Microbiota-Based Therapeutics as New Standard-of-Care Treatment for Recurrent Clostridioides difficile Infection.

Background: Clostridioides difficile (C. difficile) is a spore-forming bacterial species that ubiquitously exists in the environment. Colonization by C. difficile is highly prevalent in infants, while fewer than 5% of adults are asymptomatic carriers. Disruption of the microbiome, such as through antibiotic treatment, triggers the germination of bacterial spores into numerous vegetative cells. These cells then produce enterotoxins that result in watery diarrhea and colonic inflammation. If left untreated, C. difficile infection (CDI) can lead to pseudomembranous colitis with the potentially life-threatening complication of toxic megacolon. Summary: Over the past few decades, the incidence, morbidity, and mortality associated with CDIs have increased. They have emerged as the primary cause of nosocomial gastrointestinal infections in industrialized countries, posing a significant burden on healthcare systems. Despite antibiotics often being the cause of CDIs, they remain the standard treatment. However, a considerable number of patients treated with antibiotics will experience recurrent CDI (rCDI). Microbiota-based therapies targeting the core issue of CDI - antibiotic-induced dysbiosis - hold promise for rCDI treatment. While data for probiotics are insufficient, numerous studies have highlighted the effectiveness of fecal microbiota transplantation (FMT) as a safe and viable therapeutic option for rCDI. This approach is now endorsed by multiple guidelines. Nonetheless, regulatory prerequisites, such as comprehensive stool donor screening, restrict the widespread adoption of FMT beyond specialized centers. Recently, the US Food and Drug Administration has approved two commercial microbiota-based therapeutics to prevent CDI recurrence. These therapeutics are available by prescription in the USA. RBX2660 (REBYOTA™) comprises a diverse consortium of live microbes derived from human stool and is administered via enema. On the other hand, SER-109 (VOWST™) is an orally administered spore-based medication. In this review, we discuss the potential of microbiota-based treatments for rCDI against the background of medico-legal challenges associated with classical FMT. Key Messages: FMT has emerged as a highly effective cure for rCDI. Nonetheless, regulatory prerequisites and laborious preparation procedures impede its widespread use. The establishment of ready-to-use microbiota-based therapeutics in clinical practice is necessary. In the USA, the recent approval of the first two commercial medications, including a spore-based oral preparation, marks a significant step forward.

Treatment Strategies in Inflammatory Bowel Diseases.

BACKGROUND: The prevalence of inflammatory bowel disease (IBD) is rising globally. In Germany, these conditions affect 0.7% of the population, or approximately 600 000 patients. Treatment strategies have become more diversified as a result of an improved understanding of disease pathogenesis. It remains unclear how the currently available drugs should best be used in each individual patient. METHODS: This review is based on pertinent publications retrieved by a selective search in PubMed, with special attention to phase III and IV trials and to the German and European guidelines on the treatment of IBD. RESULTS: An improved understanding of the immunological mechanisms of disease underlies the current treatment strategies in patients with IBD. For those with a complex clinical course, monoclonal antibodies against pro-inflammatory cytokines (TNF, IL-12/IL-23, IL-23) and cell adhesion molecules (α4ß7) are of established therapeutic value, along with "small molecules" such as JAK inhibitors and sphingosine-1-phosphate receptor modulators. The numerous studies that have been performed, only a few of which have been head-to-head comparison trials, and the (network) meta-analyses that have been published to date do not imply that any single one of these drugs can be considered the universal, primary treatment for all patients with IBD. In this review, we discuss the available substances and certain important differential-therapeutic aspects of the treatment of IBD. CONCLUSION: The treatment of a patient with IBD must take his or her prior treatment(s) and comorbidities into account, along with individual patient characteristics and treatment goals. Rational decision-making is required on the basis of the mechanism of action and the side-effect profile of the various drugs that are now available for use.

Immunomodulator comedication promotes the reversal of anti-drug antibody-mediated loss of response to anti-TNF therapy in inflammatory bowel disease.

