Ileum Of Patients Research Articles

Prospective characterization of intestinal MRI intravoxel incoherent motion in pediatric and young adultpatients with newly diagnosed small bowel Crohn's disease.

MRI diffusion-weighted imaging (DWI) is commonly used in MR enterography protocols for assessment of intestinal inflammation in patients with Crohn's disease. The intravoxel incoherent motion (IVIM) approach to DWI has been proposed as a more objective approach, providing quantitative parameters that reflect water diffusivity (D), blood flow (D*), and perfusion fraction (f). We aimed to determine if DWI-IVIM metrics from the terminal ileum in patients with newly diagnosed Crohn's disease differ from healthy participants and change in response to biologic medical therapy. In this prospective case-control study, 20 consecutive pediatric patients (mean age = 14years ± 2 [SD]; eight females) with newly diagnosed ileal Crohn's disease and 15 pediatric healthy participants (mean age = 18years ± 4 [SD]; eight females) underwent research MRI examinations of the small bowel between 12/2018 and 10/2021. Participants with Crohn's disease underwent MR studies at baseline, 6weeks, and 6months following initiation of anti-TNF-alpha therapy, whereas control participants underwent one research MRI examination. The MRI protocol included a DWI-IVIM sequence with nine b-values and the IVIM parameters (D, D*, and f) were extracted. Unpaired t-tests and mixed-effects models were used for analyses. Mean IVIM D (P < 0.001), D* (P = 0.004), and f (P = 0.001) metrics were lower for Crohn's patients at the time of diagnosis compared to healthy participants. Mean IVIM f value increased over time in response to medical therapy (mean f at baseline, 22% ± 6%; 6weeks, 25% ± 7%; 6months, 29% ± 10%; P = 0.016). Mean IVIM D* value increased over time in response to treatment (mean D* at baseline, 10.9 ± 3.0 × 10-3 mm2/s; 6weeks, 11.8 ± 2.8 × 10-3 mm2/s; 6months, 13.3 ± 3.3 × 10-3 mm2/s; P = 0.047), while there was no significant change in mean IVIM D value (P = 0.10). MRI DWI-IVIM metrics in patients with ileal Crohn's disease change over time in response to biological therapy and help discriminate these patients from healthy participants.

Carbohydrase activity in proximal and distal segments of small intestine in health, irritated bowel syndrome and enteropathy with membrane digestion disturbance

Diagnostics of enteropathy with membrane digestion disturbance (EMDD) is based on the evaluation of carbohydrase activity in duodenal mucosa (DM). There is no information about diagnostic value of ileum mucosa (IM) carbohydrases.&#x0D; Aim. Assess the diagnostic value of carbohydrases activity in mucous membrane of duodenum and distal ileum in patients with EMDD and irritable bowel syndrome (IBS).&#x0D; Materials and methods. The study includes 12 patients with EMDD, 16 patients with IBS and 12 relatively healthy persons (control group). Study design included analysis of food tolerance and evaluation of activity of sucrase, glucoamylase, maltase and lactase in mucous membrane of duodenum and ileum based on the method of Dahlquist with N.I. Belostotsky modification. Microsoft Office Excel and Statistica 10.0 were used for statistical evaluation of material. Mann–Whitney criterion was used to evaluate the differences between quantitative parameters. The differences were considered to be significant at p0.05. Control group was used for comparison.&#x0D; Results. In control group the carbohydrases activity in duodenum mucosa was not significantly statistically different from the one in IM. In patients of the IBS group, the activity of glucoamylase and maltase in the IM was significantly higher than in the duodenum: glucoamylase by 2.2 times, maltase by 2 times. In patients with EMDD the activity of glucoamylase, maltase and sucrase in IM is higher than in DM by 5.2, 3.8 and 2 times respectively, which signifies the formation of compensatory reaction of adaptive shift, initially described by A.M. Ugolev in 1985.&#x0D; Conclusion. The evaluation of carbohydrase activity in IM provides more information about pathogenesis of EMDD and its differential diagnosis with IBS.

Single nuclear RNA sequencing of terminal ileum in patients with cirrhosis demonstrates multi-faceted alterations in the intestinal barrier

Patients with cirrhosis have intestinal barrier dysfunction but the role of the individual cell types in human small intestine is unclear. We performed single-nuclear RNA sequencing (snRNAseq) in the pinch biopsies of terminal ileum of four age-matched men [56 years, healthy control, compensated, early (ascites and lactulose use) and advanced decompensated cirrhosis (ascites and rifaximin use)]. Cell type proportions, differential gene expressions, cell-type specific pathway analysis using IPA, and cellular crosstalk dynamics were compared. Stem cells, enterocytes and Paneth cells were lowest in advanced decompensation. Immune cells like naive CD4 + T cells were lowest while ITGAE + cells were highest in advanced decompensation patients. MECOM had lowest expression in stem cells in advanced decompensation. Defensin and mucin sulfation gene (PAPSS2) which can stabilize the mucus barrier expression were lowest while IL1, IL6 and TNF-related genes were significantly upregulated in the enterocytes, goblet, and Paneth cells in decompensated subjects. IPA analysis showed higher inflammatory pathways in enterocytes, stem, goblet, and Paneth cells in decompensated patients. Cellular crosstalk analysis showed that desmosome, protease-activated receptors, and cadherin-catenin complex interactions were most perturbed in decompensated patients. In summary, the snRNAseq of the human terminal ileum in 4 subjects (1 control and three cirrhosis) identified multidimensional alteration in the intestinal barrier with lower stem cells and altered gene expression focused on inflammation, mucin sulfation and cell–cell interactions with cirrhosis decompensation.

