A Real-World Prospective Cohort Study of Patients With Newly Diagnosed Crohn's Disease Treated by a Multidisciplinary Team: 1-Year Outcomes.

Background: Real-world data on outcomes of patients with newly diagnosed Crohn's disease (ndCD) is limited. We aimed to assess the achievement of corticosteroid-free clinical remission (CS-free CR) and other therapeutic targets 1 year after diagnosis in a cohort of patients with ndCD treated by a multidisciplinary team (MDT). Methods: A prospective observational cohort study was conducted on consecutive treatment-naïve adults with ndCD. Patients received management at the treating physician's discretion, along with a tailored nutritional plan provided by an inflammatory bowel disease (IBD)-oriented dietitian. Patients were guided and educated by an IBD nurse, with flexible communication access to the IBD team. Therapeutic targets were assessed at 1 year. Multivariable logistic regression was used to evaluate predictors of CS-free CR. Results: Seventy-six patients (50% female) with a median age of 27 (22-39) years were eligible. Over 75% of patients were assessed by IBD-oriented dietitians and the IBD nurse. Within a median of 4.3 (2.5-6.7) months from diagnosis 60.5% initiated biologics (96% anti- tumor necrosis factor). Dietary intervention was applied to 77.6% of the cohort, either monotherapy (33.9%) or add-on (66.1%). At 1 year, 64.5% of patients achieved sustained CS-free CR, 56.6% biochemical remission, 55.8% endoscopic response, 44.2% endoscopic remission, 30.8% deep remission, and in 39.5% there was an improvement in health-related quality of life (HRQoL). Predictors for CS-free CR were uncomplicated phenotype (B1/P0), lower body mass index, and lower patient-reported outcome 2 scores at diagnosis. Conclusions: In a real-world setting at a tertiary medical center, a cohort of ndCD patients treated by an MDT resulted in favorable 1-year outcomes. Over 60% achieved CS-free CR, along with significant improvements in biomarkers and HRQoL.

Serologic Response and Safety after a Third Dose of the COVID-19 BNT162b2 Vaccine in Patients with Inflammatory Bowel Diseases.

Vaccines are pivotal for control of the coronavirus disease (COVID-19) pandemic. Patients with inflammatory bowel diseases (IBDs) treated with antitumor necrosis factor (TNF)-α have lower serologic response after two COVID-19 vaccine doses. Data regarding a third vaccine dose are scarce. An Israeli multicenter prospective observational study recruited 319 subjects: 220 with IBD (79 treated with anti-TNFα) and 99 healthy control (HC) participants. All patients received two mRNA-BNT162b2 vaccines (Pfizer/BioNTech), 80% of whom received a third vaccine dose. Evaluation included disease activity, anti-spike (S) and nucleocapsid (N) antibody levels, anti-TNFα drug levels, and adverse events (AEs). All participants showed significant serologic response one month after receiving a third dose. However, three months later, the anti-S levels decreased significantly in patients treated with anti-TNFα compared with the non-anti-TNFα and HC groups. A correlation between serologic response to the third vaccine dose and anti-TNF drug levels was not found. No significant AE or IBD exacerbation was observed. Importantly, lower serologic response after the third vaccine dose predicted infection. A third dose of BNT162b2 is effective and safe in patients with IBD. Lower serologic response predicted infection, even in seropositive subjects. Lower serologic responses and their rapid decline suggest a fourth vaccine dose in this patient population.

Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis.

