resonance imaging and a linear accelerator, allowing for daily treatment adaptation. This study aimed to assess the impact of radiologist-delivered training in magnetic resonance (MR) contouring of relevant structures within the male pelvis. METHODS: Two radiation oncologists, two radiation oncology registrars and seven radiation therapists completed contouring on 10 male pelvis MR datasets both pre- and post-training. A 2-hour MR anatomy training session was delivered by a radiologist, who also provided the 'gold standard' contours. The pre- and post-training contours were compared against the gold standard with Dice similarity coefficient (DSC) and Hausdorff distances calculated; and the pre- and post-confidence scores and timing were compared. RESULTS: The improvement in DSC were significant in prostate, rectum and seminal vesicles, with a post-training median DSC of 0.87 ± 0.06, 0.92 ± 0.04 and 0.80 ± 0.14, respectively. The median Hausdorff improved with a median of 1.46 ± 0.78 mm, 0.52 ± 0.32 mm and 1.11 ± 0.86 mm for prostate, rectum and seminal vesicles, respectively. Bladder concordance was high both pre- and post-training. Urethra contours improved post-training, however, remained difficult to contour with a median post-DSC of 0.51 ± 0.24. Overall, confidence scoring improved (P < 0.001) and timing decreased by an average of 4.4 ± 16.4 min post-training. CONCLUSION: Radiologist-delivered training improved concordance of male pelvis contouring on MR datasets. Further work is required in the identification of urethra on MRs. These findings are of importance in the MRL adaptive workflow.

A comparison of measured and treatment planning system out-of-field dose for a 1.5 T MR linac.

Objective.Dose due to the electron streaming effect (ESE) is a significant contribution to out-of-field dose on the Elekta Unity MR-Linac. The aim of this work is to provide a systematic comparison of calculated and measured streaming dose for this system.Approach.Beams 1.0 × 1.0 cm2to 5.0 × 5.0 cm2, gantry 90.0°, 1000 MU, were incident on an in-house phantom. At the beam entrance and exit surfaces of the phantom, ESE was generated in theY-direction (IEC 61217). EBT3 film, orientated within theX-Zplane and at 14.0 mm depth in a solid water block, was used to determine ESE dose 5.0 cm beyond the phantom. The experimental arrangement was simulated in the Monaco v5.4 treatment planning system (TPS), utilising a CT phantom dataset with differing relative electron densities (RED) for the surrounding air. Horizontal (Xdirection) and vertical (Zdirection) film dose profiles were compared to the corresponding TPS profiles.Main results. For each field, the maximum ESE dose was observed at the beam exit, the magnitude of which decreases with decreasing field size. For the 5.0 × 5.0 cm2field, the exit and entry ESE doses were 19.6% and 7.0% of theDmaxdose to water, respectively. Across horizontal profiles, differences (simulated-measured) were reduced with smaller fields and lower RED. The maximum absolute profile difference was 1.7% of theDmaxdose to water for optimal RED and isocentre location. In vertical profiles an offset consistent with the Lorentz force was observed relative to theX-Yisoplane.Significance. For the fields investigated, maximum absolute differences (simulated-measured) ≤ 5.2% occurred in peak regions of ESE, at the beam entrance and exit from the phantom. Generally, there is good agreement between Monaco simulated and measured ESE. Simulated out-of-field dose is sensitive to the RED assigned to air structures and unforced RED optimises out-of-field dose calculation accuracy.

Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling.

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

Lower serum levels of IL-1ß and IL-6 cytokines in adolescents with anorexia nervosa and their association with gut microbiota in a longitudinal study.