#4049 NEFECON® TREATMENT LIKELY MODULATES DOWNSTREAM PATHWAYS OF KIDNEY INFLAMMATION AND FIBROSIS IN IGA NEPHROPATHY

Abstract Background and Aims The lifetime risk of kidney failure in patients with IgA nephropathy (IgAN) is substantial. Since the original description of the disease, over 50 years ago, there has been little progress in how we manage this important cause of kidney failure in young adults. KDIGO 2021 Clinical Practice Guidelines recommend goal-directed supportive care as the treatment with the strongest evidence base for the management of all patients with IgAN. The NEFIGAN trial (NCT01738035) tested the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver budesonide to the gut-associated lymphoid tissue-rich distal ileum in patients with IgAN in addition to optimised supportive care. The trial comprised a 6-month run-in, 9-month treatment, and a 3-month follow-up phase. Forty-eight patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day, and 50 patients received placebo. The headline result of the study was that Nefecon® 16 mg/day, added to optimised RAS blockade, reduced proteinuria and stabilised eGFR in patients with IgAN. These findings have now been replicated in the NefIgArd study, which reported in 2021 and provided the basis for the recent FDA and EMA approval of Nefecon® as a treatment for patients with IgAN at high risk of progressive disease. In this study, we determined the composition of urinary proteins from patients treated with placebo and 16 mg of Nefecon® in the NEFIGAN trial using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Method Urine samples from 18 patients from each of the placebo and 16-mg arms of the NEFIGAN trial collected at the start of treatment (SOT) and end of treatment (EOT) were analysed. Patients were only included if they had received at least 8 months of treatment and their urine sample was taken up to 2 days after the completion of tapering. Urine samples were treated to remove abundant albumin and immunoglobulins. The remaining proteins were desalted and then precipitated with 5% trichloroacetic acid and washed twice with ice-cold acetone. The air-dried pellets were dissolved in 45 µL of 100 mM triethylammonium bicarbonate buffer, reduced with 2 mM TCEP (tris(2-carboxyethyl)phosphine), and alkylated with 15 mM chloroacetamide. 500 ng of Sequencing Grade Trypsin was added for enzymatic digestion, carried out overnight at 37°C. The samples were dried to completeness and re-solubilised in 10 µL of MS sample buffer (3% acetonitrile, 0.1% formic acid). LC-MS/MS analysis was performed on an Q Exactive mass spectrometer equipped with a Digital PicoView source and coupled to a nanoAcquity UPLC. For protein identification and quantification, raw data were processed with FragPipe (V16) having at least two peptides per protein. Protein intensities were reported in the combined_protein.txt file generated by the FragPipe: These were Log_2-transformed and internally normalised against a group of peptides found in all samples, The normalised protein abundance at SOT was subtracted from the abundance at EOT for each patient, A probabilistic dropout model was fitted to the data to estimate fold changes between the treatment and placebo groups at EOT, The proteins were ranked using the t-statistic and a gene-set enrichment analysis was performed to determine the gene sets significantly affected by the treatment compared with the placebo group. Results Gene-ontology analysis revealed that treatment with 16 mg of Nefecon® led to a significant enrichment of multiple pathways (n = 57) involved in several processes previously shown to be important in the pathogenesis of kidney injury in IgAN. These included epigenetic pathways (n = 13), microvesicle formation (n = 3), kidney remodelling (n = 9), and regulation of local immune and inflammatory responses (n = 32). Conclusion We have previously shown that 16 mg of Nefecon® for 9 months positively impacts on multiple biomarkers of IgAN severity. These urine proteomic data support the positive impact of Nefecon® on downstream pro-inflammatory and profibrotic pathways within the kidneys. These data will be validated in the biomarker analyses currently underway as part of the NefIgArd study.

THE UTILITY OF BIOPSYING ENDOSCOPICALLY NORMAL APPEARING TERMINAL ILEUM

The terminal ileum is often evaluated during colonoscopy procedures. A common indication for endoscopic examination and biopsy of an endoscopically normal appearing terminal ileum is in patients with inflammatory bowel disease (IBD).1 In patients without IBD, indications for biopsying an endoscopically normal appearing terminal ileum are less clear and may be associated with increased cost and healthcare utilization, as well as increased levels of anxiety for patients. In this study, we aim to determine the indications for examining and biopsying an endoscopically normal appearing terminal ileum in patients without IBD and if, upon review of these biopsies, there is change in management.