Background: Intestinal epithelial cells (IECs) are the first to encounter luminal microorganisms and actively participate in intestinal immunity. We reported that IECs express the ß-glucan receptor Dectin-1, and respond to commensal fungi and ß-glucans. In phagocytes, Dectin-1 mediates LC3-associated phagocytosis (LAP) utilizing autophagy components to process extracellular cargo. Dectin-1 can mediate phagocytosis of ß-glucan-containing particles by non-phagocytic cells. We aimed to determine whether human IECs phagocytose ß-glucan-containing fungal particles via LAP. Methods: Colonic (n=18) and ileal (n=4) organoids from individuals undergoing bowel resection were grown as monolayers. Fluorescent-dye conjugated zymosan (ß-glucan particle), heat-killed- and UV inactivated C. albicans were applied to differentiated organoids and to human IEC lines. Confocal microscopy was used for live imaging and immuno-fluorescence. Quantification of phagocytosis was carried out with a fluorescence plate-reader. Results: zymosan and C. albicans particles were phagocytosed by monolayers of human colonic and ileal organoids and IEC lines. LAP was identified by LC3 and Rubicon recruitment to phagosomes and lysosomal processing of internalized particles was demonstrated by co-localization with lysosomal dyes and LAMP2. Phagocytosis was significantly diminished by blockade of Dectin-1, actin polymerization and NAPDH oxidases. Conclusions: Our results show that human IECs sense luminal fungal particles and internalize them via LAP. This novel mechanism of luminal sampling suggests that IECs may contribute to the maintenance of mucosal tolerance towards commensal fungi.

Newly Diagnosed Crohn's Disease Patients in India and Israel Display Distinct Presentations and Serological Markers: Insights from Prospective Cohorts.

Background: Crohn's disease (CD) incidence is rising in India. However, features of newly diagnosed patients with CD in this population are largely unknown. The Indo-Israeli IBD GastroEnterology paRtnership (TiiiGER) aimed to investigate differences in presentation among patients with newly diagnosed CD in India and Israel, and to explore phenotype−serotype correlations. Methods: A prospective observational cohort study of consecutive adults (>18 years) conducted in two large referral centers in India and Israel (2014−2018). Clinical data, an antiglycan serological panel, and 20 CD-associated genetic variants were analyzed. Outcomes: complicated phenotype at diagnosis and early complicated course (hospitalizations/surgeries) within 2 years of diagnosis. Results: We included 260 patients (104, Indian (65.4%, male; age, 37.8); 156 Israeli (49.4%, male; 31.8, age)). Median lag time from symptoms onset to diagnosis was 10.5 (IQR 3−38) vs. 3 (IQR 1−8) months in Indian vs. Israeli patients (p < 0.001). Complicated phenotype at diagnosis was observed in 48% of Indian and 30% of Israeli patients (p = 0.003). Complicated phenotype was associated with higher anti-Saccharomyces cerevisiae antibody (ASCA) seropositivity rate among Israeli patients (p < 0.001), but not among Indian patients. Antiglycan serology did not correlate with the tested genetic variants. Early complicated course occurred in 28 (18%) Israeli and 13 (12.5%) Indian patients. The time from diagnosis to complication was comparable (log rank p = 0.152). Antiglycan serology did not correlate with a complicated early course in either cohort. Conclusions: There are significant differences in patients presenting with newly diagnosed CD in India and Israel, including phenotype and distinct biomarkers at diagnosis. These differences suggest different genetic and environmental disease modifiers.

SARS-CoV-2 IgG Antibody Levels in Women with IBD Vaccinated during Pregnancy.

INTRODUCTION: Regulatory agencies supported vaccination of pregnant women with SARS-CoV-2 mRNA vaccines, including patients with IBD. No data exist regarding these vaccines in IBD during pregnancy. AIM: To assess the serologic response to two doses of the mRNA SARS-CoV-2 BNT162b2 vaccine in pregnant women with IBD vaccinated during pregnancy, compared to that of pregnant women without IBD, and non-pregnant women with IBD. METHODS: Anti-spike antibody levels were assessed in all women and in cord blood of consenting women. RESULTS: From December 2020 to December 2021, 139 women were assessed: pregnant with IBD-36, pregnant without IBD-61, and not pregnant with IBD-42. Antibodies were assessed in cords of two and nine newborns of women with and without IBD, respectively. Mean gestational ages at administration of the second vaccine doses were 22.0 weeks in IBD and 23.2 weeks in non-IBD, respectively. Mean (SD) duration from the second vaccine dose to serology analysis in pregnant women with IBD, without IBD, and in non-pregnant women with IBD was 10.6 (4.9), 16.4 (6.3), and 4.3 (1.0) weeks, respectively. All women mounted a serologic response. In multivariable analysis, no correlation was found between the specific group and antibody levels. In both pregnancy groups, an inverse correlation between antibody levels and the interval from the second vaccine dose was demonstrated. Cord blood antibody levels exceeded maternal levels in women with and without IBD. CONCLUSION: All patients with IBD mounted a serologic response. The interval between vaccine administration to serology assessment was the most important factor determining antibody levels. A third vaccine dose should be considered in pregnant women with IBD vaccinated at early stages of pregnancy.

