Double-negative B cells and DNASE1L3 colocalise with microbiota in gut-associated lymphoid tissue.

Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.

How achievable are STRIDE-II treatment targets in real-world practice and do they predict long-term treatment outcomes?

Objective: The second iteration of the Selecting Therapeutic Targets in Inflammatory Bowel Disease (STRIDE-II) initiative recommends use of the Simple Endoscopic Score for Crohn's disease (SES-CD) as a treatment target for patients with CD. We aimed to assess whether the STRIDE-II endoscopic endpoints are achievable and whether the degree of mucosal healing (MH) affects long-term outcomes. Design/method: We performed a retrospective observational study between 2015 and 2022. Patients with CD who had baseline and follow-up SES-CD scores after biological therapy initiation were included. The primary outcome was treatment failure, defined as the need for: (1) change of biological therapy for active disease (2) corticosteroid use (3) CD-related hospitalisation or (4) surgery. We compared rates of treatment failure with the degree of MH achieved. Patients were followed up until treatment failure or study end (August 2022). Results: 50 patients were included and followed up for median 39.9 (34.6-48.6) months. Baseline characteristics: 62% male, median age 36.4 (27.8-43.9) years, disease distribution (L1: 4, L2: 11, L3: 35, perianal: 18). The proportion of patients achieving STRIDE-II end-points were: SES-CD≤2-25 (50%) and >50% reduction in SES-CD-35 (70%). Failure to achieve SES-CD≤2 (HR 11.62; 95% CI 3.33 to 40.56, p=0.003) or >50% improvement in SES-CD (HR 30.30; 95% CI 6.93 to 132.40, p<0.0001) predicted treatment failure. Conclusion: Use of SES-CD is feasible in real-world clinical practice. Achieving an SES-CD≤2 or a greater than 50% reduction, as set out by STRIDE-II, is associated with reduced rates of overall treatment failure including CD-related surgery.

Multi-Photon Microscopy.

In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last review covers multi-photon microscopy with a brief reference to intravital imaging and Brainbow labeling. Multi-photon microscopy is often referred to as two-photon microscopy. Indeed, using two-photon microscopy is by far the most common way of imaging thick tissues; however, it is theoretically possible to use a higher number of photons, and three-photon microscopy is possible. Therefore, this review is titled "multi-photon microscopy." Another term for describing multi-photon microscopy is "non-linear" microscopy because fluorescence intensity at the focal spot depends upon the average squared intensity rather than the squared average intensity; hence, non-linear optics (NLO) is an alternative name for multi-photon microscopy. It is this non-linear relationship (or third exponential power in the case of three-photon excitation) that determines the axial optical sectioning capability of multi-photon imaging. In this paper, the necessity for two-photon or multi-photon imaging is explained, and the method of optical sectioning by multi-photon microscopy is described. Advice is also given on what fluorescent markers to use and other practical aspects of imaging thick tissues. The technique of Brainbow imaging is discussed. The review concludes with a description of intravital imaging of the mouse. © 2023 Wiley Periodicals LLC.

Lightsheet Microscopy.

In this paper, we review lightsheet (selective plane illumination) microscopy for mouse developmental biologists. There are different means of forming the illumination sheet, and we discuss these. We explain how we introduced the lightsheet microscope economically into our core facility and present our results on fixed and living samples. We also describe methods of clearing fixed samples for three-dimensional imaging and discuss the various means of preparing samples with particular reference to mouse cilia, adipose spheroids, and cochleae. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

A retrospective cohort study: pre-operative oral enteral nutritional optimisation for Crohn's disease in a UK tertiary IBD centre.

