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1.
Quantifying technical confounders in microbiome studies.
Bartolomaeus, TUP, Birkner, T, Bartolomaeus, H, Löber, U, Avery, EG, Mähler, A, Weber, D, Kochlik, B, Balogh, A, Wilck, N, et al
Cardiovascular research. 2021;(3):863-875
Abstract
AIMS: Recent technical developments have allowed the study of the human microbiome to accelerate at an unprecedented pace. Methodological differences may have considerable impact on the results obtained. Thus, we investigated how different storage, isolation, and DNA extraction methods can influence the characterization of the intestinal microbiome, compared to the impact of true biological signals such as intraindividual variability, nutrition, health, and demographics. METHODS AND RESULTS An observative cohort study in 27 healthy subjects was performed. Participants were instructed to collect stool samples twice spaced by a week, using six different methods (naive and Zymo DNA/RNA Shield on dry ice, OMNIgene GUT, RNALater, 95% ethanol, Zymo DNA/RNA Shield at room temperature). DNA extraction from all samples was performed comparatively using QIAamp Power Fecal and ZymoBIOMICS DNA Kits. 16S rRNA sequencing of the gut microbiota as well as qPCRs were performed on the isolated DNA. Metrics included alpha diversity as well as multivariate and univariate comparisons of samples, controlling for covariate patterns computationally. Interindividual differences explained 7.4% of overall microbiome variability, whereas the choice of DNA extraction method explained a further 5.7%. At phylum level, the tested kits differed in their recovery of Gram-positive bacteria, which is reflected in a significantly skewed enterotype distribution. CONCLUSION DNA extraction methods had the highest impact on observed microbiome variability, and were comparable to interindividual differences, thus may spuriously mimic the microbiome signatures of various health and nutrition factors. Conversely, collection methods had a relatively small influence on microbiome composition. The present study provides necessary insight into the technical variables which can lead to divergent results from seemingly similar study designs. We anticipate that these results will contribute to future efforts towards standardization of microbiome quantification procedures in clinical research.
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2.
Dietary Fibre Intake Is Associated with Serum Levels of Uraemic Toxins in Children with Chronic Kidney Disease.
El Amouri, A, Snauwaert, E, Foulon, A, Vande Moortel, C, Van Dyck, M, Van Hoeck, K, Godefroid, N, Glorieux, G, Van Biesen, W, Vande Walle, J, et al
Toxins. 2021;(3)
Abstract
Imbalanced colonic microbial metabolism plays a pivotal role in generating protein-bound uraemic toxins (PBUTs), which accumulate with deteriorating kidney function and contribute to the uraemic burden of children with chronic kidney disease (CKD). Dietary choices impact the gut microbiome and metabolism. The aim of this study was to investigate the relation between dietary fibre and gut-derived PBUTs in paediatric CKD. Sixty-one (44 male) CKD children (9 ± 5 years) were prospectively followed for two years. Dietary fibre intake was evaluated by either 24-h recalls (73%) or 3-day food records (27%) at the same time of blood sampling for assessment of total and free serum levels of different PBUTs using liquid chromatography. We used linear mixed models to assess associations between fibre intake and PBUT levels. We found an inverse association between increase in fibre consumption (g/day) and serum concentrations of free indoxyl sulfate (-3.1% (-5.9%; -0.3%) (p = 0.035)), free p-cresyl sulfate (-2.5% (-4.7%; -0.3%) (p = 0.034)), total indole acetic acid (IAA) (-1.6% (-3.0%; -0.3%) (p = 0.020)), free IAA (-6.6% (-9.3%; -3.7%) (p < 0.001)), total serum p-cresyl glucuronide (pCG) (-3.0% (-5.6%; -0.5%) (p = 0.021)) and free pCG levels (-3.3% (-5.8%; -0.8%) (p = 0.010)). The observed associations between dietary fibre intake and the investigated PBUTs highlight potential benefits of fibre intake for the paediatric CKD population. The present observational findings should inform and guide adaptations of dietary prescriptions in children with CKD.
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3.