Anti-TNFα Treatment Impairs Long-Term Immune Responses to COVID-19 mRNA Vaccine in Patients with Inflammatory Bowel Diseases.

Patients with inflammatory bowel disease (IBD) treated with anti-tumor-necrosis factor-alpha (TNFα) exhibited lower serologic responses one-month following the second dose of the COVID-19 BNT162b2 vaccine compared to those not treated with anti-TNFα (non-anti-TNFα) or to healthy controls (HCs). We comprehensively analyzed long-term humoral responses, including anti-spike (S) antibodies, serum inhibition, neutralization, cross-reactivity and circulating B cell six months post BNT162b2, in patients with IBD stratified by therapy compared to HCs. Subjects enrolled in a prospective, controlled, multi-center Israeli study received two BNT162b2 doses. Anti-S levels, functional activity, specific B cells, antigen cross-reactivity, anti-nucleocapsid levels, adverse events and IBD disease score were detected longitudinally. In total, 240 subjects, 151 with IBD (94 not treated with anti-TNFα and 57 treated with anti-TNFα) and 89 HCs participated. Six months after vaccination, patients with IBD treated with anti-TNFα had significantly impaired BNT162b2 responses, specifically, more seronegativity, decreased specific circulating B cells and cross-reactivity compared to patients untreated with anti-TNFα. Importantly, all seronegative subjects were patients with IBD; of those, >90% were treated with anti-TNFα. Finally, IBD activity was unaffected by BNT162b2. Altogether these data support the earlier booster dose administration in these patients.

Escherichia coli Strains from Patients with Inflammatory Bowel Diseases have Disease-specific Genomic Adaptations.

BACKGROUND AND AIMS: Escherichia coli is over-abundant in the gut microbiome of patients with inflammatory bowel disease [IBD]. Here, we aimed to identify IBD-specific genomic functions of diverse E. coli lineages. METHODS: We investigated E. coli genomes from patients with ulcerative colitis [UC], Crohn's disease [CD] or a pouch, and healthy subjects. The majority of genomes were reconstructed from metagenomic samples, including newly sequenced faecal metagenomes. Clinical metadata were collected. Functional analysis at the gene and mutation level were performed and integrated with IBD phenotypes and biomarkers. RESULTS: Overall, 530 E. coli genomes were analysed. The E. coli B2 lineage was more prevalent in UC compared with other IBD phenotypes. Genomic metabolic capacities varied across E. coli lineages and IBD phenotypes. Host mucin utilisation enzymes were present in a single lineage and depleted in patients with a pouch, whereas those involved in inulin hydrolysis were enriched in patients with a pouch. E. coli strains from patients with UC were twice as likely to encode the genotoxic molecule colibactin than strains from patients with CD or a pouch. Strikingly, patients with a pouch showed the highest inferred E. coli growth rates, even in the presence of antibiotics. Faecal calprotectin did not correlate with the relative abundance of E. coli. Finally, we identified multiple IBD-specific non-synonymous mutations in E. coli genes encoding for bacterial cell envelope components. CONCLUSIONS: Comparative genomics indicates that E. coli is a commensal species adapted to the overactive mucosal immune milieu in IBD, rather than causing it. Our results reveal mutations that may lead to attenuated antigenicity in some E. coli strains.

Lower Serologic Response to COVID-19 mRNA Vaccine in Patients With Inflammatory Bowel Diseases Treated With Anti-TNFα.