THE UTILITY OF BIOPSYING ENDOSCOPICALLY NORMAL APPEARING TERMINAL ILEUM

Abstract INTRODUCTION The terminal ileum is often evaluated during colonoscopy procedures. A common indication for endoscopic examination and biopsy of an endoscopically normal appearing terminal ileum is in patients with inflammatory bowel disease (IBD). 1 In patients without IBD, indications for biopsying an endoscopically normal appearing terminal ileum are less clear and may be associated with increased cost and healthcare utilization, as well as increased levels of anxiety for patients. In this study, we aim to determine the indications for examining and biopsying an endoscopically normal appearing terminal ileum in patients without IBD and if, upon review of these biopsies, there is change in management. METHODS This was a retrospective chart review of patients who underwent colonoscopy with terminal ileum biopsy at Mayo Clinic between 1/1/2021 and 6/30/21. Patients were excluded if they had a history of IBD, prior ileocecal resection, or an endoscopically abnormal terminal ileum. Clinical and laboratory data, as well as outcomes of interest, were obtained from the electronic medical record. Comparisons between patients were performed using Pearson’s chi-square, Student’s and Mann-Whitney tests. RESULTS A total of two hundred consecutive patients were identified as having had biopsies from their endoscopically normal appearing terminal ileum. After reviewing the charts, 69 met inclusion criteria. Of the 69 patients, the majority were female (68.1%) and white (89.9%). Nearly half the patients had a BMI of 30 or above (49.3%). The most common indication for colonoscopy among these patients was chronic diarrhea followed by abdominal pain and hematochezia. Terminal ileum mucosa was histologically abnormal in 6 patients (8.7%): four patients had chronic ileitis, one had acute ileitis, and one had acute and chronic ileitis. All patients with either chronic or acute ileitis had chronic diarrhea listed as an indication for their colonoscopy. The patient with acute and chronic ileitis underwent colonoscopy for suspected Crohn’s disease due to incidental asymptomatic findings of small bowel inflammation on CT enterography. CONCLUSIONS In patients with a normal appearing terminal ileum, clinically significant histologic abnormalities on biopsies were found in a small percentage of patients. Although our sample size is small, there may not be diagnostic and therapeutic utility to routinely biopsy endoscopically normal appearing terminal ileum in patients, even in those with chronic diarrhea. Large prospective studies are needed to further support this observation. REFERENCES 1McHugh, Jonathan B et al. “The diagnostic value of endoscopic terminal ileum biopsies.” The American journal of gastroenterology vol. 102,5 (2007): 1084-9.

The ileal fungal microbiota is altered in Crohn's disease and is associated with the disease course.

IntroductionFungal microbiota's involvement in the pathogenesis of Crohn's disease (CD) is incompletely understood. The terminal ileum is a predilection site both for primary involvement and recurrences of CD. We, therefore, assessed the mucosa-associated mycobiota in the inflamed and non-inflamed ileum in patients with CD.MethodsThe mucosa-associated mycobiota was assessed by ITS2 sequencing in a total of 168 biopsies sampled 5 and 15 cm proximal of the ileocecal valve or ileocolic anastomosis in 44 CD patients and 40 healthy controls (HC). CD patients with terminal ileitis, with endoscopic inflammation at 5 cm and normal mucosa at 15 cm and no history of upper CD involvement, were analyzed separately. The need for additional CD treatment the year following biopsy collection was recorded.ResultsCD patients had reduced mycobiota evenness, increased Basidiomycota/Ascomycota ratio, and reduced abundance of Chytridiomycota compared to HC. The mycobiota of CD patients were characterized by an expansion of Malassezia and a depletion of Saccharomyces, along with increased abundances of Candida albicans and Malassezia restricta. Malassezia was associated with the need for treatment escalation during follow-up. Current anti-TNF treatment was associated with lower abundances of Basidiomycota. The alpha diversity of the inflamed and proximal non-inflamed mucosa within the same patients was similar. However, the inflamed mucosa had a more dysbiotic composition with increased abundances of Candida sake and reduced abundances of Exophiala equina and Debaryomyces hansenii.ConclusionsThe ileal mucosa-associated mycobiota in CD patients is altered compared to HC. The mycobiota in the inflamed and proximal non-inflamed ileum within the same patients harbor structural differences which may play a role in the CD pathogenesis. Increased abundance of Malassezia was associated with an unfavorable disease course.

The Mechanosensitive Ion Channel PIEZO1 in Intestinal Epithelial Cells Mediates Inflammation through the NOD-Like Receptor 3 Pathway in Crohn's Disease.

Crohn's disease (CD) is an incurable chronic intestinal inflammatory disease with no recognized cause. It has been reported that the mechanosensitive ion channel PIEZO1 initiates proinflammatory responses. However, little is known about the role of PIEZO1 in CD. Ileum biopsies were obtained from 30 patients with CD and 15 healthy volunteers. Clinical data were collected to determine the relationship between CD and PIEZO1. First, HT29 cells were incubated with Yoda1 and GsMTx4 (Grammostola spatulata mechanotoxin 4) to activate and inhibit PIEZO1, respectively. Second, PIEZO1 knockdown was performed using small interfering RNA. Third, calcium imaging, flow cytometry, and immunofluorescence were used to detect intracellular calcium and mitochondrial function. Last, real-time quantitative polymerase chain reaction, immunoblotting, and enzyme-linked immunosorbent assay were used to quantify PIEZO1, proinflammatory cytokines, and NLRP3 (NOD-like receptor 3)-related compounds. PIEZO1 was highly expressed in the ileum of patients with CD and correlated positively with the Crohn's Disease Activity Index, platelet count, and hematocrit and fecal calprotectin levels. In HT29 cells, Yoda1 triggered calcium influx, which was inhibited by GsMTx4 treatment and small interfering RNA-mediated PIEZO1 knockdown. Increased calcium concentrations resulted in increased reactive oxygen species accumulation and decreased mitochondrial membrane potential, whereas decreased calcium concentrations caused by GsMTx4 and PIEZO1 knockdown had the opposite effect. Mechanistically, molecules in the NLRP3 pathway were activated in patients with CD and HT29 cells were stimulated by lipopolysaccharide; these effects were reversed by the knockdown of PIEZO1. Finally, PIEZO1 and NLRP3 knockdown decreased proinflammatory cytokine levels in HT29 cells. PIEZO1 in intestinal epithelial cells caused calcium influx, which resulted in mitochondrial dysfunction and activated the NLRP3 inflammasome, mediating intestinal inflammation.

