Giardia hinders growth by disrupting nutrient metabolism independent of inflammatory enteropathy.

Giardia lamblia (Giardia) is among the most common intestinal pathogens in children in low- and middle-income countries (LMICs). Although Giardia associates with early-life linear growth restriction, mechanistic explanations for Giardia-associated growth impairments remain elusive. Unlike other intestinal pathogens associated with constrained linear growth that cause intestinal or systemic inflammation or both, Giardia seldom associates with chronic inflammation in these children. Here we leverage the MAL-ED longitudinal birth cohort and a model of Giardia mono-association in gnotobiotic and immunodeficient mice to propose an alternative pathogenesis of this parasite. In children, Giardia results in linear growth deficits and gut permeability that are dose-dependent and independent of intestinal markers of inflammation. The estimates of these findings vary between children in different MAL-ED sites. In a representative site, where Giardia associates with growth restriction, infected children demonstrate broad amino acid deficiencies, and overproduction of specific phenolic acids, byproducts of intestinal bacterial amino acid metabolism. Gnotobiotic mice require specific nutritional and environmental conditions to recapitulate these findings, and immunodeficient mice confirm a pathway independent of chronic T/B cell inflammation. Taken together, we propose a new paradigm that Giardia-mediated growth faltering is contingent upon a convergence of this intestinal protozoa with nutritional and intestinal bacterial factors.

Circulating IgG Levels in SARS-CoV-2 Convalescent Individuals in Cyprus.

Long-term persistence and the heterogeneity of humoral response to SARS-CoV-2 have not yet been thoroughly investigated. The aim of this work is to study the production of circulating immunoglobulin class G (IgG) antibodies against SARS-CoV-2 in individuals with past infection in Cyprus. Individuals of the general population, with or without previous SARS-CoV-2 infection, were invited to visit the Biobank at the Center of Excellence in Biobanking and Biomedical Research of the University of Cyprus. Serum IgG antibodies were measured using the SARS-CoV-2 IgG and the SARS-CoV-2 IgG II Quant assays of Abbott Laboratories. Antibody responses to SARS-CoV-2 were also evaluated against participants' demographic and clinical data. All statistical analyses were conducted in Stata 16. The median levels of receptor binding domain (RBD)-specific IgG in 969 unvaccinated individuals, who were reportedly infected between November 2020 and September 2021, were 432.1 arbitrary units (AI)/mL (interquartile range-IQR: 182.4-1147.3). Higher antibody levels were observed in older participants, males, and those who reportedly developed symptoms or were hospitalized. The RBD-specific IgG levels peaked at three months post symptom onset and subsequently decreased up to month six, with a slower decay thereafter. IgG response to the RBD of SARS-CoV-2 is bi-phasic with considerable titer variability. Levels of IgG are significantly associated with several parameters, including age, gender, and severity of symptoms.

Associations between biomarkers of environmental enteric dysfunction and oral rotavirus vaccine immunogenicity in rural Zimbabwean infants.

BACKGROUND: Oral rotavirus vaccines (RVV) are poorly immunogenic in low-income countries. Environmental enteric dysfunction (EED) resulting from poor water, sanitation and hygiene (WASH) may contribute. We therefore tested associations between EED and RVV immunogenicity, and evaluated the effect of improved WASH on EED. METHODS: We measured nine biomarkers of EED among Zimbabwean infants born to mothers enrolled in a cluster-randomised 2 × 2 factorial trial of improved WASH and improved feeding between November 2012 and March 2015 (NCT01824940). We used multivariable regression to determine associations between EED biomarkers and RVV seroconversion, seropositivity and geometric mean titer. Log-binomial regression was used to evaluate the effect of improved WASH on EED. FINDINGS: Among 303 infants with EED biomarkers and immunogenicity data, plasma intestinal fatty-acid binding protein and stool myeloperoxidase were positively associated with RVV seroconversion; adjusted RR 1.63 (95%CI 1.04, 2.57) and 1.29 (95%CI 1.01, 1.65), respectively. There were no other associations between RVV immunogenicity and either individual biomarkers or EED domains (intestinal permeability, intestinal damage, intestinal inflammation and microbial translocation). EED biomarkers did not differ between randomised WASH and non-WASH groups. INTERPRETATION: We found no evidence that EED was associated with poor RVV immunogenicity. Contrary to our hypothesis, there was weak evidence that EED was associated with increased seroconversion. EED biomarkers were not affected by a package of household-level WASH interventions.