BACKGROUND & AIM: Patients with inflammatory bowel diseases (IBD), specifically those treated with anti-tumor necrosis factor (TNF)α biologics, are at high risk for vaccine-preventable infections. Their ability to mount adequate vaccine responses is unclear. The aim of the study was to assess serologic responses to messenger RNA-Coronavirus Disease 2019 vaccine, and safety profile, in patients with IBD stratified according to therapy, compared with healthy controls (HCs). METHODS: Prospective, controlled, multicenter Israeli study. Subjects enrolled received 2 BNT162b2 (Pfizer/BioNTech) doses. Anti-spike antibody levels and functional activity, anti-TNFα levels and adverse events (AEs) were detected longitudinally. RESULTS: Overall, 258 subjects: 185 IBD (67 treated with anti-TNFα, 118 non-anti-TNFα), and 73 HCs. After the first vaccine dose, all HCs were seropositive, whereas ∼7% of patients with IBD, regardless of treatment, remained seronegative. After the second dose, all subjects were seropositive, however anti-spike levels were significantly lower in anti-TNFα treated compared with non-anti-TNFα treated patients, and HCs (both P < .001). Neutralizing and inhibitory functions were both lower in anti-TNFα treated compared with non-anti-TNFα treated patients, and HCs (P < .03; P < .0001, respectively). Anti-TNFα drug levels and vaccine responses did not affect anti-spike levels. Infection rate (∼2%) and AEs were comparable in all groups. IBD activity was unaffected by BNT162b2. CONCLUSIONS: In this prospective study in patients with IBD stratified according to treatment, all patients mounted serologic response to 2 doses of BNT162b2; however, its magnitude was significantly lower in patients treated with anti-TNFα, regardless of administration timing and drug levels. Vaccine was safe. As vaccine serologic response longevity in this group may be limited, vaccine booster dose should be considered.

Meta-Analysis of IBD Gut Samples Gene Expression Identifies Specific Markers of Ileal and Colonic Diseases.

BACKGROUND: Inflammatory bowel diseases (IBDs) are characterized by chronic inflammation and tissue damages in limited segments of the digestive tract. Pathogenesis in the tissue and mucosal inflammation probably differs according to disease location. Our aim was to further analyze transcriptomic profiles in different locations of IBD, differentiating ulcerative colitis (UC), colonic Crohn's disease (CD), ileal CD, and pouchitis, with respect to normal colonic and ileal mucosa. We thus performed a meta-analysis focusing on specific transcriptomic signatures of ileal and colonic diseases. METHODS: We identified 5 cohorts with available transcriptomic data in ileal or colonic samples from active IBD and non-IBD control samples. The meta-analysis was performed on 1047 samples. In each cohort separately, we compared gene expression in CD ileitis and normal ileum; in CD colitis, UC, and normal colon; and finally in pouchitis and normal ileum. RESULTS: We identified specific markers of ileal (FOLH1, CA2) and colonic (REG3A) inflammation and showed that, with disease, some cells from the ileum start to express colonic markers. We confirmed by immunohistochemistry that these markers were specifically present in ileal or colonic diseases. We highlighted that, overall, colonic CD resembles UC and is distinct from ileal CD, which is in turn closer to pouchitis. CONCLUSIONS: We demonstrated that ileal and colonic diseases exhibit specific signatures, independent of their initial clinical classification. This supports molecular, rather than clinical, disease stratification, and may be used to design drugs specifically targeting ileal or colonic diseases.

We perform a meta-analysis of publicly available inflammatory bowel disease transcriptomes and identify FOLH1, REG3A, and CA2 as specific markers of ileal and colonic diseases. We demonstrate that Crohn's colitis resembles ulcerative colitis, while Crohn's ileitis resembles pouchitis.

