Investigating the Human Intestinal DNA Virome and Predicting Disease-Associated Virus-Host Interactions in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Understanding how the human virome, and which of its constituents, contributes to health or disease states is reliant on obtaining comprehensive virome profiles. By combining DNA viromes from isolated virus-like particles (VLPs) and whole metagenomes from the same faecal sample of a small cohort of healthy individuals and patients with severe myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), we have obtained a more inclusive profile of the human intestinal DNA virome. Key features are the identification of a core virome comprising tailed phages of the class Caudoviricetes, and a greater diversity of DNA viruses including extracellular phages and integrated prophages. Using an in silico approach, we predicted interactions between members of the Anaerotruncus genus and unique viruses present in ME/CFS microbiomes. This study therefore provides a framework and rationale for studies of larger cohorts of patients to further investigate disease-associated interactions between the intestinal virome and the bacteriome.

Investigating Antibody Reactivity to the Intestinal Microbiome in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): A Feasibility Study.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic disease of unknown aetiology that is characterised by disabling chronic fatigue and involves both the immune and gastrointestinal (GI) systems. Patients display alterations in GI microbiome with a significant proportion experiencing GI discomfort and pain and elevated blood biomarkers for altered intestinal permeability compared with healthy individuals. To investigate a possible GI origin of ME/CFS we designed a feasibility study to test the hypothesis that ME/CFS pathogenesis is a consequence of increased intestinal permeability that results in microbial translocation and a breakdown in immune tolerance leading to generation of antibodies reactive to indigenous intestinal microbes. Secretory immunoglobulin (Ig) A and serum IgG levels and reactivity to intestinal microbes were assessed in five pairs of severe ME/CFS patients and matched same-household healthy controls. For profiling serum IgG, we developed IgG-Seq which combines flow-cytometry based bacterial cell sorting and metagenomics to detect mucosal IgG reactivity to the microbiome. We uncovered evidence for immune dysfunction in severe ME/CFS patients that was characterised by reduced capacity and reactivity of serum IgG to stool microbes, irrespective of their source. This study provides the rationale for additional studies in larger cohorts of ME/CFS patients to further explore immune-microbiome interactions.

Role of mucin glycosylation in the gut microbiota-brain axis of core 3 O-glycan deficient mice.

Alterations in intestinal mucin glycosylation have been associated with increased intestinal permeability and sensitivity to inflammation and infection. Here, we used mice lacking core 3-derived O-glycans (C3GnT-/-) to investigate the effect of impaired mucin glycosylation in the gut-brain axis. C3GnT-/- mice showed altered microbial metabolites in the caecum associated with brain function such as dimethylglycine and N-acetyl-L-tyrosine profiles as compared to C3GnT+/+ littermates. In the brain, polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive granule cells showed an aberrant phenotype in the dentate gyrus of C3GnT-/- mice. This was accompanied by a trend towards decreased expression levels of PSA as well as ZO-1 and occludin as compared to C3GnT+/+. Behavioural studies showed a decrease in the recognition memory of C3GnT-/- mice as compared to C3GnT+/+ mice. Combined, these results support the role of mucin O-glycosylation in the gut in potentially influencing brain function which may be facilitated by the passage of microbial metabolites through an impaired gut barrier.

Accumulation of Sulforaphane and Alliin in Human Prostate Tissue.

Diets rich in cruciferous vegetables have been associated with a lower risk of incidence and progression of prostate cancer. Sulforaphane, an isothiocyanate derived from 4-methylsulphinylbutyl glucosinolate (glucoraphanin) that accumulates in certain of these vegetables, notably broccoli, has been implicated in their protective effects. Likewise, the consumption of garlic and its sulphur-containing compounds such as alliin have been associated with a reduction in risk of prostate cancer. In this study, we tested whether consuming glucoraphanin derived from broccoli seeds and alliin derived from garlic resulted in the occurrence of these potential bioactive compounds in the prostate, which may contribute to our understanding of the putative protective effects of these dietary components. We recruited 42 men scheduled for a trans-perineal prostate biopsy into a randomised, double-blinded, 2 × 2-factorial dietary supplement four-week intervention study, and 39 completed the study. The two active interventions were supplements providing glucoraphanin from broccoli (BroccoMax®) and alliin from garlic (Kwai Heartcare®). Following the intervention, prostate biopsy tissue was analysed for the presence of sulforaphane and its thiol conjugates and for alliin and associated metabolites. Sulforaphane occurred in significantly higher levels in the prostate tissue (both within the transition and peripheral zone) of men consuming the glucoraphanin containing supplements (p < 0.0001) compared to men not consuming these supplements. However, while alliin and alliin-derived metabolites were detected within the prostate, there was no significant difference in the concentrations of these compounds in the prostate of men consuming supplements derived from garlic compared to men not consuming these supplements.