Obesity in Humans Is Characterized by Gut Inflammation as Shown by Pro-Inflammatory Intestinal Macrophage Accumulation.
Rohm, TV, Fuchs, R, Müller, RL, Keller, L, Baumann, Z, Bosch, AJT, Schneider, R, Labes, D, Langer, I, Pilz, JB, et al
Frontiers in immunology. 2021;:668654
Abstract
Chronic low-grade inflammation is a hallmark of obesity and associated with cardiovascular complications. However, it remains unclear where this inflammation starts. As the gut is constantly exposed to food, gut microbiota, and metabolites, we hypothesized that mucosal immunity triggers an innate inflammatory response in obesity. We characterized five distinct macrophage subpopulations (P1-P5) along the gastrointestinal tract and blood monocyte subpopulations (classical, non-classical, intermediate), which replenish intestinal macrophages, in non-obese (BMI<27kg/m2) and obese individuals (BMI>32kg/m2). To elucidate factors that potentially trigger gut inflammation, we correlated these subpopulations with cardiovascular risk factors and lifestyle behaviors. In obese individuals, we found higher pro-inflammatory macrophages in the stomach, duodenum, and colon. Intermediate blood monocytes were also increased in obesity, suggesting enhanced recruitment to the gut. We identified unhealthy lifestyle habits as potential triggers of gut and systemic inflammation (i.e., low vegetable intake, high processed meat consumption, sedentary lifestyle). Cardiovascular risk factors other than body weight did not affect the innate immune response. Thus, obesity in humans is characterized by gut inflammation as shown by accumulation of pro-inflammatory intestinal macrophages, potentially via recruited blood monocytes. Understanding gut innate immunity in human obesity might open up new targets for immune-modulatory treatments in metabolic disease.
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Human intestinal lipid storage through sequential meals reveals faster dinner appearance is associated with hyperlipidemia.
Jacome-Sosa, M, Hu, Q, Manrique-Acevedo, CM, Phair, RD, Parks, EJ
JCI insight. 2021;(15)
Abstract
BackgroundIt is increasingly recognized that intestinal cells can store lipids after a meal, yet the effect of this phenomenon on lipid absorption patterns in insulin resistance remains unknown.MethodsThe kinetics of meal fat appearance were measured in insulin-sensitive (IS, n = 8) and insulin-resistant (IR, n = 8) subjects after sequential, isotopically labeled lunch and dinner meals. Plasma dynamics on triacylglycerol-rich (TAG-rich) lipoproteins and plasma hormones were analyzed using a nonlinear, non-steady state kinetic model.ResultsAt the onset of dinner, IS subjects showed an abrupt plasma appearance of lunch lipid consistent with the "second-meal effect," followed by slower appearance of dinner fat in plasma, resulting in reduced accumulation of dinner TAG of 48% compared with lunch. By contrast, IR subjects exhibited faster meal TAG appearance rates after both lunch and dinner. This effect of lower enterocyte storage between meals was associated with greater nocturnal and next-morning hyperlipidemia. The biochemical data and the kinetic analysis of second-meal effect dynamics are consistent with rapid secretion of stored TAG bypassing lipolysis and resynthesis. In addition, the data are consistent with a role for the diurnal pattern of plasma leptin in regulating the processing of dietary lipid.ConclusionThese data support the concept that intestinal lipid storage may be physiologically beneficial in IS subjects.Trial registrationClinicalTrials.gov NCT02020343.FundingThis study was supported by a grant from the American Diabetes Association (grant 1-13-TS-12).
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5.
Re-evaluating Blood Markers as Predictors of Outcome in Multivisceral and Intestinal Transplantation.