Alteration in Urease-producing Bacteria in the Gut Microbiomes of Patients with Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Bacterial urease is a major virulence factor of human pathogens, and murine models have shown that it can contribute to the pathogenesis of inflammatory bowel diseases [IBD]. METHODS: The distribution of urease-producing bacteria in IBD was assessed using public faecal metagenomic data from various cohorts, including non-IBD controls [n = 55], patients with Crohn's disease [n = 291] or ulcerative colitis [n = 214], and patients with a pouch [n = 53]. The ureA gene and the taxonomic markers gyrA, rpoB, and recA were used to estimate the percentage of urease producers in each sample. RESULTS: Levels of urease producers in patients with IBD and non-IBD controls were comparable. In non-IBD controls and most IBD patients, urease producers were primarily acetate-producing genera such as Blautia and Ruminococcus. A shift in the type of the dominant urease producers towards Proteobacteria and Bacilli was observed in a subset of all IBD subtypes, which correlated with faecal calprotectin levels in one cohort. Some patients with IBD had no detectable urease producers. In patients with a pouch, the probiotic-associated species Streptococcus thermophilus was more common as a main urease producer than in other IBD phenotypes, and it generally did not co-occur with other Bacilli or with Proteobacteria. CONCLUSIONS: Unlike all non-IBD controls, patients with IBD often showed a shift towards Bacilli or Proteobacteria or a complete loss of urease production. Probiotics containing the species S. thermophilus may have a protective effect against colonisation by undesirable urease-producing bacteria in a subset of patients with a pouch.

Dysbiosis in Metabolic Genes of the Gut Microbiomes of Patients with an Ileo-anal Pouch Resembles That Observed in Crohn's Disease.

Crohn's disease (CD), ulcerative colitis (UC), and pouchitis are multifactorial and chronic inflammatory bowel diseases (IBD). Pouchitis develops in former UC patients after proctocolectomy and ileal-pouch-anal anastomosis and is characterized by inflammation of the previously normal small intestine comprising the pouch. The extent to which microbial functional alteration (dysbiosis) in pouchitis resembles that of CD or UC has not been investigated, and the pathogenesis of pouchitis remains unknown. We collected 208 fecal metagenomes from 69 patients with a pouch (normal pouch and pouchitis) and compared them to publicly available metagenomes of patients with CD (n = 88), patients with UC (n = 76), and healthy controls (n = 56). Patients with pouchitis presented the highest alterations in species, metabolic pathways, and enzymes, which was correlated with intestinal inflammation. Ruminococcus gnavus strains encoding mucin-degrading glycoside hydrolases were highly enriched in pouchitis. Butyrate and secondary bile acid biosynthesis pathways were decreased in IBD phenotypes and were especially low in pouchitis. Pathways such as amino acid biosynthesis and degradation of aromatic compounds and sugars, encoded by members of the Enterobacteriaceae, were enriched in pouch and CD but not in UC. We developed microbial feature-based classifiers that can distinguish between patients with a normal pouch and pouchitis and identified species and genes that were predictive of pouchitis. We propose that the noninflamed pouch is already dysbiotic and microbially is similar to CD. Our study reveals microbial functions that outline the pathogenesis of pouchitis and suggests bacterial groups and functions that could be targeted for intervention to attenuate small intestinal inflammation present in pouchitis and CD.IMPORTANCE Crohn's disease (CD), ulcerative colitis (UC), and pouchitis are chronic inflammatory conditions of the bowel. Pouchitis develops in former UC patients after proctocolectomy and ileal-pouch-anal anastomosis and is characterized by inflammation of the previously normal small intestine comprising the pouch. The extent to which microbial dysbiosis in patients with pouchitis resembles that of CD or UC and the pathogenesis of pouchitis remains unclear. We investigated the functions in the gut microbiomes of these patients using metagenomics. We found that the noninflamed pouch is already dysbiotic and microbially is similar to CD. Our study reveals microbial functions with a potential role in pouchitis pathogenesis such as depletion in butyrate and secondary bile acid synthesis and enrichment of amino acid synthesis and degradation of aromatic compounds, encoded by members of the Enterobacteriaceae We developed microbial feature-based classifiers that can distinguish between patients with a normal pouch and pouchitis and identified species and genes that were predictive of pouchitis. We suggest species and functions that could be targeted for intervention to attenuate small intestinal inflammation present in pouchitis and CD.

Commensal fungi and their cell-wall ß-glucans direct differential responses in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that ß-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to ß-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and ß-glucans activated Syk and ERK in IEC lines. However, only ß-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with ß-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with ß-glucans alone. Finally, ex vivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than ß-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.

Early Indolent Course of Crohn's Disease in Newly Diagnosed Patients Is Not Rare and Possibly Predictable.