The role of the mucin-glycan foraging Ruminococcus gnavus in the communication between the gut and the brain.

Ruminococcus gnavus is a prevalent member of the human gut microbiota, which is over-represented in inflammatory bowel disease and neurological disorders. We previously showed that the ability of R. gnavus to forage on mucins is strain-dependent and associated with sialic acid metabolism. Here, we showed that mice monocolonized with R. gnavus ATCC 29149 (Rg-mice) display changes in major sialic acid derivatives in their cecum content, blood, and brain, which is accompanied by a significant decrease in the percentage of sialylated residues in intestinal mucins relative to germ-free (GF) mice. Changes in metabolites associated with brain function such as tryptamine, indolacetate, and trimethylamine N-oxide were also detected in the cecal content of Rg-mice when compared to GF mice. Next, we investigated the effect of R. gnavus monocolonization on hippocampus cell proliferation and behavior. We observed a significant decrease of PSA-NCAM immunoreactive granule cells in the dentate gyrus (DG) of Rg-mice as compared to GF mice and recruitment of phagocytic microglia in the vicinity. Behavioral assessments suggested an improvement of the spatial working memory in Rg-mice but no change in other cognitive functions. These results were also supported by a significant upregulation of genes involved in proliferation and neuroplasticity. Collectively, these data provide first insights into how R. gnavus metabolites may influence brain regulation and function through modulation of granule cell development and synaptic plasticity in the adult hippocampus. This work has implications for further understanding the mechanisms underpinning the role of R. gnavus in neurological disorders.

The Origin of Plasma-Derived Bacterial Extracellular Vesicles in Healthy Individuals and Patients with Inflammatory Bowel Disease: A Pilot Study.

The gastrointestinal tract harbors the gut microbiota, structural alterations of which (dysbiosis) are linked with an increase in gut permeability ("leaky gut"), enabling luminal antigens and bacterial products such as nanosized bacterial extracellular vesicles (BEVs) to access the circulatory system. Blood-derived BEVs contain various cargoes and may be useful biomarkers for diagnosis and monitoring of disease status and relapse in conditions such as inflammatory bowel disease (IBD). To progress this concept, we developed a rapid, cost-effective protocol to isolate BEV-associated DNA and used 16S rRNA gene sequencing to identify bacterial origins of the blood microbiome of healthy individuals and patients with Crohn's disease and ulcerative colitis. The 16S rRNA gene sequencing successfully identified the origin of plasma-derived BEV DNA. The analysis showed that the blood microbiota richness, diversity, or composition in IBD, healthy control, and protocol control groups were not significantly distinct, highlighting the issue of 'kit-ome' contamination in low-biomass studies. Our pilot study provides the basis for undertaking larger studies to determine the potential use of blood microbiota profiling as a diagnostic aid in IBD.

Effects of Human Milk Oligosaccharides on the Adult Gut Microbiota and Barrier Function.

Human milk oligosaccharides (HMOs) shape the gut microbiota in infants by selectively stimulating the growth of bifidobacteria. Here, we investigated the impact of HMOs on adult gut microbiota and gut barrier function using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), Caco2 cell lines, and human intestinal gut organoid-on-chips. We showed that fermentation of 2'-O-fucosyllactose (2'FL), lacto-N-neotetraose (LNnT), and combinations thereof (MIX) led to an increase of bifidobacteria, accompanied by an increase of short chain fatty acid (SCFA), in particular butyrate with 2'FL. A significant reduction in paracellular permeability of FITC-dextran probe was observed using Caco2 cell monolayers with fermented 2'FL and MIX, which was accompanied by an increase in claudin-8 gene expression as shown by qPCR, and a reduction in IL-6 as determined by multiplex ELISA. Using gut-on-chips generated from human organoids derived from proximal, transverse, and distal colon biopsies (Colon Intestine Chips), we showed that claudin-5 was significantly upregulated across all three gut-on-chips following treatment with fermented 2'FL under microfluidic conditions. Taken together, these data show that, in addition to their bifidogenic activity, HMOs have the capacity to modulate immune function and the gut barrier, supporting the potential of HMOs to provide health benefits in adults.

