An absolute quantification method for selected MFGM proteins in infant formula using targeted proteomics.

Milk fat globule membrane (MFGM) proteins are receiving increased attention due to their reported benefits for human health, particularly in infant populations. Challenges exist in MFGM protein quantification due to their low quantities, complex chemistry, interactions with other matrix components, and the high matrix complexity. In this study, a subset of four MFGM proteins were selected as relevant targets for identification and quantification in an infant formula (IF) matrix: butyrophilin, mucin 1, xanthine dehydrogenase/oxidase, and perilipin 2. An analytical protocol for absolute quantification of these four MFGM proteins in IF was successfully developed and validated. Additionally, the feasibility to apply the method in selected MFGM ingredients was also demonstrated. The method demonstrated good performance; high accuracy and robustness, low LOQ and uncertainty, while controlling the matrix effect. This study represents a report of an analytical method to quantify selected MFGM proteins in IF.

Exploiting human immune repertoire transgenic mice for protective monoclonal antibodies against antimicrobial resistant Acinetobacter baumannii.

The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era.

The impact of complexation or complex coacervation of lactoferrin and osteopontin on simulated infant gastrointestinal digestion, intestinal inflammation, and in vivo bone development.

Lactoferrin (LF) and osteopontin (OPN) are bioactive milk proteins which can form heteroprotein complexes and complex coacervates. This research studied the effect of LF-OPN complexation and complex coacervation on the simulated infant gastrointestinal digestion of LF with subsequent examination of gut and bone health bioactivities in preclinical models. In an infant digestion model, the proteolytic profile of LF was unaltered by the pre-association of LF and OPN. Gastric proteolysis of LF was increased when the model gastric pH was reduced from 5.3 to 4.0, but less so when complexed with OPN. In a model of intestinal inflammation, undigested (79% inhibition) and gastric digestates (26% inhibition) of LF, but not gastrointestinal digestates, inhibited lipopolysaccharide (LPS)-induced NF-κB activation in intestinal epithelial cells. LF-OPN complexation sustained the inhibitory effect (21-43% of the undigested effect, depending on the type of complex) of LF after gastrointestinal digestion, suggesting that the peptides produced were different. In a neonatal rodent model used to study bone development, coacervating LF and OPN improved bone structures with a significant increase of trabecular proportion (BV/TV increase by 21.7%). This resulted in an 11.3% increase in stiffness of bones. Feeding the LF and OPN proteins in coacervate format also increased the levels of OPN, P1NP and M-CSF in blood, signifying a more pronounced impact on bone development. This research demonstrated that LF-OPN complexation and complex coacervation can delay simulated infant gastrointestinal digestion of LF and protect or improve the bioactivity of the proteins.

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum ( T. pallidum ) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted Green Fluorescent Protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum , better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes. Graphical abstract: By employing genetic engineering, T. pallidum was modified to express GFP, and Sf1Ep cells to express mCherry on the cytoplasmic membrane and BFP in the nucleus. These new resources for syphilis research will facilitate experimental designs to better define the complex interplay between T. pallidum and the host during infection.

Overexpression of soluble epoxide hydrolase reduces post-ischemic recovery of cardiac contractile function.

Cytochromes P450 can metabolize endogenous fatty acids, such as arachidonic acid, to bioactive lipids such as epoxyeicosatrienoic acids (EETs) that have beneficial effects. EETs protect hearts against ischemic damage, heart failure or fibrosis; however, their effects are limited by hydrolysis to less active dihydroxy oxylipins by soluble epoxide hydrolase (sEH), encoded by the epoxide hydrolase 2 gene (EPHX2, EC 3.3.2.10). Pharmacological inhibition or genetic disruption of sEH/EPHX2 have been widely studied for their impact on cardiovascular diseases. Less well studied is the role of increased EPHX2 expression, which occurs in a substantial human population that carries the EPHX2 K55R polymorphism or after induction by inflammatory stimuli. Herein, we developed a mouse model with cardiomyocyte-selective expression of human EPHX2 (Myh6-EPHX2) that has significantly increased total EPHX2 expression and activity. Myh6-EPHX2 hearts exhibit strong, cardiomyocyte-selective expression of EPHX2. EPHX2 mRNA, protein, and epoxide hydrolysis measurements suggest that Myh6-EPHX2 hearts have 12-fold increase in epoxide hydrolase activity relative to wild type (WT) hearts. This increased activity significantly decreased epoxide:diol ratios in vivo. Isolated, perfused Myh6-EPHX2 hearts were not significantly different from WT hearts in basal parameters of cardiac function; however, compared to WT hearts, Myh6-EPHX2 hearts demonstrated reduced recovery of heart contractile function after ischemia and reperfusion (I/R). This impaired recovery after I/R correlated with reduced activation of PI3K/AKT and GSK3ß signaling pathways in Myh6-EPHX2 hearts compared to WT hearts. In summary, the Myh6-EPHX2 mouse line represents a novel model of cardiomyocyte-selective overexpression of EPHX2 that has detrimental effects on cardiac function.