FC050: Nefecon® Selectively Modifies the Composition of Circulating Immune Complexes in IGA Nephropathy

Abstract BACKGROUND AND AIMS IgA nephropathy (IgAN) is characterized by the mesangial deposition of IgA-containing immune complexes (IgA-IC). There is, however, no clear association between extent of mesangial IgA-IC deposition and disease severity in IgAN, suggesting that the composition of the IgA-IC, rather than the actual amount, may be critical in determining pathogenic consequences. The NEFIGAN trial (NCT01738035) assessed the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver the drug to the gut-associated lymphoid tissue (GALT)-rich distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. Here, we report the composition of IgA-IC in the serum of patients treated with Nefecon® 16 mg/day or placebo in the NEFIGAN trial, assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study was supported by a grant from Calliditas Therapeutics AB. METHOD Using Jacalin Agarose and elution with 0.1 M d-galactose, IgA-ICs were affinity-purified from serum of patients who received placebo (n=18) or Nefecon® 16 mg/day (n=18) at the start of treatment (SOT) and end of treatment (EOT). All protein samples were reduced and alkylated prior to digestion with trypsin, dried using a Savant SpeedVac (Thermo Fisher Scientific, UK) and dissolved in 40 µL of 0.1% formic acid. Each sample, spiked with iRT standard peptides (Biognosys, Switzerland), was loaded onto an Evotip (Evosep Biosystems, Denmark) column. Mass spectrometry analyses were performed on the Evosep One (Evosep) platform, coupled to the TIMS TOF Pro (Bruker, US). For protein identification and quantification, raw data were processed with FragPipe (V16), based on at least two peptides per protein. The protein intensities thus generated were Log_2-transformed and internally normalized using IGHA1 protein as standard. For each patient, the normalized protein amount at SOT was subtracted from the amount at EOT. A probabilistic dropout model was applied to the data to estimate fold changes between treatment and placebo groups at EOT. The proteins were ranked using t-statistic, and a gene set enrichment analysis was performed to determine the gene sets significantly affected by Nefecon® 16 mg/day versus placebo. RESULTS Gene ontology analysis revealed that treatment with Nefecon® 16 mg/day led to a significant protein enrichment in IgA-IC associated with pathways driving biological processes involved in the negative regulation of gene expression and transcription, gene silencing and epigenetics. In parallel, treatment with Nefecon® was also associated with an enrichment of proteins associated with extracellular vesicle and exosome formation. CONCLUSION For the first time, we show that it is possible to pharmacologically modify the composition of circulating IgA-IC in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively altered the protein composition of IgA-IC in a way that is likely to have significant consequences on the downstream mesangial response to IgA-IC accumulation; this may, in part, explain the antiproteinuric effect of this approach and the longer-term kidney function protection that has been reported in both the NEFIGAN and NefIgArd clinical trials. Studies are ongoing to precisely delineate the impact of these compositional changes on the pathogenicity of IgA-IC in IgAN.

FC049: Nefecon® (Budesonide) is an Epigenetic Modifier in IGA Nephropathy

Abstract BACKGROUND AND AIMS In recent years, the dysregulated expression of microRNAs (miRs)—small molecules of noncoding RNA that epigenetically fine-tune post-transcriptional gene expression—has been increasingly implicated in the pathogenesis of IgA nephropathy (IgAN). Extracellular miRs, contained within extracellular vesicles such as exosomes and microvesicles, are thought to function as important intercellular and interorgan modes of communication in health and disease. In the current study, next-generation sequencing (NGS) and subsequent real-time quantitative polymerase chain reaction (RT-qPCR) validation were performed to identify serum miRs associated with a high risk of future kidney function decline in IgAN. We then went on to determine whether treatment with Nefecon® 16 mg/day for 9 months was capable of modifying the serum levels of miRs associated with high-risk IgAN. This study was supported by a grant from Calliditas Therapeutics AB. METHOD NGS was performed by Qiagen (Germany) on serum from patients with IgAN (10 at high risk and 10 at low risk of progression) or membranous nephropathy (MN, n=10), as well as healthy individuals (HS, n=10). Sequencing data were validated by RT-qPCR using TaqMan miR PCR assays in independent sets of serum samples (20 per cohort). Using the Total Exosome Isolation (from serum) reagent (Thermo Fisher Scientific, UK), extracellular exosomes were isolated from the serum of participants of the NEFIGAN trial from the placebo group (n=40) and Nefecon® 16 mg/day group (n=40) at the start of treatment (SOT) and end of treatment (EOT). RNA was isolated and levels of miRs-483-5p and -122-5p were measured by RT-qPCR. The NEFIGAN trial (NCT01738035) was a randomized, double-blind, placebo-controlled Phase 2b trial designed to assess the safety and efficacy of Nefecon® a novel targeted-release formulation of budesonide, designed to deliver the drug to the distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. RESULTS miRs-483-5p and -122-5p were significantly overexpressed in the serum of patients with IgAN at high risk of progression compared with those who had stable IgAN or HS, but not compared with patients with MN. Both miRs were also found to be significantly increased in extracellular exosomes in high-risk IgAN compared with low-risk IgAN, HS and MN. Interestingly, levels of exosomal miRs-483-5p and -122-5p did not correlate with total serum IgA, IgA1, galactose-deficient IgA1 or IgA–IgG immune complex levels, suggesting a novel mechanism of action. Treatment with 16 mg/day of Nefecon® led to a significant reduction in the exosomal levels of miR-122-5p, while no change in miR-483-5p levels was observed in the treated group. CONCLUSION These data reveal a dysregulation of systemic exosomal miR expression in IgAN, and demonstrate for the first time that it is possible to pharmacologically modify the miR content of circulating exosomes in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively downregulated exosomal miR-122-5p, while having no effect on exosomal miR-483-5p levels, arguing against a generalized ‘anti-miR’ effect of Nefecon®. Studies are ongoing to both precisely delineate the cellular source(s) of these exosomes and determine the downstream impact of these changes on glomerular and tubulointerstitial biology.