BACKGROUND: Low-quality evidence suggests that pre-operative exclusive enteral nutrition (E/EN) can improve postoperative outcomes in patients with Crohn's disease (CD). It is not standard practice in most centres. AIMS: To test the hypothesis that pre-operative EN in patients undergoing ileal/ileocolonic surgery for CD is associated with improved postoperative outcome. METHODS: We performed a single centre retrospective observational study comparing surgical outcomes in patients receiving pre-operative EN (≥600 kcal/day for ≥2 weeks) with those who received no nutritional optimisation. Consecutive adult patients undergoing ileal/ileocolonic resection from 2008 to 2020 were included. The primary outcome was postoperative complications <30 days. Secondary outcomes included EN tolerance, specific surgical complications, unplanned stoma formation, length of stay, length of bowel resected, readmission and biochemical/anthropometric changes. RESULTS: 300 surgeries were included comprising 96 without nutritional optimisation and 204 optimised cases: oral EN n = 173, additional PN n = 31 (4 of whom had received nasogastric/nasojejunal EN). 142/204 (69.6%) tolerated EN. 125/204 (61.3%) initiated EN in clinic. Patients in the optimised cohort were younger at operation and diagnosis, with an increased frequency of penetrating disease and exposure to antibiotics or biologics, and were more likely to undergo laparoscopic surgery. The optimised cohort had favourable outcomes on multivariate analysis: all complications [OR 0.29; 0.15-0.57, p < 0.001], surgical complications [OR 0.41; 95% CI 0.20-0.87, p = 0.02], non-surgical complications [OR 0.24 95% CI 0.11-0.52, p < 0.001], infective complications [OR 0.32; 95% CI 0.16-0.66, p = 0.001]. CONCLUSIONS: Oral EN was reasonably well tolerated and associated with a reduction in 30-day postoperative complications. Randomised controlled trials are required to confirm these findings.

Relationship Between Thiopurine S-Methyltransferase Genotype/Phenotype and 6-Thioguanine Nucleotide Levels in 316 Patients With Inflammatory Bowel Disease on 6-Thioguanine.

BACKGROUND: In inflammatory bowel disease (IBD), conventional thiopurine users cease treatment in 60% of cases within 5 years, mostly because of adverse events or nonresponse. In this study, the authors aimed to investigate the role of 6-thioguanine nucleotide (TGN) measurements, geno/phenotyping of thiopurine S-methyltransferase (TPMT), and their mutual relationship with TG therapy in IBD. METHODS: An international retrospective, multicenter cohort study was performed at 4 centers in the Netherlands (Máxima Medical Centre) and the United Kingdom (Guy's and St. Thomas' Hospital, Queen Elizabeth Hospital, and East Surrey Hospital). RESULTS: Overall, 526 6-TGN measurements were performed in 316 patients with IBD. The median daily dosage of TG was 20 mg/d (range 10-40 mg/d), and the median duration of TG use was 21.1 months (SD, 28.0). In total, 129 patients (40.8%) had a known TPMT status. In the variant-type and wild-type TPMT genotype metabolism groups, median 6-TGN values were 1126 [interquartile range (IQR) 948-1562] and 467.5 pmol/8 × 10E8 red blood cells (RBCs) (IQR 334-593). A significant difference was observed between the 2 groups (P = 0.0001, t test). For TPMT phenotypes, in the slow, fast, and normal metabolism groups, the median 6-TGN values were 772.0 (IQR 459-1724), 296.0 (IQR 200-705), and 774.5 pmol/8 × 10E8 RBCs (IQR 500.5-981.5), with a significant difference observed between groups (P < 0.001, analysis of variance). CONCLUSIONS: Our findings indicated that TPMT measurements at TG initiation can be useful but are not necessary for daily practice. TPMT genotypes and phenotypes are both associated with significant differences in 6-TGN levels between metabolic groups. However, the advantage of TG remains that RBC 6-TGN measurements are not crucial to monitor treatments in patients with IBD because these measurements did not correlate with laboratory result abnormalities. This presents as a major advantage in countries where patients cannot access these diagnostic tests.

Super-Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology.