Characterizing the breast cancer lipidome and its interaction with the tissue microbiota.

Breast cancer is the most diagnosed cancer amongst women worldwide. We have previously shown that there is a breast microbiota which differs between women who have breast cancer and those who are disease-free. To better understand the local biochemical perturbations occurring with disease and the potential contribution of the breast microbiome, lipid profiling was performed on non-tumor breast tissue collected from 19 healthy women and 42 with breast cancer. Here we identified unique lipid signatures between the two groups with greater amounts of lysophosphatidylcholines and oxidized cholesteryl esters in the tissue from women with breast cancer and lower amounts of ceramides, diacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. By integrating these lipid signatures with the breast bacterial profiles, we observed that Gammaproteobacteria and those from the class Bacillus, were negatively correlated with ceramides, lipids with antiproliferative properties. In the healthy tissues, diacylglyerols were positively associated with Acinetobacter, Lactococcus, Corynebacterium, Prevotella and Streptococcus. These bacterial groups were found to possess the genetic potential to synthesize these lipids. The cause-effect relationships of these observations and their contribution to disease patho-mechanisms warrants further investigation for a disease afflicting millions of women around the world.

Postnatal prebiotic supplementation in rats affects adult anxious behaviour, hippocampus, electrophysiology, metabolomics, and gut microbiota.

We have shown previously that prebiotic (Bimuno galacto-oligosacharides, B-GOS®) administration to neonatal rats increased hippocampal NMDAR proteins. The present study has investigated the effects of postnatal B-GOS® supplementation on hippocampus-dependent behavior in young, adolescent, and adult rats and applied electrophysiological, metabolomic and metagenomic analyses to explore potential underlying mechanisms. The administration of B-GOS® to suckling, but not post-weaned, rats reduced anxious behavior until adulthood. Neonatal prebiotic intake also reduced the fast decay component of hippocampal NMDAR currents, altered age-specific trajectories of the brain, intestinal, and liver metabolomes, and reduced abundance of fecal Enterococcus and Dorea bacteria. Our data are the first to show that prebiotic administration to rats during a specific postnatal period has long-term effects on behavior and hippocampal physiology. The study also suggests that early-life prebiotic intake may affect host brain function through the reduction of stress-related gut bacteria rather than increasing the proliferation of beneficial microbes.

Sex Disparities and Neutralizing-Antibody Durability to SARS-CoV-2 Infection in Convalescent Individuals.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has now caused over 2 million deaths worldwide and continues to expand. Currently, much is unknown about functionally neutralizing human antibody responses and durability to SARS-CoV-2 months after infection or the reason for the discrepancy in COVID-19 disease and sex. Using convalescent-phase sera collected from 101 COVID-19-recovered individuals 21 to 212 days after symptom onset with 48 additional longitudinal samples, we measured functionality and durability of serum antibodies. We also evaluated associations of individual demographic and clinical parameters with functional neutralizing antibody responses to COVID-19. We found robust antibody durability out to 6 months, as well as significant positive associations with the magnitude of the neutralizing antibody response and male sex and in individuals with cardiometabolic comorbidities. IMPORTANCE In this study, we found that neutralizing antibody responses in COVID-19-convalescent individuals vary in magnitude but are durable and correlate well with receptor binding domain (RBD) Ig binding antibody levels compared to other SARS-CoV-2 antigen responses. In our cohort, higher neutralizing antibody titers are independently and significantly associated with male sex compared to female sex. We also show for the first time that higher convalescent antibody titers in male donors are associated with increased age and symptom grade. Furthermore, cardiometabolic comorbidities are associated with higher antibody titers independently of sex. Here, we present an in-depth evaluation of serologic, demographic, and clinical correlates of functional antibody responses and durability to SARS-CoV-2 which supports the growing literature on sex discrepancies regarding COVID-19 disease morbidity and mortality, as well as functional neutralizing antibody responses to SARS-CoV-2.