PURPOSE: Loss of therapeutic response (LOR) due to anti-drug antibodies (ADA) against tumor necrosis factor (TNF) inhibitors is common in patients with inflammatory bowel disease (IBD). We aimed to investigate whether immunomodulator comedication can reverse the immunogenic LOR to TNF inhibitors in IBD. METHODS: In this real-world retrospective cohort study, 123 IBD patients with neutralizing ADA to infliximab or adalimumab and concomitant subtherapeutic trough levels were screened for clinical LOR. Subsequent ADA and trough level measurements and clinical outcomes were analyzed for patients who received either immunomodulator comedication or dose intensification of infliximab or adalimumab to overcome LOR. RESULTS: Following immunogenic LOR, the initial anti-TNF regimen was optimized in 33 patients. In univariable and multivariable logistic regression analyses, immunomodulator comedication was identified as the crucial factor for regaining clinical remission and ADA clearance. Detectable trough levels (≥ 0.98 or ≥ 1.00 mg/L, respectively) had optimal predictive performance for both endpoints in receiver operating characteristics curves [area under the curve 0.86 (95% confidence interval 0.68-1.00) for regaining clinical remission, 0.87 (0.71-1.00) for ADA clearance]. Furthermore, 11/20 patients (55%) on a comedication with azathioprine or methotrexate and 2/13 patients (15%) receiving anti-TNF dose intensification exclusively (P = 0.032) exhibited ADA elimination, regain of therapeutic trough levels, and clinical remission. Regain of clinical remission alone was achieved in 17/20 (85%) patients receiving comedication and 2/13 (15%) patients receiving anti-TNF dose intensification (P = 1.6 × 10-4). CONCLUSION: Immunogenic LOR to infliximab or adalimumab in IBD can be successfully reversed using immunomodulator comedication.

Detection of duodenal villous atrophy on endoscopic images using a deep learning algorithm.

BACKGROUND AND AIMS: Celiac disease with its endoscopic manifestation of villous atrophy (VA) is underdiagnosed worldwide. The application of artificial intelligence (AI) for the macroscopic detection of VA at routine EGD may improve diagnostic performance. METHODS: A dataset of 858 endoscopic images of 182 patients with VA and 846 images from 323 patients with normal duodenal mucosa was collected and used to train a ResNet18 deep learning model to detect VA. An external dataset was used to test the algorithm, in addition to 6 fellows and 4 board-certified gastroenterologists. Fellows could consult the AI algorithm's result during the test. From their consultation distribution, a stratification of test images into "easy" and "difficult" was performed and used for classified performance measurement. RESULTS: External validation of the AI algorithm yielded values of 90%, 76%, and 84% for sensitivity, specificity, and accuracy, respectively. Fellows scored corresponding values of 63%, 72%, and 67% and experts scored 72%, 69%, and 71%, respectively. AI consultation significantly improved all trainee performance statistics. Although fellows and experts showed significantly lower performance for difficult images, the performance of the AI algorithm was stable. CONCLUSIONS: In this study, an AI algorithm outperformed endoscopy fellows and experts in the detection of VA on endoscopic still images. AI decision support significantly improved the performance of nonexpert endoscopists. The stable performance on difficult images suggests a further positive add-on effect in challenging cases.

Disease recurrence in patients with Crohn's disease after biologic therapy or surgery: a meta-analysis.

BACKGROUND: Relapse is a problem in patients with Crohn's disease (CD) after medical therapy (including biologics) and after surgery to treat acute inflammation. It is unclear whether the recurrence rate over time is higher after surgical therapy than after continuous drug treatment. AIM: We sought to compare clinical relapse rates and the need for re-interventions (resection or therapeutic endoscopic intervention) in patients with CD. METHODS: A meta-analysis was performed according to PRISMA guidelines. RESULTS: The need for one of the three re-interventions (surgery, biologics or both) increased over time. The recurrence rates in patients after ileocecal resection were lower than the rates under biologic therapy. The odds ratio for clinical recurrence under biologics versus after surgical treatment was 2.50 (95% confidence interval [CI] 1.53-4.08, p-value < 0.001). The odds ratio for surgical recurrence under biologics versus after surgery was 3.60 (95% CI 1.06-12.3, p-value 0.041). CONCLUSION: These findings support surgical resection as a treatment option in patients with CD with limited disease.

[Fecal microbiota transplantation: indications, risks and opportunities]. / Fäkaler Mikrobiomtransfer ­ Indikationen, Risiken und Chancen.