Super-resolution (diffraction unlimited) microscopy was developed 15 years ago; the developers were awarded the Nobel Prize in Chemistry in recognition of their work in 2014. Super-resolution microscopy is increasingly being applied to diverse scientific fields, from single molecules to cell organelles, viruses, bacteria, plants, and animals, especially the mammalian model organism Mus musculus. In this review, we explain how super-resolution microscopy, along with fluorescence microscopy from which it grew, has aided the renaissance of the light microscope. We cover experiment planning and specimen preparation and explain structured illumination microscopy, super-resolution radial fluctuations, stimulated emission depletion microscopy, single-molecule localization microscopy, and super-resolution imaging by pixel reassignment. The final section of this review discusses the strengths and weaknesses of each super-resolution technique and how to choose the best approach for your research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

An observational study of switching infliximab biosimilar: no adverse impact on inflammatory bowel disease control or drug levels with first or second switch.

BACKGROUND: Biologics account for a significant cost in inflammatory bowel disease (IBD) management; however, switching from infliximab originator to its biosimilars has enabled cost saving without compromising disease control. The effects on IBD activity and infliximab trough levels of a second switch to another biosimilar are, however, uncertain. AIMS: To assess the effects on disease activity and infliximab trough levels associated with switching from infliximab biosimilar CT-P13 to another biosimilar SB2 and compare outcomes in those switching for the first and second time. METHODS: IBD patients on CT-P13, including some previously switched from originator, were prospectively followed during a switch to SB2. C-reactive protein (CRP), trough infliximab level and clinical disease activity indices were collected at baseline, Infusion 3 or 4 ('early' after switch), and 1 year. RESULTS: One hundred eighty-six patients (n = 99 second switch) on stable infliximab dosing underwent switching. Compared with baseline, there was no significant change in CRP, clinical disease activity scores or median trough infliximab level at the early time point among first-switch (baseline vs early: 5.7 vs 6.6 µg/mL, P = 0.05) and second-switch (4.3 vs 4.9 µg/mL, P = 0.07) patients nor at 1 year (median infliximab trough levels, baseline vs 1 year, in first-switch [5.7 vs 5.7 µg/mL, P = 0.37] and second-switch [4.3 vs 4.7 µg/mL, P = 0.06] patients). The proportion of patients in clinical remission did not significantly change at the early (92% vs 91% at baseline, P = 0.75) or 1 year (95% vs 91% at baseline, P = 0.16) time points. There was no significant difference in time to loss of response between patients switching for the first or second time (P = 0.69). CONCLUSIONS: Switching from one infliximab biosimilar to another had no adverse impact on infliximab trough levels, and clinical and biochemical disease activity, regardless of whether switching for the first or second time.

Human marginal zone B cell development from early T2 progenitors.

B cells emerge from the bone marrow as transitional (TS) B cells that differentiate through T1, T2, and T3 stages to become naive B cells. We have identified a bifurcation of human B cell maturation from the T1 stage forming IgMhi and IgMlo developmental trajectories. IgMhi T2 cells have higher expression of α4ß7 integrin and lower expression of IL-4 receptor (IL4R) compared with the IgMlo branch and are selectively recruited into gut-associated lymphoid tissue. IgMhi T2 cells also share transcriptomic features with marginal zone B cells (MZBs). Lineage progression from T1 cells to MZBs via an IgMhi trajectory is identified by pseudotime analysis of scRNA-sequencing data. Reduced frequency of IgMhi gut-homing T2 cells is observed in severe SLE and is associated with reduction of MZBs and their putative IgMhi precursors. The collapse of the gut-associated MZB maturational axis in severe SLE affirms its existence in health.

Anti-Mycobacterium paratuberculosis (MAP) therapy for Crohn's disease: an overview and update.

The role of Mycobacterium avium subspecies paratuberculosis (MAP) in the pathogenesis of Crohn's disease (CD) has been strongly debated for many years. MAP is the known aetiological agent of Johne's disease, a chronic enteritis affecting livestock. At present, due to the paucity of high-quality data, anti-MAP therapy (AMT) is not featured in international guidelines as a treatment for CD. Although the much-quoted randomised trial of AMT did not show sustained benefits over placebo, questions have been raised regarding trial design, antibiotic dosing and the formulation used. There are several lines of evidence supporting the CD and MAP association with uncontrolled and controlled trials demonstrating effectiveness, including a retrospective review of cases treated at our own institution. Here, we provide an overview of the evidence supporting and refuting AMT in CD before focussing on updates of the current research in the field, including the ongoing trials with the novel RHB-104 formulation and the MAP vaccine trial. While controversial, gastroenterologists are often asked about long-term combination antibiotic therapy for CD. There has been broadcast and social media coverage surrounding this, particularly with regard to current trials. Although patients should not be deterred from treatments of proven effectiveness, this review aims to help with commonly asked questions and highlights our own approach for the use of anti-MAP in specific circumstances.