Introduction: Anorexia nervosa (AN) is an often chronic and debilitating psychiatric disease whose etiology is not completely understood. Recently, a potential role of inflammation has emerged in other psychiatric diseases, such as depression, PTSD and schizophrenia. The first results in adults with AN seemed to confirm a low-grade proinflammatory state until recent studies presented more differential findings. Studying adolescents with a shorter illness duration and fewer confounding factors might help elucidate the role of inflammation in the underlying pathophysiology of AN; however, the few available studies in adolescents remain ambiguous, and no longitudinal data are available in this age range. Methods: We examined the proinflammatory cytokines Tumor Necrosis Factor-alpha (TNF-α), Interleukin (IL)-1ß, IL-6, IL-15, and the cytokine-receptor IL-6 Receptor alpha (IL-6 Rα) in the serum of twenty-two hospitalized female adolescent patients with AN longitudinally at admission and discharge and compared their results to nineteen healthy controls (HC). We also collected clinical data and stool samples that were analyzed with 16S rRNA amplicon sequencing to explore potential influencing factors of cytokine changes. Results: TNF-α serum levels were significantly elevated in patients with AN at admission, while IL-1ß and IL-6 levels were lower at admission and discharge than in HC. After treatment, we also found significantly elevated levels of IL-6 Rα compared to HC, while IL-15 did not show significant changes. Exploratory analyses revealed positive associations of cytokine and genus-level changes between admission and discharge for IL-1ß (Bacteroides) and IL-15 (Romboutsia), and negative associations for IL-15 (Anaerostipes) and TNF-α (uncultured Lachnospiraceae). Conclusion: We confirmed a previous finding of elevated levels of TNF-α also in adolescents with AN; however, the reduced IL-1ß and IL-6 levels differed from the mostly increased levels found in adults. A mixed pro- and anti-inflammatory state appears to be present in adolescents, potentially due to their shorter illness duration. The gut microbiota, with its regulatory function on cytokine production, might play a role in mediating these inflammatory processes in AN and could offer targets for new therapeutic approaches.

Performance evaluation of an LED flatbed scanner for triple channel film dosimetry with EBT3 and EBT-XD film.

We investigate the properties of a light emitting diode (LED) flatbed scanner for use with EBT3 and EBT-XD film types in a clinical radiochromic film (RCF) dosimetry program with modern treatment techniques. The flatbed scanner was characterised in terms of lateral and longitudinal response, X-Y scaling integrity, scanning reproducibility, scanner warm up dependence and film orientation dependence. The preferred lateral response artefact (LRA) corrections are investigated for the LED light source. Supporting evidence is provided regarding the dose independent nature of the corrections while also providing results suggesting a potential film type independence. Results from 2D gamma analysis of four patient treatments were compared between the new 12000XL and existing 10000XL model. Lastly, a dose uncertainty analysis was performed for the film-scanner system combination. It may be concluded that the lateral response variation requires correction while the longitudinal response variation is insignificant. The linear scaling in the lateral and longitudinal directions are within 0.5% and the scanner reproducibility is stable. Scanner warm up dependence no longer exists, and effort should be made to maintain all film orientation in a study set within 15°. The LRA corrections are as reported substantially dose independent and there is evidence to support film type independence. Comparative gamma analysis of patient specific dose maps between the EPSON 10000XL (xenon fluorescent lamp) and 12000XL (LED) scanners showed that results are indistinguishable for both film types across the two scanner models when the necessary corrections are applied. Dose uncertainty is in agreement with the literature and can be kept below 3% with necessary corrections applied.

A dietary carbohydrate - gut Parasutterella - human fatty acid biosynthesis metabolic axis in obesity and type 2 diabetes.

Recent rodent microbiome experiments suggest that besides Akkermansia, Parasutterella sp. are important in type 2 diabetes and obesity development. In the present translational human study, we aimed to characterize Parasutterella in our European cross-sectional FoCus cohort (n = 1,544) followed by validation of the major results in an independent Canadian cohort (n = 438). In addition, we examined Parasutterella abundance in response to a weight loss intervention (n = 55). Parasutterella was positively associated with BMI and type 2 diabetes independently of the reduced microbiome α/ß diversity and low-grade inflammation commonly found in obesity. Nutritional analysis revealed a positive association with the dietary intake of carbohydrates but not with fat or protein consumption. Out of 126 serum metabolites differentially detectable by untargeted HPLC-based MS-metabolomics, L-cysteine showed the strongest reduction in subjects with high Parasutterella abundance. This is of interest, since Parasutterella is a known high L-cysteine consumer and L-cysteine is known to improve blood glucose levels in rodents. Furthermore, metabolic network enrichment analysis identified an association of high Parasutterella abundance with the activation of the human fatty acid biosynthesis pathway suggesting a mechanism for body weight gain. This is supported by a significant reduction of the Parasutterella abundance during our weight loss intervention. Together, these data indicate a role for Parasutterella in human type 2 diabetes and obesity, whereby the link to L-cysteine might be relevant in type 2 diabetes development and the link to the fatty acid biosynthesis pathway for body weight gain in response to a carbohydrate-rich diet in obesity development.