Fecal microbiome transfer (FMT) involving the transfer of the microbiome of healthy stool donors to patients with various diseases has been performed in Germany in clinical studies and individual treatment attempts. There is no doubt that FMT is an effective therapeutic principle for recurrent Clostridium difficile infection and ulcerative colitis. From a medico-legal point of view, it should be stressed that, in Germany, the microbiome to be transferred is regarded as a drug, the manufacture of which is subject to the Medicines Act and the risk information from the Federal Institute for Drugs and Medical Devices. The background of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the potential risk of transmitting pathogens must also be considered. There is an obligation to notify the competent state authorities to perform FMTs in the context of individual treatment attempts. In the context of the limited availability and the fundamental problem of infection, future studies aim to identify the therapeutically active components in the microbiome. Recombinant production is the aim. Initial results represent preliminary steps, as these concepts are not yet established in clinical practice.

Intestinal Wnt in the transition from physiology to oncology.

Adult stem cells are necessary for self-renewal tissues and regeneration after damage. Especially in the intestine, which self-renews every few days, they play a key role in tissue homeostasis. Therefore, complex regulatory mechanisms are needed to prevent hyperproliferation, which can lead in the worst case to carcinogenesis or under-activation of stem cells, which can result in dysfunctional epithelial. One main regulatory signaling pathway is the Wnt/ß-catenin signaling pathway. It is a highly conserved pathway, with ß-catenin, a transcription factor, as target protein. Translocation of ß-catenin from cytoplasm to nucleus activates the transcription of numerous genes involved in regulating stem cell pluripo-tency, proliferation, cell differentiation and regulation of cell death. This review presents a brief overview of the Wnt/ß-catenin signaling pathway, the regulatory mechanism of this pathway and its role in intestinal homeostasis. Additionally, this review highlights the molecular mechanisms and the histomorphological features of Wnt hyperactivation. Furthermore, the central role of the Wnt signaling pathway in intestinal carcinogenesis as well as its clinical relevance in colorectal carcinoma are discussed.

Transfer of FRozen Encapsulated multi-donor Stool filtrate for active ulcerative Colitis (FRESCO): study protocol for a prospective, multicenter, double-blind, randomized, controlled trial.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease with significant morbidity and mortality. Although the precise cause remains unknown, disturbances in the intestinal microbial community have been linked to its pathogenesis. Randomized controlled trials in UC and relapsing Clostridioides difficile infection (CDI) have established fecal microbiota (FM) transfer (FMT) as an effective therapy. In this context, preliminary results indicated that the transfer of sterile fecal microbiota filtrates (<0.2 µm; FMF, FMFT) of donor stool also drives gastrointestinal microbiota changes and eliminates symptoms in CDI patients. However, along with the success of FMT, regulatory agencies issued safety alerts following reports of serious adverse events due to transmission of enteric pathogens through FMT. To reduce this risk, we established an extensive test protocol for our donors and quarantine regulations for the produced capsules, but alternative concepts are desirable. METHODS: Our project is a randomized, controlled, longitudinal, prospective, three-arm, multicenter, double-blind study to determine the safety and efficacy of repeated long-term, multi-donor FM or FMF transfers compared to placebo using oral, frozen capsules in 174 randomized patients with mild to moderate active UC. The primary outcome will be clinical remission at week 12. DISCUSSION: This proposal aims to examine (a) the efficacy of encapsulated transfer of FM and FMF as a therapy for mild to moderate UC, (b) the short- and long-term safety of FMT and FMFT in patients with UC, and (c) the microbial and immunologic changes that occur after FMT and FMFT to help understand how and why it affects inflammatory bowel disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT03843385 . DRKS (Deutsches Register für Klinische Studien) DRKS00020471.

Iron Deficiency in Inflammatory Bowel Disease Is Associated With Low Levels of Vitamin D Modulating Serum Hepcidin and Intestinal Ceruloplasmin Expression.

