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Abstract Background The composition of the gut microbiome is a key determinant in inflammatory bowel disease. Consequently, modifying the composition of the gut microbiota has the potential to transform the treatment of ulcerative colitis (UC). The clinical use of faecal microbiota transplantation (FMT) has become widespread, especially for the treatment of C. difficile associated diseases, as a method of changing the composition of the gut microbiome from a diseased to a healthy state. FMT is, by its nature, an undefined and variable therapy due to differences between donors. Microbiotica has taken a "patient-first" discovery approach to identify a defined consortium, which is being developed as a live bacterial therapeutic for treatment of mild-to-moderate UC patients in a Phase 1b clinical trial, the COMPOSER-1 study, in 2024. Methods A clinical trial in UC patients (Costello et al., 2019), in which patients were treated with FMT from healthy donors, showed a 32% clinical remission rate in the active treatment group, as compared to 9% in the placebo arm. Donor, pre-treatment recipient, and post-FMT recipient samples were analysed using shotgun metagenomic sequencing. All samples were analysed using Microbiotica’s precision microbiome analysis platform to identify bacteria associated with patient response. These bacteria were assessed in cellular assays using relevant human cell/cell lines including: Caco2, dendritic cells, M1 macrophages, and CD4+ T-cells. Results We defined a signature of therapeutic response based on bacteria that engrafted from the donor into UC patients and were associated with clinical benefit. A sub-species level analysis identified 8 bacteria, which have been developed into a defined live bacterial therapeutic, MB310. The consortium bacteria enhanced the barrier integrity of an epithelial cell monolayer, and protected the barrier from inflammatory challenge by LPS. In vitro, the bacteria also have an anti-inflammatory effect when incubated with different primary innate immune cells, dendritic cells and M1 macrophages. Specific MB310 bacteria, either directly or via metabolites, are able to regulate T-cell responses. Conclusion In summary, we have identified a consortium of bacteria using patient-first discovery in a successful FMT study that is being developed as a treatment for UC. The consortium of 8 bacteria is able to impact multiple disease-relevant mechanisms including epithelial barrier integrity and immunomodulation. MB310 is being advanced to a first-in-human study in 2024 to explore safety and initial signs of efficacy. Costello, et al. (2019) Jama 321.2: 156-164.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

IntroductionThere is increasing attention on the integration of targeted agents for oesophageal adenocarcinoma (OA) therapy. The most notable example of success of oesophagogastric targeted therapy was the addition of a HER2 targeting agent in the Phase III ToGA study. However, there is limited data on the expression of HER2 in the progression from Barrett's (BE) to OA. This study aimed to clarify expression in this sequence, and to show binding of C6.5, a single chain HER2 targeting human antibody Fv fragment (scFv), to known HER2 expressing OA cell lines in vitro. Efficacy of antibody based therapies can be enhanced by scFv's which penetrate tumours more quickly and demonstrate better tumour: normal tissue specificity.Methods33 paraffin embedded oesophageal tissue specimens were selected from patients with squamous (n=4), non-dysplastic BE (NDBE; n=4), low grade dysplasia (LGD; n=6), high grade dysplasia (HGD; n=8) and OA (n=12). Sections were immunostained with the automated Oracle Bond system (Leica, UK) for consistency. Staining was then scored by 2 expert pathologists according to the proportion of cells in the tissue staining positively as negative (<5%), borderline (5%–10%) or positive (>10%). In phase 2, binding of C6.5 scFv to the cancer cell lines SKOV-3 (ovarian), OE-19, OE-33 (oesophageal) and HT-29 (colon) was identified with flow cytometry using a mouse secondary followed by tertiary anti-mouse FITC. Data were then analysed with FlowJo software.ResultsSignificant expression of HER2 (>10% cells positive according to National Guidelines) was only seen in HGD (25%) and cancer (25%) specimens. Borderline staining (5%–10%) was seen in LGD (17%), HGD (13%) and cancer (8%). All NDBE and the remaining LGD, HGD and cancer samples were negative. Flow cytometry demonstrated C6.5 binding to SKOV-3, OE19 and OE-33 cells but not HT-29 cells.ConclusionThis study demonstrated that significant HER2 expression is only seen in roughly a quarter of patients with HGD and OA and not in NDBE or LGD. We also found that the HER2 targeting scFv C6.5 binds to breast cancer and OA cell lines but not colon cancer, the negative control. HER2 targeting therapies could therefore be postulated for patients with BE with HGD and OA, and these may be enhanced with scFv's such as C6.5.Competing interestsNone declared.