Cardiac GR Mediates the Diurnal Rhythm in Ventricular Arrhythmia Susceptibility.

BACKGROUND: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.

From lab-based to in-line: Analytical tools for the characterization of whey protein denaturation and aggregation-A review.

Whey protein denaturation and aggregation have long been areas of research interest to the dairy industry, having significant implications for process performance and final product functionality and quality. As such, a significant number of analytical techniques have been developed or adapted to assess and characterize levels of whey protein denaturation and aggregation, to either maximize processing efficiency or create products with enhanced functionality (both technological and biological). This review aims to collate and critique these approaches based on their analytical principles and outline their application for the assessment of denaturation and aggregation. This review also provides insights into recent developments in process analytical technologies relating to whey protein denaturation and aggregation, whereby some of the analytical methods have been adapted to enable measurements in-line. Developments in this area will enable more live, in-process data to be generated, which will subsequently allow more adaptive processing, enabling improved product quality and processing efficiency. Along with the applicability of these techniques for the assessment of whey protein denaturation and aggregation, limitations are also presented to help assess the suitability of each analytical technique for specific areas of interest.

Extracellular vesicles could be a putative posttranscriptional regulatory mechanism that shapes intracellular RNA levels in Plasmodium falciparum.

Plasmodium falciparum secretes extracellular vesicles (PfEVs) that contain parasite-derived RNA. However, the significance of the secreted RNA remains unexplored. Here, we compare secreted and intracellular RNA from asexual cultures of six P. falciparum lines. We find that secretion of RNA via extracellular vesicles is not only periodic throughout the asexual intraerythrocytic developmental cycle but is also highly conserved across P. falciparum isolates. We further demonstrate that the phases of RNA secreted via extracellular vesicles are discernibly shifted compared to those of the intracellular RNA within the secreting whole parasite. Finally, transcripts of genes with no known function during the asexual intraerythrocytic developmental cycle are enriched in PfEVs compared to the whole parasite. We conclude that the secretion of extracellular vesicles could be a putative posttranscriptional RNA regulation mechanism that is part of or synergise the classic RNA decay processes to maintain intracellular RNA levels in P. falciparum.

Post-vaccine epidemiology of serotype 3 pneumococci identifies transformation inhibition through prophage-driven alteration of a non-coding RNA.

BACKGROUND: The respiratory pathogen Streptococcus pneumoniae (the pneumococcus) is a genetically diverse bacterium associated with over 101 immunologically distinct polysaccharide capsules (serotypes). Polysaccharide conjugate vaccines (PCVs) have successfully eliminated multiple targeted serotypes, yet the mucoid serotype 3 has persisted despite its inclusion in PCV13. This capsule type is predominantly associated with a single globally disseminated strain, GPSC12 (clonal complex 180). METHODS: A genomic epidemiology study combined previous surveillance datasets of serotype 3 pneumococci to analyse the population structure, dynamics, and differences in rates of diversification within GPSC12 during the period of PCV introductions. Transcriptomic analyses, whole genome sequencing, mutagenesis, and electron microscopy were used to characterise the phenotypic impact of loci hypothesised to affect this strain's evolution. RESULTS: GPSC12 was split into clades by a genomic analysis. Clade I, the most common, rarely underwent transformation, but was typically infected with the prophage ϕOXC141. Prior to the introduction of PCV13, this clade's composition shifted towards a ϕOXC141-negative subpopulation in a systematically sampled UK collection. In the post-PCV13 era, more rapidly recombining non-Clade I isolates, also ϕOXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the ~100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage ϕOXC141 was found to modify csRNA3, a non-coding RNA that inhibits the induction of transformation. This alteration was identified in ~30% of all pneumococci and was particularly common in the unusually clonal serotype 1 GPSC2 strain. RNA-seq and quantitative reverse transcriptase PCR experiments using a genetically tractable pneumococcus demonstrated the altered csRNA3 was more effective at inhibiting production of the competence-stimulating peptide pheromone. This resulted in a reduction in the induction of competence for transformation. CONCLUSION: This interference with the quorum sensing needed to induce competence reduces the risk of the prophage being deleted by homologous recombination. Hence the selfish prophage-driven alteration of a regulatory RNA limits cell-cell communication and horizontal gene transfer, complicating the interpretation of post-vaccine population dynamics.