INTRODUCTION: Iron deficiency and vitamin D deficiency are common comorbidities in inflammatory bowel disease (IBD). Accumulating evidence indicates that active 1,25-dihydroxyvitamin D (1,25(OH)D) may enhance iron absorption by suppressing hepcidin. We investigated the influence of vitamin D on iron metabolism in patients with IBD and on the expression of genes facilitating intestinal epithelial iron absorption. METHODS: Iron parameters and serum levels of 25-hydroxyvitamin D (25(OH)D), 1,25(OH)D, and hepcidin were measured in 104 adult patients with IBD (67 with Crohn's disease and 37 with ulcerative colitis). Genes involved in iron absorption were tested for induction by 1,25(OH)D in Caco-2 cells, which resemble the small intestinal epithelium. RESULTS: In multiple regression models controlling for age, sex, body mass index, smoking status, disease activity, and C-reactive protein levels, low 25(OH)D levels were associated with iron deficiency in patients with IBD (ß [SE] = -0.064 [0.030], P = 0.029). Vitamin D sufficiency was associated with increased levels of ferritin (ß [SE] = 0.25 [0.11], P = 0.024) and transferrin saturation (ß [SE] = 8.41 [4.07], P = 0.044). Higher 1,25(OH)D:25(OH)D ratios were associated with lower hepcidin levels (ß [SE] = -4.31 [1.67], P = 0.012). Especially in Crohn's disease, increased 1,25(OH)D correlated with higher transferrin saturation (ß [SE] = 0.43 [0.18], P = 0.027). Furthermore, 1,25(OH)D strongly induced the expression of the ferroxidase ceruloplasmin in Caco-2 cells. DISCUSSION: Low vitamin D levels in IBD correlate with iron deficiency. Vitamin D may ameliorate iron deficiency, potentially by downregulating hepcidin and upregulating ceruloplasmin, enhancing intestinal iron absorption.

Head-to-head study of oxelumab and adalimumab in a mouse model of ulcerative colitis based on NOD/Scid IL2Rγnull mice reconstituted with human peripheral blood mononuclear cells.

This study's aim was to demonstrate that the combination of patient immune profiling and testing in a humanized mouse model of ulcerative colitis (UC) might lead to patient stratification for treatment with oxelumab. First, immunological profiles of UC patients and non-UC donors were analyzed for CD4+ T cells expressing OX40 (CD134; also known as TNFRSF4) and CD14+ monocytes expressing OX40L (CD252; also known as TNFSF4) by flow cytometric analysis. A significant difference was observed between the groups for CD14+ OX40L+ (UC: n=11, 85.44±21.17, mean±s.d.; non-UC: n=5, 30.7±34.92; P=0.02), whereas no significant difference was detected for CD4+ OX40+. CD14+ OX40L+ monocytes were correlated significantly with T helper 1 and 2 cells. Second, NOD/Scid IL2Rγ null mice were reconstituted with peripheral blood mononuclear cells from UC donors exhibiting elevated levels of OX40L, and the efficacy of oxelumab was compared with that of adalimumab. The clinical, colon and histological scores and the serum concentrations of IL-6, IL-1ß and glutamic acid were assessed. Treatment with oxelumab or adalimumab resulted in significantly reduced clinical, colon and histological scores, reduced serum concentrations of IL-6 and reduced frequencies of splenic human effector memory T cells and switched B cells. Comparison of the efficacy of adalimumab and oxelumab by orthogonal partial least squares discrimination analysis revealed that oxelumab was slightly superior to adalimumab; however, elevated serum concentrations of glutamic acid suggested ongoing inflammation. These results suggest that oxelumab addresses the pro-inflammatory arm of inflammation while promoting the remodeling arm and that patients exhibiting elevated levels of OX40L might benefit from treatment with oxelumab.

Development of a uniform, very aggressive disease phenotype in all homozygous carriers of the NOD2 mutation p.Leu1007fsX1008 with Crohn's disease and active smoking status resulting in ileal stenosis requiring surgery.

BACKGROUND: NOD2 variants are the strongest genetic predictors for susceptibility to Crohn's disease (CD). However, the clinical value of NOD2 on an individual patient level remains controversial. We aimed to define the predictive power of the major NOD2 mutations regarding complicated CD in a large single center cohort. METHODS: 1076 CD patients were prospectively genotyped for the three common CD-associated NOD2 mutations rs2066844, rs2066845, and rs2066847, followed by detailed genotype-phenotype analyses. RESULTS: Overall, 434 CD patients (40.3%) carried at least one of the three main NOD2 mutations. A significantly higher minor allele frequency (15.6%) of the NOD2 frameshift mutation p.Leu1007fsX1008 (rs2066847) was seen in patients with aggressive disease compared to 8.2% in patients with mild disease (p = 2.6 x 10-5). Moreover, a total of 54 CD patients (5.0%) were homozygous for this NOD2 frameshift mutation. 100% of these patients had ileal disease compared to 82% of NOD2 wild-type carriers (p<0.0001). In homozygous carriers of the NOD2 frameshift mutation, 87% presented with ileal stenosis, 68.5% had fistulas, and 72.2% required CD-related surgery despite immunosuppressive therapy in 87% of these patients. All homozygous carriers of the 1007fs mutation who were active smokers had ileal stenosis and required CD-related surgery. CONCLUSION: Homozygosity for Leu1007fsX1008 is an excellent biomarker for predicting complicated CD on an individual patient level. Active smoking and homozygosity for this mutation is associated with a 100% risk for developing ileal stenosis requiring CD-related surgery. In these patients, smoking cessation and early initiation of immunosuppressive strategies may be beneficial.