Evaluation of MU2net as an online secondary dose check for MR guided radiation therapy with the Elekta unity MR linac.

During the adaptive workflow associated with MRgRT, a secondary dose calculation is required and MU2net (DOSIsoft, France) is one commercial option. The suitability of MU2net to be used in conjunction with the online Monaco treatment planning system of the Elekta Unity (Elekta AB, Stockholm, Sweden), is evaluated in this work. Monaco and MU2net point doses are compared for various fields on and off axis and at different SSDs. To investigate the comparative effects of attenuation due to the cryostat, couch and posterior coil, measured, MU2net and Monaco dose outputs at the isocentre, as a function of gantry angle, were compared. Point doses for the beams of nine step and shoot IMRT (SSIMRT) test plans (courtesy Elekta) were calculated with Monaco v5.4 and compared to corresponding doses computed with MU2net. In addition, Monaco v5.4 and MU2net point doses were compared for 1552 beams treated on the Unity at our facility. For the on-axis fields investigated the agreement between MU2net and measured data is acceptable. MU2net and Monaco point doses for the Elekta SSIMRT test plans were within ± 5.0% and ± 6.4% for beams delivered from gantry zero and at planned beam angles, respectively. For the 1552 beams delivered approximately 80.0% of MU2net and Monaco point doses agree within ± 5.0%, therefore it is recommended to correlate MU2net Dose Reference Points (DRPs) with pre and post treatment dosimetry verification. Computational accuracy of MU2net could be enhanced with improved modelling of attenuation due to the couch, cryostat and posterior MR imaging coil.

Commissioning measurements on an Elekta Unity MR-Linac.

Magnetic resonance-guided radiotherapy technology is relatively new and commissioning publications, quality assurance (QA) protocols and commercial products are limited. This work provides guidance for implementation measurements that may be performed on the Elekta Unity MR-Linac (Elekta, Stockholm, Sweden). Adaptations of vendor supplied phantoms facilitated determination of gantry angle accuracy and linac isocentre, whereas in-house developed phantoms were used for end-to-end testing and anterior coil attenuation measurements. Third-party devices were used for measuring beam quality, reference dosimetry and during treatment plan commissioning; however, due to several challenges, variations on standard techniques were required. Gantry angle accuracy was within 0.1°, confirmed with pixel intensity profiles, and MV isocentre diameter was < 0.5 mm. Anterior coil attenuation was approximately 0.6%. Beam quality as determined by TPR20,10 was 0.705 ± 0.001, in agreement with treatment planning system (TPS) calculations, and gamma comparison against the TPS for a 22.0 × 22.0 cm2 field was above 95.0% (2.0%, 2.0 mm). Machine output was 1.000 ± 0.002 Gy per 100 MU, depth 5.0 cm. During treatment plan commissioning, sub-standard results indicated issues with machine behaviour. Once rectified, gamma comparisons were above 95.0% (2.0%, 2.0 mm). Centres which may not have access to specialized equipment can use in-house developed phantoms, or adapt those supplied by the vendor, to perform commissioning work and confirm operation of the MRL within published tolerances. The plan QA techniques used in this work can highlight issues with machine behaviour when appropriate gamma criteria are set.

Deficiency in X-linked inhibitor of apoptosis protein promotes susceptibility to microbial triggers of intestinal inflammation.

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)­, receptor-interacting protein kinase 1 (RIPK1)­, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell­derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell­derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

Characterisation of a synthetic diamond detector for end-to-end dosimetry in stereotactic body radiotherapy and radiosurgery.