Defining the early stages of intestinal colonisation by whipworms.

Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche.

Serum Buprenorphine Concentrations and Behavioral Activity in Mice After a Single Subcutaneous Injection of Simbadol, Buprenorphine SR-LAB, or Standard Buprenorphine.

Buprenorphine, an analgesic commonly used in rodent surgery, requires repeated dosing every 4 to 6 h in order to provide adequate analgesia. However, redosing requires repeated handling, which may itself cause stress. Buprenorphine SR-LAB, which reportedly maintains serum levels of buprenorphine greater than 1 ng/mL for 48 to 72 h, is commercially available. However, the viscosity of the product and small dosing volumes make accurate dosing a challenge. Simbadol is a concentrated formulation of buprenorphine hydrochloride labeled for use in cats with recommended dosing frequency of every 24 h. We measured serum concentrations over time after a single injection of this product in C57BL/6NCrl mice and compared it to standard buprenorphine (Buprenex) and Buprenorphine SR-LAB. Male and female mice were injected subcutaneously with one of the 3 buprenorphine formulations at a dose of 1 mg/kg at time 0. Groups of mice (n = 8) were euthanized at 1, 4, 8, 12, 16 h for all groups and 24 h for the Simbadol and the Buprenorphine SR-LAB. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine concentrations of buprenorphine in each serum sample. High concentrations were observed in both Simbadol and standard buprenorphine groups one hour after injection (>50 ng/mL). These groups had similar buprenorphine concentration curves, including rates of decline. The standard buprenorphine group had mean concentrations less than 1 ng/mL by 12 h and the Simbadol group by 16 h. In contrast, the Buprenorphine SR-LAB group remained above the 1 ng/mL therapeutic threshold throughout the 24 h. In addition, clinical signs, including increased activity, that lasted for up to an hour after the injection in the Simbadol and standard buprenorphine groups. We conclude that Simbadol does not offer dosing advantages over the standard buprenorphine formulation when given at 1 mg/kg. Buprenorphine SR-LAB maintained a steady concentration of buprenorphine above 1 ng/mL for at least 24 h, and as such is a superior choice for providing long-term analgesia.

Host adaptation in gut Firmicutes is associated with sporulation loss and altered transmission cycle.

BACKGROUND: Human-to-human transmission of symbiotic, anaerobic bacteria is a fundamental evolutionary adaptation essential for membership of the human gut microbiota. However, despite its importance, the genomic and biological adaptations underpinning symbiont transmission remain poorly understood. The Firmicutes are a dominant phylum within the intestinal microbiota that are capable of producing resistant endospores that maintain viability within the environment and germinate within the intestine to facilitate transmission. However, the impact of host transmission on the evolutionary and adaptive processes within the intestinal microbiota remains unknown. RESULTS: We analyze 1358 genomes of Firmicutes bacteria derived from host and environment-associated habitats. Characterization of genomes as spore-forming based on the presence of sporulation-predictive genes reveals multiple losses of sporulation in many distinct lineages. Loss of sporulation in gut Firmicutes is associated with features of host-adaptation such as genome reduction and specialized metabolic capabilities. Consistent with these data, analysis of 9966 gut metagenomes from adults around the world demonstrates that bacteria now incapable of sporulation are more abundant within individuals but less prevalent in the human population compared to spore-forming bacteria. CONCLUSIONS: Our results suggest host adaptation in gut Firmicutes is an evolutionary trade-off between transmission range and colonization abundance. We reveal host transmission as an underappreciated process that shapes the evolution, assembly, and functions of gut Firmicutes.

The impact of thermal processing on the simulated infant gastrointestinal digestion, bactericidal and anti-inflammatory activity of bovine lactoferrin - An in vitro study.