Sex disparities and neutralizing antibody durability to SARS-CoV-2 infection in convalescent individuals.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has now caused over 2 million deaths worldwide and continues to expand. Currently, much is unknown about functionally neutralizing human antibody responses and durability to SARS-CoV-2. Using convalescent sera collected from 101 COVID-19 recovered individuals 21-212 days after symptom onset with forty-eight additional longitudinal samples, we measured functionality and durability of serum antibodies. We also evaluated associations between individual demographic and clinical parameters with functional neutralizing antibody responses to COVID-19. We found robust antibody durability out to six months, as well as significant positive associations with the magnitude of the neutralizing antibody response and male sex. We also show that SARS-CoV-2 convalescent neutralizing antibodies are higher in individuals with cardio-metabolic comorbidities.

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

Effects of improved water, sanitation, and hygiene and improved complementary feeding on environmental enteric dysfunction in children in rural Zimbabwe: A cluster-randomized controlled trial.

BACKGROUND: Environmental enteric dysfunction (EED) may be an important modifiable cause of child stunting. We described the evolution of EED biomarkers from birth to 18 months in rural Zimbabwe and tested the independent and combined effects of improved water, sanitation, and hygiene (WASH), and improved infant and young child feeding (IYCF), on EED. METHODOLOGY AND FINDINGS: The Sanitation Hygiene Infant Nutrition Efficacy (SHINE) trial was a 2x2 factorial cluster-randomised trial of improved IYCF and improved WASH on child stunting and anaemia at 18 months of age. 1169 infants born to HIV-negative mothers provided plasma and faecal specimens at 1, 3, 6, 12, and 18 months of age. We measured EED biomarkers that reflect all domains of the hypothesized pathological pathway. Markers of intestinal permeability and intestinal inflammation declined over time, while markers of microbial translocation and systemic inflammation increased between 1-18 months. Markers of intestinal damage (I-FABP) and repair (REG-1ß) mirrored each other, and citrulline (a marker of intestinal epithelial mass) increased from 6 months of age, suggesting dynamic epithelial turnover and regeneration in response to enteric insults. We observed few effects of IYCF and WASH on EED after adjustment for multiple comparisons. The WASH intervention decreased plasma IGF-1 at 3 months (ß:0.89, 95%CI:0.81,0.98) and plasma kynurenine at 12 months (ß: 0.92, 95%CI:0.87,0.97), and increased plasma IGF-1 at 18 months (ß:1.15, 95%CI:1.05,1.25), but these small WASH effects did not translate into improved growth. CONCLUSIONS: Overall, we observed dynamic trends in EED but few effects of IYCF or WASH on biomarkers during the first 18 months after birth, suggesting that these interventions did not impact EED. Transformative WASH interventions are required to prevent or ameliorate EED in low-income settings.

Enteropathogenic Escherichia coli Infection Induces Diarrhea, Intestinal Damage, Metabolic Alterations, and Increased Intestinal Permeability in a Murine Model.