BACKGROUND AND PURPOSE: Synthetic diamond detectors offer real time measurement of dose in radiotherapy applications which require high spatial resolution. Additional considerations and corrections are required for measurements where the diamond detector is orientated at various angles to the incident beam. This study investigated diamond detectors for end-to-end testing of Stereotactic Body Radiotherapy (SBRT) and Stereotactic Radiosurgery (SRS) in the context of dosimetry audits. MATERIAL AND METHODS: Seven individual diamond detectors were investigated and compared with respect to warm up stability, dose-rate dependence, linearity, detector shadowing, energy response, cross-calibration, angular dependence and positional sensitivity in SBRT and SRS. RESULTS: Large variation in the cross calibration factors was found between the seven individual detectors. For each detector, the energy dependence in the cross calibration factor was on average <0.6% across the beam qualities investigated (Co-60 Gamma Knife, and MV beams with TPR20,10 0.684-0.733). The angular corrections for individual fields were up to 5%, and varied with field size. However, the average angular dependence for all fields in a typical SRS treatment delivery was <1%. The overall measurement uncertainty was 3.6% and 3.1% (2σ) for an SRS and SBRT treatment plan respectively. CONCLUSION: Synthetic diamond detectors were found to be reliable and robust for end-to-end dosimetry in SBRT and SRS applications. Orientation of the detector relative to the beam axis is an important consideration, as significant corrections are required for angular dependence.

The Microbiome Tumor Axis: How the Microbiome Could Contribute to Clonal Heterogeneity and Disease Outcome in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers. It is characterized by a poor prognosis with a 5-year survival rate of only around 10% and an ongoing increase in death rate. Due to the lack of early and specific symptoms, most patients are diagnosed at an advanced or even metastasized stage, essentially limiting curative treatment options. However, even curative resection of the primary tumor and adjuvant therapy often fails to provide a long-term survival benefit. One reason for this dismal situation can be seen in the evolution of therapy resistances. Furthermore, PDAC is characterized by high intratumor heterogeneity, pointing towards an abundance of cancer stem cells (CSCs), which are regarded as essential for tumor initiation and drug resistance. Additionally, it was shown that the gut microbiome is altered in PDAC patients, promotes Epithelial-Mesenchymal-Transition (EMT), determines responses towards chemotherapy, and affects survival in PDAC patients. Given the established links between CSCs and EMT as well as drug resistance, and the emerging role of the microbiome in PDAC, we postulate that the composition of the microbiome of PDAC patients is a critical determinant for the abundance and plasticity of CSC populations and thus tumor heterogeneity in PDAC. Unravelling this complex interplay might pave the way for novel treatment strategies.

Considerations when introducing MRI into a radiation therapy environment.

This issue of Journal of Medical Radiation Sciences includes two papers presenting different uses of magnetic resonance (MR) in radiation therapy (RT). With the advancement of MR-simulators and Magnetic resonance linear accelerators (MRL), in addition to the use of diagnostic MR becoming more common place in the radiotherapy setting, there are a number of challenges to be considered. In this article, we present the perspectives of radiation therapists and medical physicists involved in the commissioning of an MRL in our centre. Image shows in-house 3D printed supports mounted on the vendor-supplied QA platform. The supports locate an array so that it is centred in the radiation field.

Genome-wide association study in 8,956 German individuals identifies influence of ABO histo-blood groups on gut microbiome.

The intestinal microbiome is implicated as an important modulating factor in multiple inflammatory1,2, neurologic3 and neoplastic diseases4. Recent genome-wide association studies yielded inconsistent, underpowered and rarely replicated results such that the role of human host genetics as a contributing factor to microbiome assembly and structure remains uncertain5-11. Nevertheless, twin studies clearly suggest host genetics as a driver of microbiome composition11. In a genome-wide association analysis of 8,956 German individuals, we identified 38 genetic loci to be associated with single bacteria and overall microbiome composition. Further analyses confirm the identified associations of ABO histo-blood groups and FUT2 secretor status with Bacteroides and Faecalibacterium spp. Mendelian randomization analysis suggests causative and protective effects of gut microbes, with clade-specific effects on inflammatory bowel disease. This holistic investigative approach of the host, its genetics and its associated microbial communities as a 'metaorganism' broaden our understanding of disease etiology, and emphasize the potential for implementing microbiota in disease treatment and management.

An alternative to the use of lead for patient treatment shielding in superficial radiotherapy.