Cheung, D, Garcia, J, Beduschi, T, Langshaw, A, Arheart, K, Wunsch, C, Vianna, R, Gonzalez, IA
Transplantation proceedings. 2021;(2):696-704
Abstract
BACKGROUND Multivisceral transplant (MVTx) and isolated intestinal transplant (ITx) are complex surgical procedures. The subsequent proinflammatory state in the immediate postoperative period makes interpretation of blood markers difficult. METHOD We aimed to establish the course of various blood markers after MVTx/ITx, and to evaluate their use as diagnostic markers of complications. This was a single center prospective cohort. We analyzed blood markers collected preoperatively, on alternate days for the first postoperative week, and then weekly for 4 weeks. This study was in compliance with The Declaration of Helsinki. RESULTS Over a 16-month period (July 2017-October 2018), 20 subjects aged 2 to 67 years with a median age of 24.5 years received MVTx/ITx. Twelve recipients (60%) had an infection. Neutrophil lymphocyte count ratio (NLCR) was higher than established upper limits of normal, regardless of infection status. NLCR and white blood cell count were useful to identify infected MVTx/ITx recipients, with P values <.05 for 2 and 1 of 7 time points post transplant, respectively. Higher preoperative eosinophil% predicted future acute cellular rejection (P value .023). CONCLUSIONS This is the first study to extensively track the course of blood markers post MVTx/ITx and identified NLCR and white blood cell count as potential diagnostic blood markers of infection.
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6.
Overweight and underweight status are linked to specific gut microbiota and intestinal tricarboxylic acid cycle intermediates.
Wan, Y, Yuan, J, Li, J, Li, H, Yin, K, Wang, F, Li, D
Clinical nutrition (Edinburgh, Scotland). 2020;(10):3189-3198
Abstract
BACKGROUND & AIMS Intestinal short-chain fatty acids have been demonstrated to modulate host energy metabolism and are elevated in overweight and obese individuals. We hypothesized that other intestinal energy products especially tricarboxylic acid (TCA) cycle intermediates might also related to overweight status. In addition, little information is available regarding to the potential relationship between gut microbiota and underweight status. Therefore, the aim of this study was to investigate whether gut microbiota and intestinal energy metabolites differ in underweight, normal weight, and overweight individuals, and their correlations with host cardiometabolic risk factors. SUBJECTS/METHODS Gut microbiome, intestinal energy metabolites, circulating cardiometabolic risk factors, and proinflammatory markers were determined in 29 underweight, 67 normal weight, and 67 overweight adults. RESULTS The fecal concentrations of succinic acid, fumaric acid, malic acid, propionic acid, and adipic acid were significantly increased in the overweight individuals in parallel with a higher relative abundance of Veillonellacea after adjusting for multiple comparisons (all p < 0.05). The intestinal concentration of TCA cycle intermediate succinic acid was positively associated with body weight (r = 0.28, p = 0.04), and malic acid were in positive association with circulating total cholesterol, low-density lipoprotein cholesterol, and interleukin-1β (all r > 0.25, p < 0.05). Compared with the normal weight individuals, the gut microbial α-diversity was lower in the overweight (p = 0.007 for Shannon index and p = 0.009 for Ace index) and underweight (p = 0.05 for Shannon index and p = 0.08 for Ace index) groups. However, no significant differences in the overall gut microbiota composition were observed among the three groups. CONCLUSIONS Our findings revealed that low gut microbiota diversity was associated with both overweight and underweight status. Intestinal TCA cycle intermediates were associated with overweight development and might be potential markers for future studies related to gut microbiota and host cardiometabolic health.
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7.
Predicting intestinal recovery after necrotizing enterocolitis in preterm infants.