Characterization of Specific Signatures of the Oral Cavity, Sputum, and Ileum Microbiota in Patients With Crohn's Disease.

BackgroundCrohn’s disease (CD) is a chronic nonspecific inflammatory bowel disease (IBD) with an increasing incidence worldwide. The etiology of CD is still obscure, but microbial dysbiosis has been recognized as an essential factor contributing to CD. However, few studies have revealed the microbiome’s signatures and reciprocal correlations between multiple sites in patients with CD over different disease stages. This study investigated the specific microbial architectures of the oral cavity, sputum, and ileum in patients with CD in the active and remission stages.MethodsMicrobial samples from the oral cavity, sputum, and ileum were collected from patients with CD in the active and remission stages and healthy controls. The microbial composition was assessed by 16S ribosomal RNA (rRNA) gene sequencing. In addition, bioinformatics methods were used to demonstrate the microbial signatures, functional changes, and correlations between microbiota and clinical data in CD.ResultsCompared with healthy controls, a distinct microbiota dysbiosis in the oral cavity, sputum, and ileum of patients with CD was identified, characterized by alterations in microbiota biodiversity and composition. The oral cavity and sputum microbiota showed significantly lower microbial diversity in patients with CD than in healthy controls. In terms of microbiota composition, the microbiota changes in the oral cavity of patients with CD were similar to those in the sputum, while they were different from those in the ileum. In the oral cavity and sputum of patients with CD, a lower relative abundance of Firmicutes and Actinobacteria was observed compared to healthy controls, which was most prominent in the active stage. In contrast, an increased relative abundance of Fusobacteria, Porphyromonas, and Haemophilus was observed in patients with CD. The predicted metabolic pathways involved in the oral cavity, sputum, and ileum were similar, predominantly involving metabolism, environmental information processing, and genetic information processing.ConclusionThe results revealed the alterations of microbiota architecture in the oral cavity, sputum, and ileum of patients with CD, which varied across disease stages. Studying microbiota dysbiosis may bring new insights into the etiology of CD and lead to novel treatments.

Injuries of the thoracic and lumbar spine due to juvenile osteoporosis

Complex examination of 123 children with back pain and wedge-shaped vertebra body deformity (according to X-ray data) showed that those changes were the manifestation of juvenile osteoporosis in 10,5%) of cases. Diagnosis of juvenile osteoporosis was confirmed by the decrease of bone mass by 10%) and over versus the norm. It was detected using DEXA. Conformity of that bone loss to osteoporosis was proved by comparative histomorphometry of bioptate from the upper flaring portion of the ileum in patients with BMD loss. Comparison of histomorphometry data and activity of the bone marrow stromal cell clonning showed that BMD loss was connected with the decrease of bone formation; the later was stipulated by genetic defect of osteoblasts cellprecursors. Those changes preceded to bone mass loss that could be detected by invasive or noninvasive methods. The possibility of pharmacological correction of bone mass deficit in children was shown.

Transcriptional regulation of chemokine network by biologic monotherapy in ileum of patients with Crohn’s disease

BackgroundCrohn’s disease (CD) exacerbation is marked by an intense cellular trafficking. We set out to determine the specific impact of biologic therapies on regulating chemokine network gene expression in healthy, mildly and severely inflamed tissue of CD patients. MethodsTwenty CD patients on biologics (adalimumab, ustekinumab, vedolizumab) or untreated undergoing colonoscopy due to clinical symptoms of flare. Healthy, mildly and severely inflamed ileum biopsies from each patient were collected. Chemokines and receptors gene expression was analyzed and a STRING analysis for functional enrichment was performed. ResultsThe chemokine network exhibited wide transcriptional differences among tissues in active untreated patients, whereas all biologic treatments reduced these differences and homogenized their transcriptional activity. In mildly inflamed tissue, all treatments showed gene upregulation while ustekinumab additionally maintained the downregulation of genes such as CCL2, CCL3, CCL17 or CCL23, involved in T cell chemotaxis, inflammatory monocyte and NK trafficking. In severely inflamed tissue, all treatments shared a downregulatory effect on chemokines controlling T cell response (i.e. CXCL16, CXCR3). Adalimumab and vedolizumab significantly reduced the expression of genes promoting antigen presentation by DCs and the initiation of leukocyte extravasation (i.e. CXCL12, CCL25, CCR7). Ustekinumab significantly reduced genes positively regulating Th1 cytokine production and IL-8 mediated signaling (i.e. IL1B, XCL1, CXCR1, CXCR2). ConclusionBiologic therapies differentially target the chemokine network gene expression profile in the ileal tissue of active CD patients. These results may contribute to better understanding cell homing and to defining future personalized therapeutic strategies for CD patients.