BACKGROUND & AIMS: The early stages of Crohn's disease (CD) course are heterogeneous, and it is a challenge to predict the course of disease in patients with new diagnosis. METHODS: We performed an observational longitudinal study of 156 adults (79 male; median age, 27.7 years; 57 treatment naïve) with newly diagnosed CD (within 6 months of enrollment), referred from medical centers and community clinics in Israel from 2013 through 2017. Study participants each received semi-annual scheduled evaluations. Indolent disease was defined as a disease course without need for strict interventions to control complicated course of CD (hospitalization or surgery, or decision to start steroid, immunomodulator, or biologic therapy). Cox regression and receiver operating characteristic analyses were used to identify factors associated with early indolent or complicated course of CD. We validated our findings in an independent cohort of patients with CD from a separate medical center in Israel in 2018. RESULTS: Over a median follow-up period of 17.2 months (interquartile range, 8.8-23.8 months), 52 patients (33.3%) had an indolent course of CD, 29 (18.5%) required hospitalizations, and 75 (48%) were recommended to start steroid, immunomodulator, or biologic therapies. The median time to first intervention was 3.4 months (95% CI, 2.4-4.4). We developed a model based on clinical factors that identified 4 factors associated with complicated course in treatment-naïve patients: body mass index <25 kg/m2 (hazard ratio [HR], 2.45; 95% CI, 1.07-5.43; P = .033), serum level of vitamin B12 <350 pg/mL (HR, 2.78; 95% CI, 1.21-6.41; P = .016), white blood cells ≥7 × 103/µL (HR, 2.419; 95% CI, 1.026-5.703; P = .044), and serum level of ALT ≥25 IU/L (HR, 2.680; 95% CI, 1.186-6.058; P = .018). This model discriminated between patients with vs without a complicated course of disease with 90% and 89% accuracy at 6 and 12 months after diagnosis, respectively. A validation cohort demonstrated a discriminatory ability of 79% at 3 months after diagnosis, and a nomogram was constructed. CONCLUSIONS: In an observational longitudinal study of 156 patients with newly diagnosed CD, we found that one third have an early indolent course of disease. We identified factors that can be measured at diagnosis to identify patients at risk for an early complicated course-these might be used in patient management and selection of treatment.

Predominantly Antibiotic-resistant Intestinal Microbiome Persists in Patients With Pouchitis Who Respond to Antibiotic Therapy.

BACKGROUND & AIMS: Pouchitis that develops in patients with ulcerative colitis after total proctocolectomy and ileal pouch anal anastomosis is usually treated with antibiotics. Some patients have recurrence of flares, or become antibiotic-dependent, and require repeated courses or prolonged periods of antibiotic therapy. We investigated microbial factors associated with response to antibiotic treatment and development of antibiotic dependence in patients with pouchitis. METHODS: We performed a prospective study of 49 patients who had undergone pouch surgery at a tertiary center. Disease activity was determined based on clinical, endoscopic, and histologic criteria. Pouch phenotype was defined as recurrent-acute pouchitis (n = 6), chronic pouchitis and Crohn's-like disease of the pouch (n = 27), normal pouch from patient with ulcerative colitis (n = 10), and normal pouch from patient with familial adenomatous polyposis (n = 6). Fecal samples (n = 234) were collected over time during or in the absence of antibiotic treatment (ciprofloxacin and/or metronidazole). Thirty-three patients were treated with antibiotics, for a median of 425 days of cumulative antibiotic therapy, during follow-up. Calprotectin was measured and fecal DNA was sequenced using shotgun metagenomics and analyzed with specifically designed bioinformatic pipelines. Bacterial strains were isolated from fecal samples. We assessed their ciprofloxacin resistance and ability to induce secretion of inflammatory cytokines by HT-29 intestinal epithelial cells. RESULTS: Most antibiotic-treated patients (79%) had a clinical response to each course of antibiotics; however, 89% of those who completed a 4-week course relapsed within 3 months. Median calprotectin levels decreased by 40% in response to antibiotics. Antibiotic treatment reduced disease-associated bacteria such as Clostridium perfringens, Ruminococcus gnavus, and Klebsiella pneumoniae, but also beneficial species, such as Faecalibacterium prausnitzii. The microbiomes of antibiotic-responsive patients were dominated by facultative anaerobic genera (Escherichia, Enterococcus, and Streptococcus), with multiple ciprofloxacin-resistance mutations in drug target genes and confirmed drug resistance. However, these strains had lower potential for virulence and did not induce secretion of inflammatory cytokines by epithelial cells. After antibiotic cessation, patients had an abrupt shift in microbiome composition, with blooms of oral and disease-associated bacteria. In addition, antibiotic treatment enriched for strains that acquired multidrug resistance loci, encoding enzymes that confer resistance to nonrelated antibiotics, including extended-spectrum beta-lactamases. CONCLUSIONS: The efficacy of antibiotic treatment of pouchitis might be attributed to the establishment of an antibiotic-resistant microbiome with low inflammatory potential. This microbiome might provide resistance against colonization by bacteria that promote inflammation. To avoid progression to antibiotic-dependent disease and its consequences, strategies such as short-term alternating antibiotics and nutrition- and microbiome-based interventions should be considered.