Kuik, SJ, Kalteren, WS, Mebius, MJ, Bos, AF, Hulscher, JBF, Kooi, EMW
Pediatric research. 2020;(5):903-909
Abstract
BACKGROUND Intestinal recovery after NEC is difficult to predict in individuals. We evaluated whether several biomarkers predict intestinal recovery after NEC in preterm infants. METHODS We measured intestinal tissue oxygen saturation (rintSO2) and collected urinary intestinal-fatty acid binding protein (I-FABPu) levels 0-24 h and 24-48 h after NEC onset, and before and after the first re-feed. We assessed intestinal recovery in two ways: time to full enteral feeding (FEFt; below or equal/above group's median) and development of post-NEC complications (recurrent NEC/post-NEC stricture). We determined whether the rintSO2, its range, and I-FABPu differed between groups. RESULTS We included 27 preterm infants who survived NEC (Bell's stage ≥ 2). Median FEFt was 14 [IQR: 12-23] days. Biomarkers only predicted intestinal recovery after the first re-feed. Mean rintSO2 ≥ 53% combined with mean rintSO2range ≥ 50% predicted FEFt < 14 days with OR 16.7 (CI: 2.3-122.2). The rintSO2range was smaller (33% vs. 51%, p < 0.01) and I-FABPu was higher (92.4 vs. 25.5 ng/mL, p = 0.03) in case of post-NEC stricture, but not different in case of recurrent NEC, compared with infants without complications. CONCLUSION The rintSO2, its range, and I-FABPu after the first re-feed after NEC predicted intestinal recovery. These biomarkers have potential value in individualizing feeding regimens after NEC.
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8.
Microbiome Signatures Associated With Steatohepatitis and Moderate to Severe Fibrosis in Children With Nonalcoholic Fatty Liver Disease.
Schwimmer, JB, Johnson, JS, Angeles, JE, Behling, C, Belt, PH, Borecki, I, Bross, C, Durelle, J, Goyal, NP, Hamilton, G, et al
Gastroenterology. 2019;(4):1109-1122
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Abstract
BACKGROUND & AIMS The intestinal microbiome might affect the development and severity of nonalcoholic fatty liver disease (NAFLD). We analyzed microbiomes of children with and without NAFLD. METHODS We performed a prospective, observational, cross-sectional study of 87 children (age range, 8-17 years) with biopsy-proven NAFLD and 37 children with obesity without NAFLD (controls). Fecal samples were collected and microbiome composition and functions were assessed using 16S ribosomal RNA amplicon sequencing and metagenomic shotgun sequencing. Microbial taxa were identified using zero-inflated negative binomial modeling. Genes contributing to bacterial pathways were identified using gene set enrichment analysis. RESULTS Fecal microbiomes of children with NAFLD had lower α-diversity than those of control children (3.32 vs 3.52, P = .016). Fecal microbiomes from children with nonalcoholic steatohepatitis (NASH) had the lowest α-diversity (control, 3.52; NAFLD, 3.36; borderline NASH, 3.37; NASH, 2.97; P = .001). High abundance of Prevotella copri was associated with more severe fibrosis (P = .036). Genes for lipopolysaccharide biosynthesis were enriched in microbiomes from children with NASH (P < .001). Classification and regression tree model with level of alanine aminotransferase and relative abundance of the lipopolysaccharide pathway gene encoding 3-deoxy-d-manno-octulosonate 8-phosphate-phosphatase identified patients with NASH with an area under the receiver operating characteristic curve value of 0.92. Genes involved in flagellar assembly were enriched in the fecal microbiomes of patients with moderate to severe fibrosis (P < .001). Classification and regression tree models based on level of alanine aminotransferase and abundance of genes encoding flagellar biosynthesis protein had good accuracy for identifying case children with moderate to severe fibrosis (area under the receiver operating characteristic curve, 0.87). CONCLUSIONS In an analysis of fecal microbiomes of children with NAFLD, we associated NAFLD and NASH with intestinal dysbiosis. NAFLD and its severity were associated with greater abundance of genes encoding inflammatory bacterial products. Alterations to the intestinal microbiome might contribute to the pathogenesis of NAFLD and be used as markers of disease or severity.
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Investigating Intestinal Glucagon After Roux-en-Y Gastric Bypass Surgery.