The Combination of Patient Profiling and Preclinical Studies in a Mouse Model Based on NOD/Scid IL2Rγ null Mice Reconstituted With Peripheral Blood Mononuclear Cells From Patients With Ulcerative Colitis May Lead to Stratification of Patients for Treatment With Adalimumab.

BACKGROUND: To date, responsiveness to tumor necrosis factor alpha inhibitors in ulcerative colitis (UC) patients is not predictable. This is partially due to a lack of understanding of the underlying inflammatory processes. The aim of this study was to identify immunological subgroups of patients with UC and to test responsiveness to adalimumab in these subgroups in the mouse model of ulcerative colitis (UC), which is based on NOD/scid IL-2Rγ null (NSG) mice reconstituted with peripheral blood mononuclear cells (PBMCs; NSG-UC). METHODS: The immunological profiles of 40 UC patients and 16 non-UC donors were determined by flow cytometric analysis of PBMCs in a snapshot and longitudinal study and analyzed by principal component, orthogonal partial least square discrimination (oPLS-DA), and hierarchical clustering analysis. NSG mice were reconstituted 5 times at consecutive time points with PBMCs from a single donor and were analyzed for frequencies of human leukocytes and histological phenotype. The response to adalimumab of 2 identified subgroups was tested in the NSG-UC model. We used the clinical, colon, and histological score, serum levels of glutamic and aspartic acid, and IL-6 and IL-1ß. Response was analyzed by oPLS-DA. RESULTS: Analysis revealed a distinction between UC and non-UC donors. Hierarchical clustering identified 2 major subgroups in UC patients. Group I was characterized by TH17 and M1 monocytes, group II by TH2/TH1, and switched B cells. These subgroups reflect the dynamics of inflammation as patients. NSG-UC mice achieved an immunological phenotype reflecting the patient's immunological phenotype. oPLS-DA revealed that NSG-UC mice reconstituted with PBMCs from group II responded better to adalimumab. CONCLUSIONS: The combination of profiling and testing of therapeutics in the NSG-UC model may lead to individualized and phase-dependent therapies.

Lipocalin-2 Is a Disease Activity Marker in Inflammatory Bowel Disease Regulated by IL-17A, IL-22, and TNF-α and Modulated by IL23R Genotype Status.

BACKGROUND: Lipocalin-2 (LCN2) is a potent bacteriostatic protein. We aimed to investigate its role as a disease activity marker in patients with inflammatory bowel disease (IBD) and its induction by the Th17 cytokines IL-17A, IL-22, and TNF-α in colonic epithelial cells. Moreover, we analyzed the influence of IBD-associated IL23R alleles on LCN2 serum levels in IBD patients. METHODS: LCN2 serum levels were determined in 131 IBD patients (71 with Crohn's disease [CD], 60 with ulcerative colitis [UC]) and 63 healthy controls. IBD patients were genotyped for 10 IBD-associated IL23R polymorphisms. LCN2 expression after stimulation with IL-17A, IL-22, and TNF-α was measured in human colonic epithelial cell lines. RESULTS: A significant upregulation of serum LCN2 in active IBD (median [IQR], 36.84 [21.17-73.74] ng/mL; P = 0.01) compared with healthy controls (24.22 [17.76-35.25] ng/mL) was confined to active UC (42.21 [28.97-73.74] ng/mL; P = 0.0006). LCN2 proved to be a marker of UC disease activity (area under the curve 0.75, sensitivity 0.83, specificity 0.63; P = 0.0002). IL-17A showed a synergistic effect with IL-22 and TNF-α in inducing colonic epithelial expression of LCN2 and its essential transcription factor IKBZ. In CD, LCN2 concentrations were significantly modulated by IL23R genotype status with homozygous carriers of IBD risk-increasing alleles showing particularly low LCN2 levels. CONCLUSIONS: Serum LCN2 proves to be a biomarker of active UC. Lower LCN2 levels in CD patients carrying IBD risk-increasing IL23R variants may result from a restricted upregulation of LCN2 due to an impaired Th17 immune response.