P094 Transcriptomic and small RNA sequencing profile in ileal resections from complicated Crohn′s Disease patients

Abstract Background Crohn′s disease is a chronic inflammatory disorder of gastrointestinal tract that is classified into three different behaviours: the inflammatory (B1), the stenotic (B2) or the penetrating (B3). We pretend to identify differences in transcriptomic and non-long coding RNA expression profiles associated to damaged and no-damaged surgical ileal resections from complicated CD patients. Methods We conducted both RNA and small RNA sequencing profiling on ileal surgical resections from CD patients with structuring (n=10) or penetrating (n=10) behaviour; from each patient we obtained a sample from affected tissue (B2A and B3A, respectively) and the paired non-affected ileum (B2C and B3C, respectively). Ten ileal resections (control samples) were obtained from non-affected ileum of patients with right colorectal cancer (IL). Enrichment scores of the GO terms and KEGG pathways were used to functional analysis investigated in this study. Gene expression and miRNA profiles were validated by RT-qPCR. Results Our comparative bioinformatic analysis of RNA sequencing showed that the comparation of IL and the non-affected ileum from CD patients (B2C+B3C) revealed that only 11 genes were significantly altered and any miRNA with significative expression. The comparative analysis between B2A+B3A vs B2C+B3C showed: a) 2562 genes differentially regulated and the GO enrichment analysis determined that inflammation, cell activation, and regulation of the extracellular matrix (EM) were the main biological processes altered (Figure 1a). KEGG analysis showed that cytokine-receptor interaction and MAPK and Ras signalling were the most affected pathways (Figure 1b). Among the top ten up-regulated genes we found several immunoglobulins (such as IGHG4, IGHG3, and IGHG1), proteins of the extracellular matrix EM (such as EPYC, COMP, and CHI3L1), and transcription factors (such as PRRX1). When the analysis was performed by CD behaviour, results revealed that most of these genes were significantly increased in both, B2 and B3 CD; b) 103 miRNAs with differential expression, 56 were up-regulated and 47 were down-regulated. Negative and significant correlations were detected between the RNA expression of ECM components and some miRNA expression. Figure 1. Bar plot of enrichment analysis with differential expressed genes between B2A+B3A vs B2C+B3C. A) Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) of Gene Ontology (GO) enrichment analysis. B) Enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Conclusion The correlation detected between extracellular matrix components and some miRNA suggest an epigenetic regulation in the affected ileum of complicated CD patients.

Colonization of fecal microbiota from patients with neonatal necrotizing enterocolitis exacerbates intestinal injury in germfree mice subjected to necrotizing enterocolitis-induction protocol via alterations in butyrate and regulatory T cells

BackgroundNecrotizing enterocolitis (NEC) remains a life-threatening disease in neonates. Numerous studies have shown a correlation between the intestinal microbiota and NEC, but the causal link remains unclear. This study aimed to demonstrate the causal role of gut microbiota in NEC and explore potential mechanisms involved.MethodsEighty-one fecal samples from patients with NEC and eighty-one matched controls (matched to the NEC infants by gestational age, birth weight, date of birth, mode of delivery and feeding patterns) were collected. To explore if altered gut microbiota contributes to the pathogenesis of NEC, fecal microbiota transplantation (FMT) was carried out in germ-free (GF) mice prior to a NEC-induction protocol that included exposure to hypoxia and cold stress. Butyric acid was also administered to demonstrate its role in NEC. The fecal microbiota from patients and mice were analyzed by 16S rRNA gene sequencing analysis. Short chain fatty acid (SCFA) levels were measured by gas chromatography-mass spectrometry (GC–MS). The ontogeny of T cells and regulatory T cells (Tregs) in lamina propria mononuclear cells (LPMC) from the ileum of patients and mice were isolated and analyzed by flow cytometry.The transcription of inflammatory cytokines was quantified by qRT-PCR.ResultsNEC patients had increased Proteobacteria and decreased Firmicutes and Bacteroidetes compared to fecal control samples, and the level of butyric acid in the NEC group was lower than the control group. FMT in GF mice with samples from NEC patients achieved a higher histological injury scores when compared to mice that received FMT with control samples. Alterations in microbiota and butyrate levels were maintained in mice following FMT. The ratio of Treg/CD4+T (Thelper) cells was reduced in both NEC patients and mice modeling NEC following FMT.ConclusionsThe microbiota was found to have NEC and the microbial butyrate-Treg axis was identified as a potential mechanism for the observed effects.