Lead shielding is commonly used in the delivery of superficial radiotherapy albeit that the toxicity of this substance is of concern. The feasibility of using a non-toxic alternative, AttenuFlex™, is assessed using Xstrahl and Sensus treatment units. A series of lead and AttenuFlex™ circular cut outs and applicators were used with superficial beams (1.0-8.5 mm Al HVL) to measure percentage depth dose (PDD), output factors (OF) and surface dose correction factors (DCF). X-ray transmission for each material was determined for each beam quality. For these measurements an Advanced Markus chamber either embedded within a virtual water phantom (PDD, OF, transmission) or placed on the surface of the phantom with entrance window downstream (DCF), was used. The depth of the phantom is 10 cm for PDD and surface OF measurements. DCF(t) measurements were obtained with underlying lead or AttenuFlex™ at depth t = 0.1-10 cm. Additionally, using EBT3 film fluorescent surface doses, to non-target tissue, due to underlying lead or AttenuFlex™ were compared. PDDs and OFs for both materials were within ± 1%. Lead and AttenuFlex™ transmission differences were clinically acceptable, all transmission values were < 5% and non-target doses were comparable. The variation of DCF(t) for lead and AttenuFlex™ exhibit a minima for all beams. In the minima region energy and applicator dependent differences between DCF(lead) and DCF(AttenuFlex™) are observed. These differences do not preclude the use of AttenuFlex™ as an alternative to lead in superficial therapy.

S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants.

BACKGROUND & AIMS: After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. METHODS: We collected stool samples (n = 517) from full-term (n = 72) and preterm infants (n = 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9-/- mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. RESULTS: Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. CONCLUSION: S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.

NOD2 Influences Trajectories of Intestinal Microbiota Recovery After Antibiotic Perturbation.

BACKGROUND & AIMS: Loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) impair the recognition of the bacterial cell wall component muramyl-dipeptide and are associated with an increased risk for developing Crohn's disease. Likewise, exposure to antibiotics increases the individual risk for developing inflammatory bowel disease. Here, we studied the long-term impact of NOD2 on the ability of the gut bacterial and fungal microbiota to recover after antibiotic treatment. METHODS: Two cohorts of 20-week-old and 52-week-old wild-type (WT) C57BL/6J and NOD2 knockout (Nod2-KO) mice were treated with broad-spectrum antibiotics and fecal samples were collected to investigate temporal dynamics of the intestinal microbiota (bacteria and fungi) using 16S ribosomal RNA and internal transcribed spacer 1 sequencing. In addition, 2 sets of germ-free WT mice were colonized with either WT or Nod2-KO after antibiotic donor microbiota and the severity of intestinal inflammation was monitored in the colonized mice. RESULTS: Antibiotic exposure caused long-term shifts in the bacterial and fungal community composition. Genetic ablation of NOD2 was associated with delayed body weight gain after antibiotic treatment and an impaired recovery of the bacterial gut microbiota. Transfer of the postantibiotic fecal microbiota of Nod2-KO mice induced an intestinal inflammatory response in the colons of germ-free recipient mice compared with respective microbiota from WT controls based on histopathology and gene expression analyses. CONCLUSIONS: Our data show that the bacterial sensor NOD2 contributes to intestinal microbial community composition after antibiotic treatment and may add to the explanation of how defects in the NOD2 signaling pathway are involved in the etiology of Crohn's disease.

Environmental Microbial Factors Determine the Pattern of Inflammatory Lesions in a Murine Model of Crohn's Disease-Like Inflammation.

Crohn's disease (CD) patients can be grouped into patients suffering from ileitis, ileocolitis, jejunoileitis, and colitis. The pathophysiological mechanism underlying this regional inflammation is still unknown. Although most murine models of inflammatory bowel disease (IBD) develop inflammation in the colon, there is an unmet need for novel models that recapitulate the spontaneous and fluctuating nature of inflammation as seen in CD. Recently, mice with an intestinal epithelial cell-specific deletion for Caspase-8 (Casp8ΔIEC mice), which are characterized by cell death-driven ileitis and disrupted Paneth cell homeostasis, have been identified as a novel model of CD-like ileitis. Here we uncovered that genetic susceptibility alone is sufficient to drive ileitis in Casp8ΔIEC mice. In sharp contrast, environmental factors, such as a disease-relevant microbial flora, determine colonic inflammation. Accordingly, depending on the microbial environment, isogenic Casp8ΔIEC mice either exclusively developed ileitis or suffered from pathologies in several parts of the gastrointestinal tract. Colitis in these mice was characterized by massive epithelial cell death, leading to spread of commensal gut microbes to the extra-intestinal space and hence an aberrant activation of the systemic immunity. We further uncovered that Casp8ΔIEC mice show qualitative and quantitative changes in the intestinal microbiome associated with an altered mucosal and systemic immune response. In summary, we identified that inflammation in this murine model of CD-like inflammation is characterized by an immune reaction, presumably directed against a disease-relevant microbiota in a genetically susceptible host, with impaired mucosal barrier function and bacterial clearance at the epithelial interface.