Gut microbiota density influences host physiology and is shaped by host and microbial factors.

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohn's disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Cell-centred meta-analysis reveals baseline predictors of anti-TNFα non-response in biopsy and blood of patients with IBD.

OBJECTIVE: Although anti-tumour necrosis factor alpha (anti-TNFα) therapies represent a major breakthrough in IBD therapy, their cost-benefit ratio is hampered by an overall 30% non-response rate, adverse side effects and high costs. Thus, finding predictive biomarkers of non-response prior to commencing anti-TNFα therapy is of high value. DESIGN: We analysed publicly available whole-genome expression profiles of colon biopsies obtained from multiple cohorts of patients with IBD using a combined computational deconvolution-meta-analysis paradigm which allows to estimate immune cell contribution to the measured expression and capture differential regulatory programmes otherwise masked due to variation in cellular composition. Insights from this in silico approach were experimentally validated in biopsies and blood samples of three independent test cohorts. RESULTS: We found the proportion of plasma cells as a robust pretreatment biomarker of non-response to therapy, which we validated in two independent cohorts of immune-stained colon biopsies, where a plasma cellular score from inflamed biopsies was predictive of non-response with an area under the curve (AUC) of 82%. Meta-analysis of the cell proportion-adjusted gene expression data suggested that an increase in inflammatory macrophages in anti-TNFα non-responding individuals is associated with the upregulation of the triggering receptor expressed on myeloid cells 1 (TREM-1) and chemokine receptor type 2 (CCR2)-chemokine ligand 7 (CCL7) -axes. Blood gene expression analysis of an independent cohort, identified TREM-1 downregulation in non-responders at baseline, which was predictive of response with an AUC of 94%. CONCLUSIONS: Our study proposes two clinically feasible assays, one in biopsy and one in blood, for predicting non-response to anti-TNFα therapy prior to initiation of treatment. Moreover, it suggests that mechanism-driven novel drugs for non-responders should be developed.

Genotype-Serotype Interactions Shed Light on Genetic Components of Inflammatory Bowel Diseases.

Background: We evaluated the impact of variations in ATG16L1 and NOD2 and genes on serologic responses in patients with inflammatory bowel disease (IBD). Methods: We recruited 308 IBD patients: 130 with Crohn's disease (CD), 67 with ulcerative colitis (UC), 111 with UC and an ileal pouch (UC-pouch), and 74 healthy controls. NOD2 variants (1007fs, G908R, R702W) and the ATG16L1 A300T variant were analyzed. The antiglycan antibodies anti-Saccharomyces cerevisiae (ASCA), antilaminaribioside (ALCA), antichitobioside (ACCA), and antimannobioside carbohydrate (AMCA) were analyzed by enzyme-linked immunosorbent assay. Results: Antichitobioside was positive in 28% of patients with CD carrying the ATG16L1 A300T variant (either heterozygote or homozygote) compared with only 3% in those without the variant (P < 0.001). Anti-Saccharomyces cerevisiae was positive in 86% of patients with CD carrying the NOD2 1007fs variant compared with 36% in those without the variant (P < 0.001). UC-pouch patients with the NOD2 1007fs variant had elevated ASCA and ALCA levels compared with those without the variant (50% vs 7%, P = 0.004, and 50% vs 8%, P = 0.006, respectively). Importantly, ATG16L1 A300T and NOD2 variants were not associated with serologic responses in healthy controls and unoperated UC patients. Multivariate analysis demonstrated that these genetic variants are the main factors associated with specific antiglycan antibody levels in CD and pouch patients. Conclusions: Genetic variants may have disease-specific phenotypic (serotypic) effects. This implies that genetic risk factors may also be disease modifiers.