How do potentially inappropriate medications and polypharmacy affect mortality in frail and non-frail cognitively impaired older adults? A cohort study.

OBJECTIVES: To test whether the use of potentially inappropriate central nervous system acting medications, proton pump inhibitors (PPIs) or polypharmacy are associated with mortality in cognitively impaired older adults and whether frailer people are at greater risk of harm. SETTING: A cohort study nested within the Cognitive Function and Ageing Study II, a population representative cohort study of the older population in Cambridgeshire, Nottingham and Newcastle, UK. PARTICIPANTS: A total of 1154 cognitively impaired participants, aged 65 years or older. EXPOSURES: Any use of antipsychotics, antidepressants, other anticholinergic medication, benzodiazepines or PPIs, polypharmacy (5-9) and hyperpolypharmacy (≥10 reported medications) were ascertained at baseline. Frailty was assessed using the Fried criteria. PRIMARY OUTCOME: Mortality up to 8 years follow-up. HRs associated with potentially inappropriate medication (PIM), frailty and their interaction were estimated adjusting for covariates. RESULTS: Within the sample, 44% were taking one or more PIM. Apart from antipsychotics (adjusted HR=3.24, 95% CI 1.83 to 5.73), use of specific PIM was not associated with greater subsequent mortality. Polypharmacy (HR=1.17, 95% CI 0.95 to 1.45) and hyperpolypharmacy were associated with mortality (HR=1.60, 95% CI 1.16 to 2.22). Being frail (HR=1.90, 95% CI 1.32 to 2.72) or prefrail (HR=1.56, 95% CI 1.10 to 2.20) was associated with increased mortality. There was some evidence that the HR for polypharmacy on mortality was lower among frailer individuals, but the overall polypharmacy by frailty interaction was not statistically significant (p=0.102). CONCLUSIONS: For those with cognitive impairment, greater concern should be afforded to the number of medications than the prescription of specific classes. Frailer individuals may have a lower relative risk of mortality associated with polypharmacy than less frail individuals.

Transcriptional changes in prostate of men on active surveillance after a 12-mo glucoraphanin-rich broccoli intervention-results from the Effect of Sulforaphane on prostate CAncer PrEvention (ESCAPE) randomized controlled trial.

BACKGROUND: Epidemiological evidence suggests that consumption of cruciferous vegetables is associated with reduced risk of prostate cancer progression, largely attributed to the biological activity of glucosinolate degradation products, such as sulforaphane derived from glucoraphanin. Because there are few therapeutic interventions for men on active surveillance for prostate cancer to reduce the risk of cancer progression, dietary approaches are an appealing option for patients. OBJECTIVE: We evaluated whether consumption of a glucoraphanin-rich broccoli soup for 1 y leads to changes in gene expression in prostate tissue of men with localized prostate cancer. METHODS: Forty-nine men on active surveillance completed a 3-arm parallel randomized double-blinded intervention study for 12 mo and underwent transperineal template biopsy procedures and dietary assessment at the start and end of the study. Patients received a weekly 300 mL portion of soup made from a standard broccoli (control) or from 1 of 2 experimental broccoli genotypes with enhanced concentrations of glucoraphanin, delivering 3 and 7 times that of the control, respectively. Gene expression in tissues from each patient obtained before and after the dietary intervention was quantified by RNA sequencing followed by gene set enrichment analyses. RESULTS: In the control arm, there were several hundred changes in gene expression in nonneoplastic tissue during the 12 mo. These were associated with an increase in expression of potentially oncogenic pathways including inflammation processes and epithelial-mesenchymal transition. Changes in gene expression and associated oncogenic pathways were attenuated in men on the glucoraphanin-rich broccoli soup in a dose-dependent manner. Although the study was not powered to assess clinical progression, an inverse association between consumption of cruciferous vegetables and cancer progression was observed. CONCLUSION: Consuming glucoraphanin-rich broccoli soup affected gene expression in the prostate of men on active surveillance, consistent with a reduction in the risk of cancer progression. This trial was registered at clinicaltrials.gov as NCT01950143.