The effects of COVID-19 on IBD prescribing and service provision in a UK tertiary centre.

BACKGROUND: To quantify the effects of COVID-19 on our inflammatory bowel disease (IBD) unit, including service provision, prescribing practices and use of therapeutic drug monitoring (TDM). METHODS: We performed a single centre retrospective observational cohort study. Data was extracted from our IBD database, electronic patient records and radiology/endoscopy reporting systems between 16/3/20-17/4/20 and the corresponding period in 2019. RESULTS: A similar number of patients commenced biologic therapy before COVID-19 (n = 37) and during the pandemic (n = 36). Patients in the pre-COVID-19 cohort were older (median 36 vs 29 years, P = 0.009) with a longer median disease duration (9.3 vs 5.2 years, P = 0.02). During COVID-19 there was a nonsignificant increase in prescribing of vedolizumab (8/37, 22% vs 14/36, 39%, P = 0.13) and a higher proportion of patients were anti-TNF-naïve (3/17, 18% vs 18/24, 74%, P = 0.0004). There was a reduction in use of concomitant immunomodulators (22/29, 76% vs 4/34, 12%, P < 0.0001) and increased biologic use in thiopurine-naïve patients (3/37, 8% vs 15/36, 42%, P = 0.001). Use of TDM fell by 75% (240 vs 59 tests). Outpatient appointments fell by 68% and were conducted via telemedicine. MRI scanning, endoscopy, luminal surgery and inpatient numbers fell by 87%, 85%, 100% and 82% respectively. IBD Clinical Nurse Specialist and Pharmacist helpline contacts increased by 76% and 228% respectively. CONCLUSIONS: We observed prescribing differences during COVID-19, bypassing the initiation of immunomodulators and/or anti-TNF therapy in favour of vedolizumab with a reduction in immunomodulator prescribing. We also observed a rapid reorganisation of service provision, including a shift towards telemedicine and online solutions.

Efficacy, safety and drug survival of thioguanine as maintenance treatment for inflammatory bowel disease: a retrospective multi-centre study in the United Kingdom.

BACKGROUND: Thioguanine (TG) is a thiopurine which has been used for patients with inflammatory bowel disease (IBD), who have failed azathioprine (AZA) or mercaptopurine (MP) due to adverse events or suboptimal response. Its widespread use has been hampered due to concerns about nodular regenerative hyperplasia (NRH) of the liver. The aim of this study was to investigate the long-term efficacy and safety of low-dose TG therapy in IBD patients failing AZA and MP. METHODS: A retrospective multicentre study was performed in IBD patients who failed prior treatment with conventional thiopurines with or without following immunomodulation (thiopurine-allopurinol, biologicals, methotrexate, tacrolimus) and were subsequently treated with TG as rescue monotherapy between 2003 and 2019 at three hospitals in the United Kingdom. Clinical response, adverse events, laboratory results, imaging and liver biopsies were retrospectively collected. RESULTS: A total of 193 patients (57% female and 64% Crohn's disease) were included, with a median daily TG dose of 20 mg (range: 20-40 mg), a median treatment duration of 23 months (IQR 10-47) and a median follow-up of 36 months (IQR 22-53). The clinical response rate at 12 months was 65 and 54% remained on TG until the end of follow-up. Adverse events consisted primarily of elevated liver tests (6%), myelotoxicity (7%) and rash (5%). NRH was histologically diagnosed in two patients and two other patients (1%) developed non-cirrhotic portal hypertension. The median 6-TGN and TPMT levels were 953 pmol/8 × 105 RBC (IQR 145-1761) and 47 mu/L (IQR 34.5-96). CONCLUSIONS: Long-term follow-up suggests that TG can be an effective and well-tolerated therapy in more than half of difficult-to-treat and multi-therapy failing IBD patients. Findings of this study indicate that TG can be used safely and the occurrence of hepatotoxicity was low. The incidence rate of NRH was within the background incidence.