Solid Organ Transplantation in Patients with Inflammatory Bowel Diseases (IBD): Analysis of Transplantation Outcome and IBD Activity in a Large Single Center Cohort.

BACKGROUND: Currently, limited data of the outcome of inflammatory bowel disease (IBD) in patients after solid organ transplantation (SOT) are available. We aimed to analyze effects of SOT on the IBD course in a large IBD patient cohort. METHODS: Clinical data from 1537 IBD patients were analyzed for patients who underwent SOT (n = 31) between July 2002 and May 2014. Sub-analyses included SOT outcome parameters, IBD activity before and after SOT, and efficacy of IBD treatment. RESULTS: 4.74% of patients with ulcerative colitis (UC) and 0.84% of patients with Crohn's disease (CD) underwent SOT (p = 2.69 x 10(-6), UC vs. CD). 77.4% of patients with SOT underwent liver transplantation (LTx) with tacrolimus-based immunosuppressive therapy after SOT. All LTx were due to primary sclerosing cholangitis (PSC) or PSC overlap syndromes. Six patients (19.4%) required renal transplantation and one patient (3.2%) heart transplantation. A survival rate of 83.9% after a median follow-up period of 103 months was observed. Before SOT, 65.0% of patients were in clinical remission and 5 patients received immunosuppressive therapy (16.1%). After SOT, 61.0% of patients were in remission (p = 1.00 vs. before SOT) and 29.0% required IBD-specific immunosuppressive or anti-TNF therapy (p = 0.54 vs. before SOT). 42.9% of patients with worsening of IBD after SOT were at higher risk of needing steroid therapy for increased IBD activity (p = 0.03; relative risk (RR): 10.29; 95% CI 1.26-84.06). Four patients (13.0%) needed anti-TNF therapy after SOT (response rate 75%). CONCLUSIONS: SOT was more common in UC patients due to the higher prevalence of PSC-related liver cirrhosis in UC. Despite mainly tacrolimus-based immunosuppressive regimens, outcome of SOT and IBD was excellent in this cohort. In this SOT cohort, concomitant immunosuppressive therapy due to IBD was well tolerated.

The NOD2 Single Nucleotide Polymorphism rs72796353 (IVS4+10 A>C) Is a Predictor for Perianal Fistulas in Patients with Crohn's Disease in the Absence of Other NOD2 Mutations.

BACKGROUND: A previous study suggested an association of the single nucleotide polymorphism (SNP) rs72796353 (IVS4+10 A>C) in the NOD2 gene with susceptibility to Crohn's disease (CD). However, this finding has not been confirmed. Given that NOD2 variants still represent the most important predictors for CD susceptibility and phenotype, we evaluated the association of rs72796353 with inflammatory bowel disease (IBD) susceptibility and the IBD phenotype. METHODOLOGY: Genomic DNA from 2256 Caucasians, including 1073 CD patients, 464 patients with ulcerative colitis (UC), and 719 healthy controls, was genotyped for the NOD2 SNP rs72796353 and the three main CD-associated NOD2 mutations rs2066844, rs2066845, and rs2066847. Subsequently, IBD association and genotype-phenotype analyses were conducted. RESULTS: In contrast to the strong associations of the NOD2 SNPs rs2066844 (p=3.51 x 10(-3)), rs2066845 (p=1.54 x 10(-2)), and rs2066847 (p=1.61 x 10(-20)) with CD susceptibility, no significant association of rs72796353 with CD or UC susceptibility was found. However, in CD patients without the three main CD-associated NOD2 mutations, rs72796353 was significantly associated with the development of perianal fistulas (p=2.78 x 10(-7), OR 5.27, [95% CI 2.75-10.12] vs. NOD2 wild-type carriers). CONCLUSION/SIGNIFICANCE: Currently, this study represents the largest genotype-phenotype analysis of the impact of the NOD2 variant rs72796353 on the disease phenotype in IBD. Our data demonstrate that in CD patients the IVS4+10 A>C variant is strongly associated with the development of perianal fistulas. This association is particularly pronounced in patients who are not carriers of the three main CD-associated NOD2 mutations, suggesting rs72796353 as additional genetic marker for the CD disease behaviour.