Correction: Metabolite quantification of faecal extracts from colorectal cancer patients and healthy controls.

[This corrects the article DOI: 10.18632/oncotarget.26022.].

Metabolite quantification of faecal extracts from colorectal cancer patients and healthy controls.

Colorectal cancer (CRC), a primary cause of morbidity and mortality worldwide is expected to rise in the coming years. A better understanding of the metabolic changes taking place during the disease progression is needed for effective improvements of screening strategies and treatments. In the present study, Nuclear Magnetic Resonance (NMR) metabolomics was used to quantify the absolute concentrations of metabolites in faecal extracts from two cohorts of CRC patients and healthy controls. The quantification of over 80 compounds revealed that patients with CRC had increased faecal concentrations of branched chain fatty acids (BCFA), isovalerate and isobutyrate plus valerate and phenylacetate but diminished concentrations of amino acids, sugars, methanol and bile acids (deoxycholate, lithodeoxycholate and cholate). These results suggest that alterations in microbial activity and composition could have triggered an increase in utilisation of host intestinal slough cells and mucins and led to an increase in BCFA, valerate and phenylacetate. Concurrently, a general reduction in the microbial metabolic function may have led to reduced levels of other components (amino acids, sugars and bile acids) normally produced under healthy conditions. This study provides a thorough listing of the most abundant compounds found in human faecal waters and presents a template for absolute quantification of metabolites. The production of BCFA and phenylacetate in colonic carcinogenesis warrants further investigations.

Prospective study of falls and risk factors for falls in adults with advanced cancer.

PURPOSE: Retrospective studies of inpatients with cancer suggest that a cancer diagnosis confers a high risk of falls. In adults with advanced cancer, we aimed to prospectively document the incidence of falls, identify the risk factors, and determine if falls in this population occur predominantly in older patients. PATIENTS AND METHODS: Patients admitted consecutively to community and inpatient palliative care services with metastatic or locoregionally advanced cancer who were mobile without assistance were recruited. Risk-factor assessment was conducted on initial encounter. Patients underwent follow-up via weekly telephone contact for 6 months or until time of fall or death. Relationship between covariates and time to fall was examined using hazard ratios (HRs) derived from univariate and multivariate Cox proportional hazards models. RESULTS: Of 185 participants (52.4% men; mean age 68 ± standard deviation of 12.6 years), 50.3% fell; 35 (53%) of 66 participants age < 65 years and 58 (48.7%) of 119 age ≥ 65 years fell; 61.3% of falls occurred in the community; 42% resulted in injury. Median time to fall was 96 days (95% CI, 64.66 to 127.34). Primary brain tumor or brain metastasis (HR 2.5; P = .002), number of falls in the preceding 3 months (HR, 1.27; P = .005), severity of depression (HR, 1.12; P = .012), benzodiazepine dose (HR, 1.05; P = .004), and cancer-related pain (HR, 1.96; P = .024) were independently associated with time to fall in multivariate analysis. CONCLUSION: Fifty percent of adults with advanced cancer, regardless of age, will experience a fall associated with high risk of physical injury. There is a compelling need to assess the efficacy of assessment and management of modifiable fall risk factors in patients with advanced cancer.

Occurrence of medical co-morbidity in mild cognitive impairment: implications for generalisation of MCI research.

BACKGROUND: diagnosis of mild cognitive impairment (MCI) typically excludes individuals with medical co-morbidity. Interest in MCI screening raises the questions of what are the best criteria to identify a representative sample and what factors are associated with MCI progression to dementia. OBJECTIVES: to compare the pattern of disease co-morbidity across different cognitive groups and to examine the role of health co-morbidity as a risk factor for dementia progression from MCI. METHODS: individuals from the MRC Cognitive Function and Ageing Study were classified as having no cognitive impairment (NCI), MCI, other cognitive impairment no dementia (OCIND) or dementia. At 2 years dementia status was assessed. FINDINGS: over 50% of individuals in each group reported one or more medical condition. The pattern of disease prevalence was similar in the NCI, MCI and OCIND groups. Anaemia was the only health factor associated with an increased risk of dementia progression from MCI. CONCLUSION: classification of MCI using medical exclusions would exclude the majority of the population from a MCI diagnosis. This has implications for treatment decisions and clinical trial recruitment. This could not only make recruitment more difficult but also limit the generalisability of trial results. Medical co-morbidity does not help to distinguish progressive from non-progressive MCI.