Increase in Processing Factors Is Involved in Skewed MicroRNA Expression in Patients with Ulcerative Colitis Who Develop Small Intestine Inflammation after Pouch Surgery.

Background: A large-scale increase in microRNA (miRNA) expression was observed in patients with ulcerative colitis who underwent pouch surgery and developed inflammation of the pouch (pouchitis). In this study, we assessed miRNA expression in these patients and investigated how regulation of its expression changes in the setting of pouchitis. Methods: Autologous samples that included mucosal biopsies, peripheral blood cells, and plasma were collected from the patients. Candidate primary and mature miRNA expressions were analyzed by quantitative polymerase chain reaction. A human intestinal epithelial cell line was used to test DICER activity, and the expression of key miRNA processing factors was analyzed by Western blot. miRNA-424 and its potential target serotonin reuptake transporter (SERT) expressions were examined by quantitative reverse transcription polymerase chain reaction and Western blot in human pouch tissues and in a human intestinal epithelial cell line stimulated with inflammatory cytokines TNF-α, IL-1ß, and INF-γ. Results: Candidate miRNA expression and protein expression of DICER-1, EXPORTIN-5, and AGO-2 were increased in association with pouch inflammation. Similarly, inflammatory cytokines increased protein expression of DICER-1, EXPORTIN-5, and AGO-2 and DICER activity in the epithelial cell line. The miRNA-424 expression increased whereas SERT expression decreased in the patients' mucosa. Similarly, incubation of the epithelial cell line with inflammatory cytokines resulted in increased miRNA-424 and decreased SERT mRNA and protein expression. Conclusions: The miRNA expression and processing are augmented in the inflamed intestinal mucosa of patients with pouchitis. These alterations are accompanied by increased expression of proteins involved in miRNA processing, suggesting that pouch inflammation contributes to miRNA processing and expression.

Transforming growth factor ß signaling controls activities of human intestinal CD8(+)T suppressor cells.

BACKGROUND & AIMS: In healthy individuals, interactions between intestinal epithelial cells and lamina propria lymphocytes give rise to a population of CD8(+) T cells with suppressor functions (Ts cells). Disruption of Ts cell activities can lead to mucosal inflammation. We investigated what factors were required for expansion of the Ts cell population or loss of their activity in patients with Crohn's disease (CD). METHODS: We developed a method to generate Ts cell lines from freshly isolated lamina propria lymphocytes from patients with ulcerative colitis (UC), patients with CD, or healthy individuals (controls). Cells were stimulated with a monoclonal antibody against CD3, interleukin (IL)-7, and IL-15. After 14 days in culture, CD8(+)T cells were purified and cultured with IL-7 and IL-15. The resulting Ts cells were analyzed for suppressor activity, expression of surface markers, and cytokine secretion profiles. RNA was isolated from the 3 groups of Ts cells and used in microarray analyses. RESULTS: Ts cells from patients with UC and controls suppressed proliferation of CD4(+) T cells; the suppression required cell contact. In contrast, Ts cells from patients with CD had a reduced capacity to suppress CD4(+) T-cell proliferation. The difference in suppressive ability was not associated with surface or intracytoplasmic markers or secretion of cytokines. Microarray analysis identified changes in expression of genes regulated by transforming growth factor (TGF)-ß that were associated with the suppressive abilities of Ts cells. We found that TGF-ß or supernatants from Ts cells of patients with CD reduced the suppressor activity of control Ts cells. CONCLUSIONS: Ts cells isolated from patients with CD have a reduced ability to suppress proliferation of CD4(+)T cells compared with control Ts cells. TGF-ß signaling reduces the suppressor activity of Ts cells.