Enteropathogenic E. coli (EPEC) are recognized as one of the leading bacterial causes of infantile diarrhea worldwide. Weaned C57BL/6 mice pretreated with antibiotics were challenged orally with wild-type EPEC or escN mutant (lacking type 3 secretion system) to determine colonization, inflammatory responses and clinical outcomes during infection. Antibiotic disruption of intestinal microbiota enabled efficient colonization by wild-type EPEC resulting in growth impairment and diarrhea. Increase in inflammatory biomarkers, chemokines, cellular recruitment and pro-inflammatory cytokines were observed in intestinal tissues. Metabolomic changes were also observed in EPEC infected mice with changes in tricarboxylic acid (TCA) cycle intermediates, increased creatine excretion and shifts in gut microbial metabolite levels. In addition, by 7 days after infection, although weights were recovering, EPEC-infected mice had increased intestinal permeability and decreased colonic claudin-1 levels. The escN mutant colonized the mice with no weight loss or increased inflammatory biomarkers, showing the importance of the T3SS in EPEC virulence in this model. In conclusion, a murine infection model treated with antibiotics has been developed to mimic clinical outcomes seen in children with EPEC infection and to examine potential roles of selected virulence traits. This model can help in further understanding mechanisms involved in the pathogenesis of EPEC infections and potential outcomes and thus assist in the development of potential preventive or therapeutic interventions.

Characterizing the metabolic perturbations induced by activity-based anorexia in the C57Bl/6 mouse using 1H NMR spectroscopy.

BACKGROUND & AIMS: Anorexia nervosa (AN) is a severe psychological and potentially life-threatening eating disorder. The activity-based anorexia (ABA) mouse model is commonly used to investigate physiological abnormalities associated with this disorder. Characterizing the holistic biochemical alterations induced by anorexia is essential to understanding AN pathophysiology as well as to define biomarkers for prognosis. METHODS: To unravel the adaptive biochemical mechanisms occurring in this model in response to self-starvation, the urinary, plasma and fecal metabolic phenotypes of mice under different experimental conditions were compared. This included control mice with and without physical activity (CT and CTPA mice), a group with limited food access (LFA), and a group with both limited food access and physical activity (ABA). Using 1H nuclear magnetic resonance (NMR) spectroscopy, several biochemical perturbations were observed. RESULTS: Physical activity altered the abundance of 14 fecal metabolites, including those involved in gut microbial metabolism and proteolysis. Food restriction disrupted a wide range of metabolic pathways including gut microbial metabolism, proteolysis and fatty acid breakdown (24 urinary and 6 plasma metabolites). The combined impact of food restriction and physical activity resulted in the same pattern of metabolic disruption (24 urine, 6 plasma). CONCLUSIONS: This work defined the metabolic signatures of ABA mice and provides novel insights into biological adaptations of mice in response to both food restriction and physical activity. These results should be further confirmed in AN patients.

A murine model of diarrhea, growth impairment and metabolic disturbances with Shigella flexneri infection and the role of zinc deficiency.

Shigella is one of the major enteric pathogens worldwide. We present a murine model of S. flexneri infection and investigate the role of zinc deficiency (ZD). C57BL/6 mice fed either standard chow (HC) or ZD diets were pretreated with an antibiotic cocktail and received S. flexneri strain 2457T orally. Antibiotic pre-treated ZD mice showed higher S. flexneri colonization than non-treated mice. ZD mice showed persistent colonization for at least 50 days post-infection (pi). S. flexneri-infected mice showed significant weight loss, diarrhea and increased levels of fecal MPO and LCN in both HC and ZD fed mice. S. flexneri preferentially colonized the colon, caused epithelial disruption and inflammatory cell infiltrate, and promoted cytokine production which correlated with weight loss and histopathological changes. Infection with S. flexneri ΔmxiG (critical for type 3 secretion system) did not cause weight loss or diarrhea, and had decreased stool shedding duration and tissue burden. Several biochemical changes related to energy, inflammation and gut-microbial metabolism were observed. Zinc supplementation increased weight gains and reduced intestinal inflammation and stool shedding in ZD infected mice. In conclusion, young antibiotic-treated mice provide a new model of oral S. flexneri infection, with ZD promoting prolonged infection outcomes.