Modification of a modulated arc total body irradiation technique: Implementation and first clinical experience for paediatric patients.

INTRODUCTION: To implement the modulated arc total body irradiation (MATBI) technique within the existing infrastructure of a radiation oncology department. The technique needed to treat paediatric patients of all ages, some of whom would require general anaesthesia (GA). METHODS: The MATBI technique required minor modifications to be incorporated within existing departmental infrastructure. Ancillary equipment essential to the technique were identified and in some cases custom designed to meet health and safety criteria. GA equipment was also considered. To evaluate the effectiveness of the implemented technique, an audit of the cases clinically treated was conducted. RESULTS: A motorised treatment couch was designed to allow the patient to be positioned in stabilisation equipment at a height, then lowered to the floor to accommodate source-to-skin-distances from 180 cm to 198 cm to treat the fixed 40 cm × 40 cm field size. Treatment couch design also facilitated positioning of the bespoke two-part spoiler. While organ at risk dose is limited using a beam weight optimisation technique, the dose is further reduced using compensators placed close to the patient's skin on a 3D printed custom-made support bridge. A digital radiography system is used to verify compensator position. Fifteen patients have been treated to date for various diseases using a variety of dose fractionations ranging from 2 Gy in a single fraction to 12 Gy in 6 fractions. Five patients have required GA due to age or behavioural issues. CONCLUSION: The modified MATBI technique and the equipment required for treatment delivery has been found to be well tolerated by all patients.

Exposure to the gut microbiota drives distinct methylome and transcriptome changes in intestinal epithelial cells during postnatal development.

BACKGROUND: The interplay of epigenetic processes and the intestinal microbiota may play an important role in intestinal development and homeostasis. Previous studies have established that the microbiota regulates a large proportion of the intestinal epithelial transcriptome in the adult host, but microbial effects on DNA methylation and gene expression during early postnatal development are still poorly understood. Here, we sought to investigate the microbial effects on DNA methylation and the transcriptome of intestinal epithelial cells (IECs) during postnatal development. METHODS: We collected IECs from the small intestine of each of five 1-, 4- and 12 to 16-week-old mice representing the infant, juvenile, and adult states, raised either in the presence or absence of a microbiota. The DNA methylation profile was determined using reduced representation bisulfite sequencing (RRBS) and the epithelial transcriptome by RNA sequencing using paired samples from each individual mouse to analyze the link between microbiota, gene expression, and DNA methylation. RESULTS: We found that microbiota-dependent and -independent processes act together to shape the postnatal development of the transcriptome and DNA methylation signatures of IECs. The bacterial effect on the transcriptome increased over time, whereas most microbiota-dependent DNA methylation differences were detected already early after birth. Microbiota-responsive transcripts could be attributed to stage-specific cellular programs during postnatal development and regulated gene sets involved primarily immune pathways and metabolic processes. Integrated analysis of the methylome and transcriptome data identified 126 genomic loci at which coupled differential DNA methylation and RNA transcription were associated with the presence of intestinal microbiota. We validated a subset of differentially expressed and methylated genes in an independent mouse cohort, indicating the existence of microbiota-dependent "functional" methylation sites which may impact on long-term gene expression signatures in IECs. CONCLUSIONS: Our study represents the first genome-wide analysis of microbiota-mediated effects on maturation of DNA methylation signatures and the transcriptional program of IECs after birth. It indicates that the gut microbiota dynamically modulates large portions of the epithelial transcriptome during postnatal development, but targets only a subset of microbially responsive genes through their DNA methylation status.