The NOD2 p.Leu1007fsX1008 mutation (rs2066847) is a stronger predictor of the clinical course of Crohn's disease than the FOXO3A intron variant rs12212067.

BACKGROUND: Very recently, a sub-analysis of genome-wide association scans revealed that the non-coding single nucleotide polymorphism (SNP) rs12212067 in the FOXO3A gene is associated with a milder course of Crohn's disease (CD) (Cell 2013;155:57-69). The aim of our study was to evaluate the clinical value of the SNP rs12212067 in predicting the severity of CD by correlating CD patient genotype status with the most relevant complications of CD such as stenoses, fistulas, and CD-related surgery. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped 550 CD patients for rs12212067 (FOXO3A) and the three common CD-associated NOD2 mutations rs2066844, rs2066847, and rs2066847 and performed genotype-phenotype analyses. RESULTS: No significant phenotypic differences were found between the wild-type genotype TT of the FOXO3A SNP rs12212067 and the minor genotypes TG and GG independently from NOD2 variants. The allele frequency of the minor G allele was 12.7%. Age at diagnosis, disease duration, body mass index, surgery rate, stenoses, fistula, need for immunosuppressive therapy, and disease course were not significantly different. In contrast, the NOD2 mutant p.Leu1007fsX1008 (rs2066847) was highly associated with penetrating CD (p = 0.01), the development of fistulas (p = 0.01) and stenoses (p = 0.01), and ileal disease localization (p = 0.03). Importantly, the NOD2 SNP rs2066847 was a strong separator between an aggressive and a mild course of CD (p = 2.99×10(-5)), while the FOXO3A SNP rs12212067 did not separate between mild and aggressive CD behavior in our cohort (p = 0.35). 96.2% of the homozygous NOD2 p.Leu1007fsX1008 carriers had an aggressive disease behavior compared to 69.3% of the patients with the NOD2 wild-type genotype (p = 0.007). CONCLUSION/SIGNIFICANCE: In clinical practice, the NOD2 variant p.Leu1007fsX1008 (rs2066847), in particular in homozygous form, is a much stronger marker for a severe clinical phenotype than the FOXO3A rs12212067 SNP for a mild disease course on an individual patient level despite its important impact on the inflammatory response of monocytes.

Anti-TNF antibody-induced psoriasiform skin lesions in patients with inflammatory bowel disease are characterised by interferon-γ-expressing Th1 cells and IL-17A/IL-22-expressing Th17 cells and respond to anti-IL-12/IL-23 antibody treatment.

BACKGROUND: We analysed incidence, predictors, histological features and specific treatment options of anti-tumour necrosis factor α (TNF-α) antibody-induced psoriasiform skin lesions in patients with inflammatory bowel diseases (IBD). DESIGN: Patients with IBD were prospectively screened for anti-TNF-induced psoriasiform skin lesions. Patients were genotyped for IL23R and IL12B variants. Skin lesions were examined for infiltrating Th1 and Th17 cells. Patients with severe lesions were treated with the anti-interleukin (IL)-12/IL-23 p40 antibody ustekinumab. RESULTS: Among 434 anti-TNF-treated patients with IBD, 21 (4.8%) developed psoriasiform skin lesions. Multiple logistic regression revealed smoking (p=0.007; OR 4.24, 95% CI 1.55 to 13.60) and an increased body mass index (p=0.029; OR 1.12, 95% CI 1.01 to 1.24) as main predictors for these lesions. Nine patients with Crohn's disease and with severe psoriasiform lesions and/or anti-TNF antibody-induced alopecia were successfully treated with the anti-p40-IL-12/IL-23 antibody ustekinumab (response rate 100%). Skin lesions were histologically characterised by infiltrates of IL-17A/IL-22-secreting T helper 17 (Th17) cells and interferon (IFN)-γ-secreting Th1 cells and IFN-α-expressing cells. IL-17A expression was significantly stronger in patients requiring ustekinumab than in patients responding to topical therapy (p=0.001). IL23R genotyping suggests disease-modifying effects of rs11209026 (p.Arg381Gln) and rs7530511 (p.Leu310Pro) in patients requiring ustekinumab. CONCLUSIONS: New onset psoriasiform skin lesions develop in nearly 5% of anti-TNF-treated patients with IBD. We identified smoking as a main risk factor for developing these lesions. Anti-TNF-induced psoriasiform skin lesions are characterised by Th17 and Th1 cell infiltrates. The number of IL-17A-expressing T cells correlates with the severity of skin lesions. Anti-IL-12/IL-23 antibody therapy is a highly effective therapy for these lesions.