Therapeutic thresholds for golimumab serum concentrations during induction and maintenance therapy in ulcerative colitis: results from the GO-LEVEL study.

BACKGROUND: Significant associations between serum golimumab concentrations and favourable outcomes have been observed during both induction and maintenance therapy in ulcerative colitis (UC). However, data regarding optimal therapeutic serum golimumab concentration thresholds are limited. AIMS: To identify optimal serum golimumab concentration thresholds during induction and maintenance treatment with golimumab. METHODS: GO-LEVEL was an open label, phase IV study that included a prospective cohort of UC patients commencing golimumab, as well as a cross-sectional cohort receiving maintenance treatment. Patients commencing induction for active UC (defined as a simple clinical colitis activity index [SCCAI] >5 in addition to a raised faecal calprotectin [FC] >59µg/g or, raised C-reactive protein [CRP] [>5mg/L] or, Mayo endoscopic disease activity 2 or 3) were evaluated at weeks 6, 10 and 14. Patients receiving maintenance therapy were recruited either at the point of flare or during remission. Combined clinical-biochemical remission was defined as SCCAI ≤2 and FC <250µg/g. Serum golimumab concentrations were measured using a commercially available ELISA (LISATRACKER, Theradiag). RESULTS: Thirty-nine patients were included in the induction cohort, of whom 15 (38%) achieved combined clinical-biochemical remission at week 6. The median serum golimumab concentration of those in combined clinical-biochemical remission was significantly higher than those who were not (5.0 vs 3.1 µg/mL, respectively, P = 0.03). Receiver operating characteristic (ROC) curve analysis demonstrated 3.8 µg/mL as the optimal threshold (sensitivity 0.71, specificity 0.65, area under curve [AUC] 0.72, positive predictive value [PPV] 0.59 and negative predictive value [NPV] 0.79). Sixty-three patients were included in the maintenance cohort; 31 (49%) were in combined remission, 32 (51%) were not. The median serum golimumab concentration of those in combined remission was significantly higher (2.9 vs 2.1 µg/mL, respectively, P = 0.01). ROC curve analysis demonstrated 2.4 µg/mL as the optimal threshold (sensitivity 0.68, specificity 0.66, AUC 0.68, PPV 0.65 and NPV 0.66). CONCLUSIONS: GO-LEVEL (NCT03124121) offers further evidence regarding golimumab's exposure-response relationship. Clinicians may consider using therapeutic drug monitoring to optimise golimumab dosing aiming to achieve our suggested therapeutic thresholds of 3.8 µg/mL at week 6 and 2.4 µg/mL during maintenance.

Fundamentals of Microscopy.

The light (or optical) microscope is the icon of science. The aphorism "seeing is believing" is often quoted in scientific papers involving microscopy. Unlike many scientific instruments, the light microscope will deliver an image however badly it is set up. Fluorescence microscopy is a widely used research tool across all disciplines of biological and biomedical science. Most universities and research institutions have microscopes, including confocal microscopes. This introductory paper in a series detailing advanced light microscopy techniques explains the foundations of both electron and light microscopy for biologists and life scientists working with the mouse. An explanation is given of how an image is formed. A description is given of how to set up a light microscope, whether it be a brightfield light microscope on the laboratory bench, a widefield fluorescence microscope, or a confocal microscope. These explanations are accompanied by operational protocols. A full explanation on how to set up and adjust a microscope according to the principles of Köhler illumination is given. The importance of Nyquist sampling is discussed. Guidelines are given on how to choose the best microscope to image the particular sample or slide preparation that you are working with. These are the basic principles of microscopy that a researcher must have an understanding of when operating core bioimaging facility instruments, in order to collect high-quality images. © 2020 The Authors. Basic Protocol 1: Setting up Köhler illumination for a brightfield microscope Basic Protocol 2: Aligning the fluorescence bulb and setting up Köhler illumination for a widefield fluorescence microscope Basic Protocol 3: Generic protocol for operating a confocal microscope.

