The influence of maternal diet on intestinal adaptation in the mother and offspring

The effect of maternal nutrition on fetal and offspring growth and risk of disease in later life is well established. Animal models have shown that the small intestine can be preferentially affected by growth restriction compared with other organs, yet the effect of maternal diet on gastrointestinal (GI) development has not been well studied. Furthermore, both the maternal GI tract and microbiome undergo significant adaptations during pregnancy in response to the increased nutritional demands of the growing fetus, which may be further modulated by the maternal diet. Inadequate development of the GI tract may result in an increase in intestinal permeability, as demonstrated in inflammatory bowel diseases. Compromised gut barrier function has been linked to the development of obesity through the passage of endotoxin on Gram-negative bacteria leading to low-level inflammation of the liver and adipose tissue. The aim of this thesis was to use two animal models of maternal dietary manipulation that are representative of suboptimal nutrition characterised by the nutritional excess of macronutrients found in many Western diets. The effect of either fat in the form of palm oil and carbohydrate in the form of fructose were therefore examined with particular focus on the gut. This was undertaken in a pig model to examine the effects around the time of birth and in the juvenile offspring, whilst in a rat model both the mothers and offspring were studied. Addition of 10% fructose to drinking water in two generations of pregnant Wistar rats induced impaired glucose tolerance and raised serum triglycerides, but only in the second generation. Sequencing of the maternal microbiome in first generation dams through pregnancy revealed significant changes in microbial diversity from pre-mating to late pregnancy, both between and within dams. Consumption of the fructose diet led to further changes in microbial diversity. Offspring of fructose-fed mothers were growth restricted and had significantly shorter small intestines compared to controls. Changes in gene expression in the ileum and jejunum were indicative of raised intestinal permeability in second-generation dams, and included the epithelial tight junction genes occludin (OCLN), claudin (CLDN) and junctional adhesion molecule (JAM). In both generations the fructose diet significantly upregulated glucose transporters 2 and 5, and sodium-glucose linked transporter 1 in the ileum and jejunum suggesting that a relatively low level supplementation of fructose gradually increases the absorptive capacity of the small intestine for both glucose and fructose. This may explain the significantly impaired glucose tolerance and insulin resistance observed in second-generation offspring. To study maternal fat supplementation, sows were fed a standard commercial diet supplemented with palm oil (6.6% added to commercial feed) throughout gestation. Median birth weight offspring born to fat supplemented mothers sampled at 7 days demonstrated no growth restriction, but significant downregulation in OCLN, CLDN and JAMA expression, suggesting increased intestinal permeability. No changes in offspring body composition were observed at either time point, and effects on gene expression did not persist up to 6 months of age. In conclusion, a suboptimal diet through pregnancy in which macronutrient composition is raised has the potential to compromise gut function in the mother and offspring. The magnitude of effect can however be temporary and dependent on the animal model used as well as the macronutrient targeted

High Fructose Intake During Pregnancy in Rats Influences the Maternal Microbiome and Gut Development in the Offspring

Studies in pregnant women indicate the maternal microbiome changes during pregnancy so as to benefit the mother and fetus. In contrast, disruption of the maternal microbiota around birth can compromise normal bacterial colonisation of the infant’s gastrointestinal tract. This may then inhibit development of the gut so as to increase susceptibility to inflammation and reduce barrier function. The impact of modulating fructose intake on the maternal microbiome through pregnancy is unknown, therefore we examined the effect of fructose supplementation on the maternal microbiome together with the immediate and next generation effects in the offspring. Wistar rat dams were divided into control and fructose fed groups that received 10% fructose in their drinking water from 8 weeks of age and throughout pregnancy (10–13 weeks). Maternal fecal and blood samples were collected pre-mating (9 weeks) and during early (gestational day 4–7) and late pregnancy (gestational day 19–21). We show supplementation of the maternal diet with fructose appears to significantly modulate the maternal microbiome, with a significant reduction in Lactobacillus and Bacteroides. In offspring maintained on this diet up to pregnancy and term there was a reduction in gene expression of markers of gut barrier function that could adversely affect its function. An exacerbated insulin response to pregnancy, reduced birth weight, but increased fat mass was also observed in these offspring. In conclusion dietary supplementation with fructose modulates the maternal microbiome in ways that could adversely affect fetal growth and later gut development

Reliable computational quantification of liver fibrosis is compromised by inherent staining variation