Hospital use, institutionalisation and mortality associated with delirium.

BACKGROUND: Delirium is a disorder affecting consciousness, which gives rise to core clinical features and associated symptoms. Older patients are particularly prone, owing to higher rates of pre-existing cognitive impairment, frailty, co-morbidity and polypharmacy. OBJECTIVES: The aim of this study was to investigate the hypotheses that delirium affects the most vulnerable older adults and is associated with long-term adverse health outcome. METHODS: This prospective cohort study evaluated 278 medical patients aged > or = 75 years admitted acutely to a district general hospital in South Wales. Patients were screened for delirium at presentation and on alternate days throughout their hospital stay. Assessments also included illness severity, preadmission cognition, co-morbidity and functional status. Patients were followed for 5 years to determine rates of institutionalisation and mortality. Number of days in hospital in the 4 years prior to and 5 years after index admission were recorded. RESULTS: Delirium was detected in 103 patients and excluded in 175. Median time to death was 162 days (interquartile range 21-556) for those with delirium compared with 1,444 days (25% mortality 435 days, 75% mortality>5 years) for those without (P < 0.001). After adjusting for multiple confounders, delirium was associated with an increased risk of death (hazard ratio range 2.0-3.5; P < or = 0.002). Institutionalisation was higher in the first year following delirium (P = 0.03). While those with delirium tended to be older with more preadmission cognitive impairment, greater functional dependency and more co-morbidity, they did not spend more days in hospital in the 4 years prior to index admission. CONCLUSIONS: Delirium is associated with high rates of institutionalisation and an increased risk of death up to 5 years after index event. Prior to delirium, individuals seem to compensate for their vulnerability. The impact of delirium itself, directly or indirectly, may convert vulnerability into adverse outcome.

Inflammation and frailty measures in older people.

Inflammation in patients defined as frail by Fried's phenotypic definition may be related to sarcopenia. This study aimed to investigate inflammation in older patients across different frailty criteria. Frailty status was determined in 110 patients aged over 75 years (mean 83.9 years) according to function (dependent, intermediate, independent); Fried (three or more items of exhaustion, weight loss, slow walking speed, low handgrip strength, low physical activity) and Frailty Index (a measure of accumulated deficits). With increasing patient frailty as defined by function and by Fried phenotype, tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and C-reactive protein (CRP) increased significantly. Albumin was lowest in the frailest subjects by each definition. The greatest differences were seen between intermediate and dependent groups and between the pre-frail and frail. Adjustment for multiple covariates (age, sex, BMI category, smoking status, number of co-morbidities and number of prescribed medications) did not account for any of the observed differences in levels of inflammatory markers. The Frailty Index correlated significantly with log-transformed CRP (r= 0.221, P < 0.05), log-transformed IL-6 (r= 0.369, P < 0.01), TNF-alpha (r= 0.379, P < 0.01) and inversely with albumin (r=- 0.545, P < 0.01). This study provides further evidence linking inflammation and frailty in older people, an association that seems consistent across different frailty measures.

Maternal age-specific fetal loss rates in Down syndrome pregnancies.

OBJECTIVES: Pregnancies affected by Down syndrome (DS) have a greater risk of spontaneous fetal loss than those that are unaffected. In this article, we investigate the relationship between maternal age and the risk of spontaneous fetal loss in DS pregnancies. METHODS: Fetal loss at different maternal ages were estimated by survival analysis using follow-up of 5177 prenatally diagnosed cases. The maternal age effect on loss rate was subsequently confirmed by a re-analysis of published comparisons of the maternal age-specific prevalence of DS at different gestational ages. RESULTS: The average fetal loss rate between the time of chorionic villus sampling (CVS) and term was 32% (95% CI: 26-38), increasing from 23% (95% CI: 16-31) for women aged 25 to 44% (33-56) for women aged 45. The average fetal loss rate between the time of amniocentesis and term was 25% (21-31), increasing from 19% (14-27) to 33% (26-45) across the same age range. CONCLUSION: The fetal loss rate in DS pregnancies increases with maternal age, and this has consequences when estimating the live birth prevalence of DS in the presence of prenatal diagnosis and termination, and when assessing the performance of prenatal screening techniques.