P0344NEFECON® (BUDESONIDE) SELECTIVELY REDUCES CIRCULATING LEVELS OF BAFF (BLYS) AND SOLUBLE BCMA AND TACI IN IGA NEPHROPATHY

Abstract Background and Aims There is increasing evidence to support a pivotal role for the gut-associated lymphoid tissue (GALT) as a major source of the poorly O-galactosylated immunoglobulin A1 (IgA1) that triggers the formation of nephritogenic immune complexes in IgA nephropathy (IgAN). Critical to the regulation of IgA synthesis in GALT are two cytokines, which regulate B-cell maturation, function and survival: BAFF (B-cell activating factor [also known as BLyS (B-lymphocyte stimulator)]) and APRIL (A PRoliferation-inducing ligand). BAFF and APRIL bind to specific cell-surface receptors: TACI (transmembrane activator and calcium modulator and cyclophilin-ligand interactor), BCMA (B-cell maturation antigen) and the BAFF receptor. Several studies have shown elevated levels of BAFF and APRIL in IgAN linked to worse clinical outcomes. The therapeutic potential of selectively targeting GALT was demonstrated in the NEFIGAN trial (NCT01738035), which assessed the safety and efficacy of a novel targeted-release formulation of budesonide (NEFECON®), designed to deliver budesonide to the GALT-rich distal ileum in patients with IgAN. The trial comprised a 6-month run-in, 9-month treatment and 3-month follow-up phase: 48 patients received NEFECON® 16 mg/day, 51 patients received NEFECON® 8 mg/day and 50 patients received placebo. The headline result of the study was that NEFECON® 16 mg/day, added to optimised renin–angiotensin system blockade, reduced proteinuria and stabilised estimated glomerular filtration rate in patients with IgAN. In this study, we aimed to determine whether NEFECON® treatment altered synthesis of the BAFF and APRIL B-cell maturation modulators. Method Serum levels of BAFF, APRIL, BCMA and TACI were measured using Luminex technology. Changes in the levels of each biomarker between randomisation (T0), end of treatment (T1) and the end of study (T2) and between treatments arms were compared using a one-way analysis of variance. Significance was set at p&amp;lt;0.05. Results A significant, dose-dependent reduction in serum BAFF levels was seen with NEFECON®, which reversed on cessation of NEFECON®. There were similar significant reductions in the levels of soluble BCMA and TACI with treatment but no effect was seen on circulating levels of APRIL. These changes were mirrored by parallel changes in soluble CD27 levels and are consistent with our previous reports describing a dose-dependent reduction in circulating IgA–IgG immune complexes, secretory IgA and galactose-deficient IgA levels with NEFECON®. Conclusion Delivering budesonide to the GALT-rich distal ileum modulates key regulators of GALT B-cell maturation in IgAN. Although the samples available are not sufficient to determine that the observed changes in serum BAFF, BCMA and TACI levels are due to a direct action on GALT, it is intriguing to note that BAFF-transgenic mice develop an IgAN-like disease, principally due to IgA overexpression in the intestinal lamina propria.

P0228THE NEFIGARD TRIAL: THE EFFECT OF NEFECON® (BUDESONIDE) IN PATIENTS WITH PRIMARY IGA NEPHROPATHY AT RISK OF DEVELOPING END-STAGE RENAL DISEASE

Abstract Background and Aims Since the first description of IgA nephropathy (IgAN) over 50 years ago, it has been recognised that the mucosal immune system plays a crucial role in the pathogenesis of this common global cause of kidney failure. Recently, particular attention has focussed on the importance of the gut-associated lymphoid tissue (GALT) as the potential source of the poorly O-galactosylated IgA1 that triggers the formation of nephritogenic immune complexes in IgAN. Pathway analysis based on a large meta-analysis of genome wide association studies identified the intestinal immune network for IgA production as the most enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway in IgAN. Furthermore, separate studies have shown that the IgA in glomerular IgA deposits is indistinguishable from mucosal IgA. The therapeutic potential of selectively targeting GALT was demonstrated in the NEFIGAN trial (NCT01738035), which assessed the safety and efficacy of a novel targeted-release formulation of budesonide (NEFECON®), designed to deliver budesonide to the GALT-rich distal ileum in patients with IgAN. After 9 months’ treatment, urine protein–creatinine ratio (UPCR) was reduced by 29.3% in the NEFECON® 16 mg group compared with the placebo group. Estimated glomerular filtration rate (eGFR) dropped 4.7 ml/min/1.73 m2 in the placebo group with no deterioration seen in the NEFECON® 16 mg group. Incidence of patients reporting adverse events was similar in all groups. These data led to the design of the NefIgArd study which aims to assess the efficacy, safety, and tolerability of NEFECON® 16 mg in patients with IgAN at risk of end-stage renal disease. Method The NefIgArd study is a randomised, double-blind, placebo-controlled Phase 3 trial, with two parts – PART A a 15–35-day screening period, 9-month treatment and 3-month follow-up period; and PART B a 12-month no-treatment follow-up period (Figure). The study is recruiting across 146 nephrology clinics in 19 countries. Patients must be at least 18 years old with biopsy-confirmed primary IgAN and persistent proteinuria &amp;gt;1 g/24 h and eGFR between 35 and 90 ml/min per 1.73 m2 (CKD-EPI) despite optimised renin–angiotensin system blockade. Patients are randomised on a 1:1 ratio to NEFECON® 16 mg/day or placebo. Consistent with the Kidney Health Initiative White Paper “Proteinuria Reduction as a Surrogate End Point in Trials of IgA Nephropathy” published in early 2019, the primary outcome of Part A is to assess the effect of NEFECON® 16 mg on 24 h UPCR at 9 months compared with placebo. The Part B primary outcome is based on data presented at the NKF/FDA/EMA workshop in March 2018 that supported eGFR slope as an endpoint for full approval and will assess the effect of NEFECON® 16 mg on a 2-year eGFR-based endpoint compared with placebo. In comparison with other studies that are recruiting, we believe that the relatively short period required to provide validation of the surrogacy of proteinuria reduction will significantly reduce the risks of non-protocol treatments and loss of patients from the study that could dilute the true treatment effect of NEFECON®. Results As of 1 January 2020, 207 patients have been randomised, and Part A is expected to complete in Q4 2020, with Part B completing in 2022. To ensure the NefIgArd study results are fully translatable to the global IgAN population, NefIgArd will also open in China in 2020. Conclusion The NefIgArd study builds on the experience of the NEFIGAN trial, the largest commercially sponsored study ever completed in IgAN. The design of the NefIgArd study has used state-of-the-art data to evaluate kidney outcomes, using proteinuria as a reasonably likely surrogate of the effect of NEFECON® on long-term kidney outcomes and confirming long-term renoprotection using an NKF/FDA/EMA-suggested eGFR-based endpoint as a basis for full approval.