Biopsy remains the gold standard measure for staging liver disease, both to inform prognosis and to assess the response to a given treatment. Semiquantitative scores such as the Ishak fibrosis score are used for evaluation. These scores are utilised in clinical trials, with the US Food and Drug Administration mandating particular scores as inclusion criteria for participants and using the change in score as evidence of treatment efficacy. There is an urgent need for improved, quantitative assessment of liver biopsies to detect small incremental changes in liver architecture over the course of a clinical trial. Artificial intelligence (AI) methods have been proposed as a way to increase the amount of information extracted from a biopsy and to potentially remove bias introduced by manual scoring. We have trained and evaluated an AI tool for measuring the amount of scarring in sections of picrosirius red-stained liver. The AI methodology was compared with both manual scoring and widely available colour space thresholding. Four sequential sections from each case were stained on two separate occasions by two independent clinical laboratories using routine protocols to study the effect of inter- and intra-laboratory staining variation on these tools. Finally, we compared these methods to second harmonic generation (SHG) imaging, a stain-free quantitative measure of collagen. Although AI methods provided a modest improvement over simpler computer-assisted measures, staining variation both within and between labs had a dramatic effect on quantitation, with manual assignment of scar proportion the most consistent. Manual assessment also correlated the most strongly with collagen measured by SHG. In conclusion, results suggest that computational measures of liver scarring from stained sections are compromised by inter- and intra-laboratory staining. Stain-free quantitative measurement using SHG avoids staining-related variation and may prove more accurate in detecting small changes in scarring that may occur in therapeutic trials

Dietary interventions reduce traditional and novel cardiovascular risk markers by altering the gut microbiome and their metabolites

Aims: The current study investigates the role of diet in mediating the gut microbiome-cardiovascular association which has notyet been explored in humans.Methods and results: Using a two-arm dietary intervention study in healthy participants (N=70), we assessed the effects ofomega-3 and fibre supplementation on gut microbiome composition and short-chain fatty acid (SCFA) production. We theninvestigated how changes in gut microbiome composition correlated with changes in traditional cardiovascular risk factors(cholesterol, triglycerides, blood pressure), cytokines, and novel validated markers such as GlycA and ceramides, previously linkedto CVD incidence and mortality. Both interventions resulted in significant drops in blood pressure, cholesterol, proinflammatorycytokines, GlycA and ceramides (all

Role of Hepatocyte Senescence in the Activation of Hepatic Stellate Cells and Liver Fibrosis Progression

Hepatocyte senescence is associated with liver fibrosis. However, the possibility of a direct, causal relation between hepatocyte senescence and hepatic stellate cell (HSC) activation was the subject of this study. Liver biopsy specimens obtained from 50 patients with non-alcoholic fatty liver disease and a spectrum of liver fibrosis stages were stained for p16, αSMA, and picrosirius red (PSR). Primary human HSCs were cultured in conditioned media derived from senescent or control HepG2 cells. Expression of inflammatory and fibrogenic genes in HSCs cultured in conditioned media were studied using RT-PCR. ELISAs were undertaken to measure factors known to activate HSCs in the conditioned media from senescent and control HepG2 cells and serum samples from healthy volunteers or patients with biopsy-proven cirrhosis. There was a strong association between proportion of senescent hepatocytes and hepatic stellate cell activation. Both proportion of hepatocyte senescence and hepatic stellate cell activation were closely associated with fibrosis stage. Inflammatory and fibrogenic genes were up-regulated significantly in HSCs cultured in conditioned media from senescent HepG2 cells compared with control HepG2 cells. PDGF levels were significantly higher in the conditioned media from senescent hepatocytes than control HepG2-conditioned media, and in serum samples from patients with cirrhosis than healthy volunteers. In conclusion, this 'proof of concept' study revealed activation of human HSCs by media from senescent HepG2 cells, indicating direct involvement of factors secreted by senescent hepatocytes in liver fibrosis

Next-generation sequencing of pancreatic cyst wall specimens obtained using micro-forceps for improving diagnostic accuracy

Background and study aims: Pancreatic cysts are common incidental findings, with an estimated prevalence of 13-15% in imaging done for other reasons. Diagnosis often relies on collection of cyst fluid, but tissue sampling using micro-forceps may allow for a more reliable diagnosis and higher yield of DNA for next-generation sequencing (NGS). The primary aim was to assess the performance of NGS in identifying mucinous cyst. The secondary aims were to assess DNA yield between the cyst fluid and cyst wall tissue, complication rate and performance of conventional investigations. Patients and methods: 24 patients referred for endoscopic ultrasound were recruited. Biopsies were taken using micro-forceps and the AmpliSeq Cancer Hotspot panel was used for NGS, a PCR assay targeting several hotspots within 50 genes, including GNAS, KRAS and VHL. Results: The concentration of DNA extracted from 24 cyst wall samples was significantly higher than in the 9/24 available matched cyst fluid samples. The sensitivity, specificity and diagnostic accuracy of NGS for diagnosing mucinous cyst was 93%, 50% and 84%, standard of care -66.6%, 50% and 63.1% and for standard of care with NGS was 100%, 50% and 89.4% respectively. Cyst wall biopsy was able to diagnose 19/24 cysts (4 high risk, 7 intraductal papillary mucinous neoplasm, 4 cysts of mucinous origin and 4 benign). Conclusions: NGS data correlates well with histology and may aid in diagnosis and risk stratification of pancreatic cysts. Cyst wall biopsy performs well in diagnosing cysts but was inadequate in 5/24 patients