Metabolic targets of watercress and PEITC in MCF-7 and MCF-10A cells explain differential sensitisation responses to ionising radiation.

PURPOSE: Watercress is a rich source of phytochemicals with anticancer potential, including phenethyl isothiocyanate (PEITC). We examined the potential for watercress extracts and PEITC to increase the DNA damage caused by ionising radiation (IR) in breast cancer cells and to be protective against radiation-induced collateral damage in healthy breast cells. The metabolic events that mediate such responses were explored using metabolic profiling. METHODS: 1H nuclear magnetic resonance spectroscopy-based metabolic profiling was coupled with DNA damage-related assays (cell cycle, Comet assay, viability assays) to profile the comparative effects of watercress and PEITC in MCF-7 breast cancer cells and MCF-10A non-tumorigenic breast cells with and without exposure to IR. RESULTS: Both the watercress extract and PEITC-modulated biosynthetic pathways of lipid and protein synthesis and resulted in changes in cellular bioenergetics. Disruptions to the redox balance occurred with both treatments in the two cell lines, characterised by shifts in the abundance of glutathione. PEITC enhanced the sensitivity of the breast cancer cells to IR increasing the effectiveness of the cancer-killing process. In contrast, watercress-protected non-tumorigenic breast cells from radiation-induced damage. These effects were driven by changes in the cellular content of the antioxidant glutathione following exposure to PEITC and other phytochemicals in watercress. CONCLUSION: These findings support the potential prophylactic impact of watercress during radiotherapy. Extracted compounds from watercress and PEITC differentially modulate cellular metabolism collectively enhancing the therapeutic outcomes of radiotherapy.

Attenuation of oxidative stress-induced lesions in skeletal muscle in a mouse model of obesity-independent hyperlipidaemia and atherosclerosis through the inhibition of Nox2 activity.

Obesity leading to hyperlipidaemia and atherosclerosis is recognised to induce morphological and metabolic changes in many tissues. However, hyperlipidaemia can occur in the absence of obesity. The impact of the latter scenario on skeletal muscle and liver is not understood sufficiently. In this regard, we used the Apolipoprotein E-deficient (ApoE-/-) mouse model, an established model of hyperlipidaemia and atherosclerosis, that does not become obese when subjected to a high-fat diet, to determine the impact of Western-type diet (WD) and ApoE deficiency on skeletal muscle morphological, metabolic and biochemical properties. To establish the potential of therapeutic targets, we further examined the impact of Nox2 pharmacological inhibition on skeletal muscle redox biology. We found ectopic lipid accumulation in skeletal muscle and the liver, and altered skeletal muscle morphology and intramuscular triacylglycerol fatty acid composition. WD and ApoE deficiency had a detrimental impact in muscle metabolome, followed by perturbed gene expression for fatty acid uptake and oxidation. Importantly, there was enhanced oxidative stress in the skeletal muscle and development of liver steatosis, inflammation and oxidative protein modifications. Pharmacological inhibition of Nox2 decreased reactive oxygen species production and protein oxidative modifications in the muscle of ApoE-/- mice subjected to a Western-type diet. This study provides key evidence to better understand the pathophysiology of skeletal muscle in the context of hyperlipidaemia and atherosclerosis and identifies Nox2 as a potential target for attenuating oxidative stress in skeletal muscle in a mouse model of obesity-independent hyperlipidaemia.

Inferring Metabolic Mechanisms of Interaction within a Defined Gut Microbiota.