Jorsal, T, Wewer Albrechtsen, NJ, Christensen, MM, Mortensen, B, Wandall, E, Langholz, E, Friis, S, Worm, D, Ørskov, C, Støving, RK, et al
The Journal of clinical endocrinology and metabolism. 2019;(12):6403-6416
Abstract
CONTEXT After Roux-en-Y gastric bypass (RYGB) surgery, postprandial plasma glucagon concentrations have been reported to increase. This occurs despite concomitant improved glucose tolerance and increased circulating plasma concentrations of insulin and the glucagon-inhibiting hormone glucagon-like peptide 1 (GLP-1). OBJECTIVE To investigate whether RYGB-induced hyperglucagonemia may be derived from the gut. DESIGN AND SETTING Substudy of a prospective cross-sectional study at a university hospital in Copenhagen, Denmark. PARTICIPANTS Morbidly obese individuals undergoing RYGB (n = 8) with or without type 2 diabetes. INTERVENTIONS Three months before and after RYGB, participants underwent upper enteroscopy with retrieval of gastrointestinal mucosal biopsy specimens. Mixed-meal tests were performed 1 week and 3 months before and after RYGB. MAIN OUTCOME MEASURES The 29-amino acid glucagon concentrations in plasma and in mucosal gastrointestinal biopsy specimens were assessed using mass spectrometry-validated immunoassays, and a new monoclonal antibody reacting with immunoreactive glucagon was used for immunohistochemistry. RESULTS Postprandial plasma concentrations of glucagon after RYGB were increased. Expression of the glucagon gene in the small intestine increased after surgery. Glucagon was identified in the small-intestine biopsy specimens obtained after, but not before, RYGB. Immunohistochemically, mucosal biopsy specimens from the small intestine harbored cells costained for GLP-1 and immunoreactive glucagon. CONCLUSION Increased concentrations of glucagon were observed in small-intestine biopsy specimens and postprandially in plasma after RYGB. The small intestine harbored cells immunohistochemically costaining for GLP-1 and glucagon-like immunoreactivity after RYGB. Glucagon derived from small-intestine enteroendocrine l cells may contribute to postprandial plasma concentrations of glucagon after RYGB.
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Gut-Derived Serum Lipopolysaccharide is Associated With Enhanced Risk of Major Adverse Cardiovascular Events in Atrial Fibrillation: Effect of Adherence to Mediterranean Diet.
Pastori, D, Carnevale, R, Nocella, C, Novo, M, Santulli, M, Cammisotto, V, Menichelli, D, Pignatelli, P, Violi, F
Journal of the American Heart Association. 2017;(6)
Abstract
BACKGROUND Gut microbiota is emerging as a novel risk factor for atherothrombosis, but the predictive role of gut-derived lipopolysaccharide (LPS) is unknown. We analyzed (1) the association between LPS and major adverse cardiovascular events (MACE) in atrial fibrillation (AF) and (2) its relationship with adherence to a Mediterranean diet (Med-diet). METHODS AND RESULTS This was a prospective single-center study including 912 AF patients treated with vitamin K antagonists (3716 patient-years). The primary end point was a composite of MACE. Baseline serum LPS, adherence to Med-diet (n=704), and urinary excretion of 11-dehydro-thromboxane B2 (TxB2, n=852) were investigated. Mean age was 73.5 years; 42.9% were women. A total of 187 MACE (5.0% per year) occurred: 54, 59, and 74 in the first, second, and third tertile of LPS, respectively (log-rank test P=0.004). Log-LPS (hazard ratio 1.194, P=0.009), age (hazard ratio 1.083, P<0.001), and previous cerebrovascular (hazard ratio 1.634, P=0.004) and cardiac events (hazard ratio 1.822, P<0.001) were predictors of MACE. In the whole cohort, AF (versus sinus rhythm) (β 0.087, P=0.014) and low-density lipoprotein cholesterol (β 0.069, P=0.049) were associated with circulating LPS. Furthermore, Med-diet score (β -0.137, P<0.001) was predictive of log-LPS, with fruits (β -0.083, P=0.030) and legumes (β -0.120, P=0.002) negatively associated with log-LPS levels. Log-LPS and log-TxB2 were highly correlated (r=0.598, P<0.001). Log-LPS (β 0.574, P<0.001) and Med-diet score (β -0.218, P<0.001) were significantly associated with baseline urinary excretion of TxB2. CONCLUSIONS In this cohort of AF patients, LPS levels were predictive of MACE and negatively affected by high adherence to Med-diet. LPS may contribute to MACE incidence in AF by increasing platelet activation.