Next-generation sequencing of pancreatic cyst wall specimens obtained using Moray micro-forceps for improving diagnostic accuracy

Background and study aims Pancreatic cysts are common incidental findings, with an estimated prevalence of 13-15% in imaging done for other reasons. It is difficult to identify cysts with malignant potential. Diagnosis often relies on collection of cyst fluid, but tissue sampling using micro-forceps may allow for a more reliable diagnosis and higher yield of DNA for next-generation sequencing (NGS).Patients and methods 24 patients referred for endoscopic ultrasound were recruited. Biopsies were taken using micro-forceps and the AmpliSeq Cancer Hotspot panel was used for NGS, a PCR assay targeting several hotspots within 50 genes, including GNAS, KRAS and VHL.Results The concentration of DNA extracted from 24 cyst wall samples was significantly higher than in the 9/24 available matched cyst fluid samples. Cyst wall biopsy was able to diagnose 19/24 cysts (5 high risk, 6 intraductal papillary mucinous neoplasm and 4 benign). The sensitivity, specificity and diagnostic accuracy for standard of care was 66.6%, 50% and 63.1% respectively and for standard of care with NGS was 100%, 50% and 89.4% respectively.Conclusions Cyst wall biopsy performs well in diagnosing cysts but was inadequate in 5/24 patients. NGS data correlates well with histology and may aid in diagnosis and risk stratification of pancreatic cysts

High intake of vegetables is linked to lower white blood cell profile and the effect is mediated by the gut microbiome

Background: Chronic inflammation, which can be modulated by diet, is linked to high white blood cell counts and correlates with higher cardiometabolic risk and risk of more severe infections, as in the case of COVID-19.Methods: Here, we assessed the association between white blood cell profile (lymphocytes, basophils, eosinophils, neutrophils, monocytes and total white blood cells) as markers of chronic inflammation, habitual diet and gut microbiome composition (determined by sequencing of the 16S RNA) in 986 healthy individuals from the PREDICT-1 nutritional intervention study. We then investigated whether the gut microbiome mediates part of the benefits ofvegetable intake on lymphocyte counts.Results: Higher levels of white blood cells, lymphocytes and basophils were all significantly correlated with lower habitual intake of vegetables, with vegetable intake explaining between 3.59 and 6.58% of variation in white blood cells after adjusting for covariates and multiple testing using false discovery rate (q < 0.1). No such association was seen with fruit intake. A mediation analysis found that 20.00% of the effect of vegetable intake on lymphocyte counts was mediated by one bacterial genus, Collinsella, known to increase with the intake of processed foods and previously associated with fatty liver disease. We further correlated white blood cells to other inflammatory markers including IL6 and GlycA, fasting and post -prandial glucose levels and found a significant relationship between inflammation and diet.Conclusion: A habitual diet high in vegetables, but not fruits, is linked to a lower inflammatory profile for white blood cells, and a fifth of the effect is mediated by the genus Collinsella.Trial registration: The ClinicalTrials.gov registration identifier is NCT03479866

Nanopore sequencing from extraction-free direct PCR of dried serum spots for portable hepatitis B virus drug-resistance typing

© 2020 Background: Effective drug regimens for the treatment of hepatitis B virus (HBV) infections are essential to achieve the World Health Organisation commitment to eliminate viral hepatitis by 2030. Lamivudine (3TC) is widely used in countries with high levels of chronic HBV, however resistance has been shown to occur in up to 50 % of individuals receiving continuous monotherapy for 4 years. Telbivudine (LdT) is now more commonly used in place of lamivudine but is ineffective against 3TC-resistant HBV. Genotyping and identification of resistanceassociated substitutions (RAS) is not practical in many locations. Objectives: A novel assay was designed to enable HBV genotyping and characterisation of resistance mutations directly from serum samples stored on filter paper, using Sanger and MinION sequencing. Study design: The assay was applied to a cohort of 30 samples stored on filter paper for several years with HBV viral loads ranging from 8.2 × 108 to 635 IU/mL. A set of 6 high-titre samples were used in a proof-of-principle study using the MinION sequencer. Results: The assay allowed determination of HBV genotype and elucidation of RAS down to 600 IU/mL using a 550bp amplicon. Sequencing of a 1.2 kb amplicon using a MinION sequencer gave results consistent with Sanger sequencing and allowed the identification of minor populations of variants. Conclusions: We present two approaches for reliable HBV sequencing and RAS identification using methods suitable for resource-limited environments. This is the first demonstration of extraction-free DNA sequencing direct from DSS using MinION and these workflows are adaptable to the investigation of other DNA viruses