The diversity and number of species present within microbial communities create the potential for a multitude of interspecies metabolic interactions. Here, we develop, apply, and experimentally test a framework for inferring metabolic mechanisms associated with interspecies interactions. We perform pairwise growth and metabolome profiling of co-cultures of strains from a model mouse microbiota. We then apply our framework to dissect emergent metabolic behaviors that occur in co-culture. Based on one of the inferences from this framework, we identify and interrogate an amino acid cross-feeding interaction and validate that the proposed interaction leads to a growth benefit in vitro. Our results reveal the type and extent of emergent metabolic behavior in microbial communities composed of gut microbes. We focus on growth-modulating interactions, but the framework can be applied to interspecies interactions that modulate any phenotype of interest within microbial communities.

A prebiotic intervention study in children with autism spectrum disorders (ASDs).

BACKGROUND: Different dietary approaches, such as gluten and casein free diets, or the use of probiotics and prebiotics have been suggested in autistic spectrum disorders in order to reduce gastrointestinal (GI) disturbances. GI symptoms are of particular interest in this population due to prevalence and correlation with the severity of behavioural traits. Nowadays, there is lack of strong evidence about the effect of dietary interventions on these problems, particularly prebiotics. Therefore, we assessed the impact of exclusion diets and a 6-week Bimuno® galactooligosaccharide (B-GOS®) prebiotic intervention in 30 autistic children. RESULTS: The results showed that children on exclusion diets reported significantly lower scores of abdominal pain and bowel movement, as well as lower abundance of Bifidobacterium spp. and Veillonellaceae family, but higher presence of Faecalibacterium prausnitzii and Bacteroides spp. In addition, significant correlations were found between bacterial populations and faecal amino acids in this group, compared to children following an unrestricted diet. Following B-GOS® intervention, we observed improvements in anti-social behaviour, significant increase of Lachnospiraceae family, and significant changes in faecal and urine metabolites. CONCLUSIONS: To our knowledge, this is the first study where the effect of exclusion diets and prebiotics has been evaluated in autism, showing potential beneficial effects. A combined dietary approach resulted in significant changes in gut microbiota composition and metabolism suggesting that multiple interventions might be more relevant for the improvement of these aspects as well as psychological traits. TRIAL REGISTRATION: NCT02720900 ; registered in November 2015.

A novel mouse model of Campylobacter jejuni enteropathy and diarrhea.

Campylobacter infections are among the leading bacterial causes of diarrhea and of 'environmental enteropathy' (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease with Campylobacter has been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche for Campylobacter infection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to test specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.

Enhanced exercise and regenerative capacity in a mouse model that violates size constraints of oxidative muscle fibres.

A central tenet of skeletal muscle biology is the existence of an inverse relationship between the oxidative fibre capacity and its size. However, robustness of this relationship is unknown. We show that superimposition of Estrogen-related receptor gamma (Errγ) on the myostatin (Mtn) mouse null background (Mtn(-/-)/Errγ(Tg/+)) results in hypertrophic muscle with a high oxidative capacity thus violating the inverse relationship between fibre size and oxidative capacity. We also examined the canonical view that oxidative muscle phenotype positively correlate with Satellite cell number, the resident stem cells of skeletal muscle. Surprisingly, hypertrophic fibres from Mtn(-/-)/Errγ(Tg/+) mouse showed satellite cell deficit which unexpectedly did not affect muscle regeneration. These observations 1) challenge the concept of a constraint between fibre size and oxidative capacity and 2) indicate the important role of the microcirculation in the regenerative capacity of a muscle even when satellite cell numbers are reduced.

Effects of domestic processing methods on the phytochemical content of watercress (Nasturtium officinale).

The impact of conventional cooking and processing methods on total phenols, antioxidant activity, carotenoids and glucosinolates of watercress was evaluated. Boiling significantly decreases phenolic content, antioxidant activity and recoverable glucosinolates, however it increases the carotenoid concentrations of watercress as compared to the raw vegetable. Cooking by microwaving and steaming maintains the majority of phytochemicals in comparison to the fresh material, suggesting that they should be used as the preferred methods of watercress preparation. Boiling of watercress should be avoided to ensure maximum ingestion of watercress-derived beneficial phytochemicals.