Bacterial Mucosa-associated Microbiome in Inflamed and Proximal Noninflamed Ileum of Patients With Crohn's Disease.

BackgroundMicrobiota is most likely essential in the pathogenesis of Crohn’s disease (CD). Fecal diversion after ileocecal resection (ICR) protects against CD recurrence, whereas infusion of fecal content triggers inflammation. After ICR, the majority of patients experience endoscopic recurrence in the neoterminal ileum, and the ileal microbiome is of particular interest. We have assessed the mucosa-associated microbiome in the inflamed and noninflamed ileum in patients with CD.MethodsMucosa-associated microbiome was assessed by 16S rRNA sequencing of biopsies sampled 5 and 15 cm orally of the ileocecal valve or ileocolic anastomosis.ResultsFifty-one CD patients and forty healthy controls (HCs) were included in the study. Twenty CD patients had terminal ileitis, with endoscopic inflammation at 5 cm, normal mucosa at 15 cm, and no history of upper CD involvement. Crohn’s disease patients (n = 51) had lower alpha diversity and separated clearly from HC on beta diversity plots. Twenty-three bacterial taxa were differentially represented in CD patients vs HC; among these, Tyzzerella 4 was profoundly overrepresented in CD. The microbiome in the inflamed and proximal noninflamed ileal mucosa did not differ according to alpha diversity or beta diversity. Additionally, no bacterial taxa were differentially represented.ConclusionsThe microbiome is similar in the inflamed and proximal noninflamed ileal mucosa within the same patients. Our results support the concept of CD-specific microbiota alterations and demonstrate that neither ileal sublocation nor endoscopic inflammation influence the mucosa-associated microbiome.

P110 Identification of the tryptophan metabolite 3-hydroxyanthranilic acid as a novel tool for the differentiation of Crohn’s disease phenotypes

Abstract Background The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise question regarding the unclarified heterogeneity of disease pathomechanisms. While biomarkers for the differentiation of Crohn’s disease (CD) vs. ulcerative colitis (UC) have been suggested, specific markers for a subclassification of CD phenotypes are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterise potential biomarkers focusing on the downstream metabolites of kynurenine metabolism. The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise question regarding the unclarified heterogeneity of disease pathomechanisms. While biomarkers for the differentiation of Crohn’s disease (CD) vs. ulcerative colitis (UC) have been suggested, specific markers for a subclassification of CD phenotypes are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterise potential biomarkers focusing on the downstream metabolites of kynurenine metabolism. Methods Using immunohistochemical staining, we analysed and compared the mucosal tryptophan immune metabolism in biotic samples from patients with UC (n = 11), CD (n = 11) and healthy control (n = 12). Localisation-specific quantification of immune cell infiltration, tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. Results As expected, we found generally increased immune cell infiltrates in the tissue of all patients with IBD. However, in patients with CD, significant differences were found between regulatory T-cell markers in the ileum compared with the colon. In line with this finding, we identified kynureninase as a modulator of immunosuppressive kynurenine levels specifically in the ileum of patients with CD. Correspondingly, significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in CD ileum samples. Conclusion Highlighting the heterogeneity of the different phenotypes of CD, we identified 3-hydroxyanthranilic acid as a potential mucosal biomarker allowing the localisation-specific differentiation of ileum or colon inflammation in patients with CD. Moreover, we characterised the kynurenine-degrading enzyme kynureninase as a modulator of immunosuppression and chronic inflammation with potential therapeutic relevance.

Identification of differentially expressed genes between the colon and ileum of patients with inflammatory bowel disease by gene co-expression analysis.

ObjectiveWe aimed to identify differentially expressed genes (DEG) in patients with inflammatory bowel disease (IBD).MethodsRNA-seq data were obtained from the Array Express database. DEG were identified using the edgeR package. A co-expression network was constructed and key modules with the highest correlation with IBD inflammatory sites were identified for analysis. The Cytoscape MCODE plugin was used to identify key sub-modules of the protein–protein interaction (PPI) network. The genes in the sub-modules were considered hub genes, and functional enrichment analysis was performed. Furthermore, we constructed a drug–gene interaction network. Finally, we visualized the hub gene expression pattern between the colon and ileum of IBD using the ggpubr package and analyzed it using the Wilcoxon test.ResultsDEG were identified between the colon and ileum of IBD patients. Based on the co-expression network, the green module had the highest correlation with IBD inflammatory sites. In total, 379 DEG in the green module were identified for the PPI network. Nineteen hub genes were differentially expressed between the colon and ileum. The drug–gene network identified these hub genes as potential drug targets.ConclusionNineteen DEG were identified between the colon and ileum of IBD patients.