Effectiveness of vedolizumab dose intensification to achieve inflammatory bowel disease control in cases of suboptimal response.

BACKGROUND: Despite the proven efficacy of vedolizumab (VDZ) for ulcerative colitis (UC) and Crohn's disease (CD), suboptimal response is commonly encountered. However, data regarding the effectiveness of dose intensification (by interval shortening) to achieve response are limited. OBJECTIVES: We evaluated the effectiveness of dose intensification at achieving response in patients with a previously suboptimal response to VDZ. Additionally, we aimed to identify predictors of response to this strategy. METHODS: We performed a retrospective cohort study of patients who underwent VDZ dose intensification for suboptimal response. Clinical disease activity was evaluated at the point of dose intensification (baseline) and at weeks 12 and 24. Response was defined as Harvey-Bradshaw Index (HBI) or Simple Clinical Colitis Activity Index (SCCAI) reduction of ≥3, and remission as HBI <5 or SCCAI <3. RESULTS: A total of 36 patients received dose intensification to 4-weekly infusions: 18 CD, 14 UC and 4 inflammatory bowel disease-unclassified (analysed in the UC group). Median SCCAI scores fell from 6 (range 0-11) at baseline to 4 (0-6, p=0.008) at week 24, while HBI scores did not change significantly (4 (0-27) and 3 (0-8), p=0.092). Overall median C reactive protein (CRP) fell from 6 mg/L (1-23) to 2 mg/L (1-17, p=0.011). Of 20 patients with clinically active disease at baseline, 10 (50%) responded, of whom 4 (20%) achieved remission at week 24. Univariate analysis demonstrated low baseline CRP (p=0.045) and response at week 12 (0.020) were associated with week 24 response. CONCLUSIONS: Our findings demonstrate VDZ dose intensification to be effective at achieving clinical response in half of patients. Low baseline CRP and response at week 12 are potential predictors of week 24 response.

Genetic and Inflammatory Biomarkers Classify Small Intestine Inflammation in Asymptomatic First-degree Relatives of Patients With Crohn's Disease.

BACKGROUND & AIMS: Relatives of individuals with Crohn's disease (CD) carry CD-associated genetic variants and are often exposed to environmental factors that increase their risk for this disease. We aimed to estimate the utility of genotype, smoking status, family history, and biomarkers can calculate risk in asymptomatic first-degree relatives of patients with CD. METHODS: We recruited 480 healthy first-degree relatives (full siblings, offspring or parents) of patients with CD through the Guy's and St Thomas' NHS Foundation Trust and from members of Crohn's and Colitis, United Kingdom. DNA samples were genotyped using the Immunochip. We calculated a risk score for 454 participants, based on 72 genetic variants associated with CD, family history, and smoking history. Participants were assigned to highest and lowest risk score quartiles. We assessed pre-symptomatic inflammation by capsule endoscopy and measured 22 markers of inflammation in stool and serum samples (reference standard). Two machine-learning classifiers (elastic net and random forest) were used to assess the ability of the risk factors and biomarkers to identify participants with small intestinal inflammation in the same dataset. RESULTS: The machine-learning classifiers identified participants with pre-symptomatic intestinal inflammation: elastic net (area under the curve, 0.80; 95% CI, 0.62-0.98) and random forest (area under the curve, 0.87; 95% CI, 0.75-1.00). The elastic net method identified 3 variables that can be used to calculate odds for intestinal inflammation: combined family history of CD (odds ratio, 1.31), genetic risk score (odds ratio, 1.14), and fecal calprotectin (odds ratio, 1.04). These same 3 variables were among the 5 factors associated with intestinal inflammation in the random forest model. CONCLUSION: Using machine learning classifiers, we found that genetic variants associated with CD, family history, and fecal calprotectin together identify individuals with pre-symptomatic intestinal inflammation who are therefore at risk for CD. A tool for detecting people at risk for CD before they develop symptoms would help identify the individuals most likely to benefit from early intervention.