IRGM variants and susceptibility to inflammatory bowel disease in the German population.

BACKGROUND & AIMS: Genome-wide association studies identified the autophagy gene IRGM to be strongly associated with Crohn's disease (CD) but its impact in ulcerative colitis (UC), its phenotypic effects and potential epistatic interactions with other IBD susceptibility genes are less clear which we therefore analyzed in this study. METHODOLOGY/PRINCIPAL FINDINGS: Genomic DNA from 2060 individuals including 817 CD patients, 283 UC patients, and 961 healthy, unrelated controls (all of Caucasian origin) was analyzed for six IRGM single nucleotide polymorphisms (SNPs) (rs13371189, rs10065172 = p.Leu105Leu, rs4958847, rs1000113, rs11747270, rs931058). In all patients, a detailed genotype-phenotype analysis and testing for epistasis with the three major CD susceptibility genes NOD2, IL23R and ATG16L1 were performed. Our analysis revealed an association of the IRGM SNPs rs13371189 (p = 0.02, OR 1.31 [95% CI 1.05-1.65]), rs10065172 = p.Leu105Leu (p = 0.016, OR 1.33 [95% CI 1.06-1.66]) and rs1000113 (p = 0.047, OR 1.27 [95% CI 1.01-1.61]) with CD susceptibility. There was linkage disequilibrium between these three IRGM SNPs. In UC, several IRGM haplotypes were weakly associated with UC susceptibility (p<0.05). Genotype-phenotype analysis revealed no significant associations with a specific IBD phenotype or ileal CD involvement. There was evidence for weak gene-gene-interaction between several SNPs of the autophagy genes IRGM and ATG16L1 (p<0.05), which, however, did not remain significant after Bonferroni correction. CONCLUSIONS/SIGNIFICANCE: Our results confirm IRGM as susceptibility gene for CD in the German population, supporting a role for the autophagy genes IRGM and ATG16L1 in the pathogenesis of CD.

Analysis of IL12B gene variants in inflammatory bowel disease.

BACKGROUND: IL12B encodes the p40 subunit of IL-12, which is also part of IL-23. Recent genome-wide association studies identified IL12B and IL23R as susceptibility genes for inflammatory bowel disease (IBD). However, the phenotypic effects and potential gene-gene interactions of IL12B variants are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed IL12B gene variants regarding association with Crohn's disease (CD) and ulcerative colitis (UC). Genomic DNA from 2196 individuals including 913 CD patients, 318 UC patients and 965 healthy, unrelated controls was analyzed for four SNPs in the IL12B gene region (rs3212227, rs17860508, rs10045431, rs6887695). Our analysis revealed an association of the IL12B SNP rs6887695 with susceptibility to IBD (p = 0.035; OR 1.15 [95% CI 1.01-1.31] including a trend for rs6887695 for association with CD (OR 1.41; [0.99-1.31], p = 0.066) and UC (OR 1.18 [0.97-1.43], p = 0.092). CD patients, who were homozygous C/C carriers of this SNP, had significantly more often non-stricturing, non-penetrating disease than carriers of the G allele (p = 6.8×10(-5); OR = 2.84, 95% CI 1.66-4.84), while C/C homozygous UC patients had less often extensive colitis than G allele carriers (p = 0.029; OR = 0.36, 95% CI 0.14-0.92). In silico analysis predicted stronger binding of the minor C allele of rs6887695 to the transcription factor RORα which is involved in Th17 differentiation. Differences regarding the binding to the major and minor allele sequence of rs6887695 were also predicted for the transcription factors HSF1, HSF2, MZF1 and Oct-1. Epistasis analysis revealed weak epistasis of the IL12B SNP rs6887695 with several SNPs (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694) in the STAT4 gene which encodes the major IL-12 downstream transcription factor STAT4 (p<0.05) but there was no epistasis between IL23R and IL12B variants. CONCLUSIONS/SIGNIFICANCE: The IL12B SNP rs6887695 modulates the susceptibility and the phenotype of IBD, although the effect on IBD susceptibilty is less pronounced than that of IL23R gene variants.