Lactoferrin (LF) is a multifunctional glycoprotein which, when thermally processed, undergoes significant physicochemical changes. The link between such changes and the bioactivity of LF is not well characterised and requires much research. In this work, bovine LF solutions (1%, w/v, protein, pH 7) were thermally processed using high temperature short time conditions (72, 80, 85 or 95 °C with 15 s holding times). Following this, it was shown that LF and heat induced LF aggregates were largely resistant to simulated infant gastric, but not intestinal, digestion. Also, the efficacy of LF bactericidal activity, and inhibition of lipopolysaccharide-induced NF-κB activation were negatively impacted by thermal processing. This study confirmed that the efficacy of LF bio-functionalities was affected by the extent of heat-induced changes in protein structure whereby processing conditions of least severity (i.e. pasteurisation) had the least impact on bioactivity.

Comparative Efficacy of Two Types of Antibiotic Mixtures in Gut Flora Depletion in Female C57BL/6 Mice.

Over the last decade, interest in the role of the microbiome in health and disease has increased. The use of germ-free animals and depletion of the microbial flora using antimicrobials are 2 methods commonly used to study the microbiome in laboratory mice. Germ-free mice are born, raised, and studied in isolators in the absence of any known microbes; however, the equipment, supplies, and training required for the use of these mice can be costly and time-consuming. The use of antibiotics to decrease the microbial flora does not require special equipment, can be used for any mouse strain, and is relatively inexpensive; however, mice treated in this manner still retain microbes and they do not live in a germ-free environment. One commonly used antibiotic cocktail regimen uses ampicillin, neomycin, metronidazole, and vancomycin in the drinking water for 2 to 4 wk. We found that the palatability of this mixture is low, resulting in weight loss and leading to removal of mice from the study. The addition of sucralose to the medicated water and making wet food (mash) with the medicated water improved intake; however, the low palatability still resulted in a high number of mice requiring removal. The current study evaluated a new combination of antibiotics designed to reduce the gut microbiota while maintaining body weights. C57BL/6NCrl mice were placed on one of the following drinking water regimens: ampicillin/neomycin/metronidazole/vancomycin water (n = 16), enrofloxacin/ampicillin water ( n = 12), or standard reverse osmosis deionized water (RODI) ( n = 11). During an 8 day regimen, mice were weighed and water consumption was measured. Feces were collected before and after 8 d of treatment. Quantitative real-time PCR (real-time qPCR) for 16S bacterial ribosome was performed on each sample, and values were compared among groups. The combination of enrofloxacin and ampicillin improved water intake, together with a greater reduction in gut flora.

An invariant Trypanosoma vivax vaccine antigen induces protective immunity.

Trypanosomes are protozoan parasites that cause infectious diseases, including African trypanosomiasis (sleeping sickness) in humans and nagana in economically important livestock1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms-including antigenic variation3-that present an apparently insurmountable barrier to vaccination. Here we show, using a systematic genome-led vaccinology approach and a mouse model of Trypanosoma vivax infection4, that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell-surface protein that is localized to the flagellum membrane (which we term 'invariant flagellum antigen from T. vivax') induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to this protein could induce sterile protection and revealed several mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained socioeconomic development in countries in sub-Saharan Africa5, and provides evidence that highly protective vaccines against trypanosome infections can be achieved.

Control of oviductal fluid flow by the G-protein coupled receptor Adgrd1 is essential for murine embryo transit.

Dysfunction of embryo transport causes ectopic pregnancy which affects approximately 2% of conceptions in the US and Europe, and is the most common cause of pregnancy-related death in the first trimester. Embryo transit involves a valve-like tubal-locking phenomenon that temporarily arrests oocytes at the ampullary-isthmic junction (AIJ) where fertilisation occurs, but the mechanisms involved are unknown. Here we show that female mice lacking the orphan adhesion G-protein coupled receptor Adgrd1 are sterile because they do not relieve the AIJ restraining mechanism, inappropriately retaining embryos within the oviduct. Adgrd1 is expressed on the oviductal epithelium and the post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. Using a large-scale extracellular protein interaction screen, we identified Plxdc2 as an activating ligand for Adgrd1 displayed on cumulus cells. Our findings demonstrate that regulating oviductal fluid flow by Adgrd1 controls embryo transit and we present a model where embryo arrest at the AIJ is due to the balance of abovarial ciliary action and the force of adovarial tubal fluid flow, and in wild-type oviducts, fluid flow is gradually attenuated through Adgrd1 activation to enable embryo release. Our findings provide important insights into the molecular mechanisms involved in embryo transport in mice.

A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131.

The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum.

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling.