Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease.

BACKGROUND: Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. METHODS: Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. RESULTS: External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. CONCLUSIONS: The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.

Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease.

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.

Effects of pelargonidin-3-O-glucoside and its metabolites on lipopolysaccharide-stimulated cytokine production by THP-1 monocytes and macrophages.

Epidemiological evidence suggests cardioprotective effects of anthocyanin consumption. This study examined the predominant strawberry anthocyanin, pelargonidin-3-O-glucoside (Pg-3-glc), and three of its plasma metabolites (protocatechuic acid [PCA], 4-hydroxybenzoic acid, and phloroglucinaldehyde [PGA]) for effects on the production of selected cytokines by lipopolysaccharide-stimulated THP-1 monocytes and macrophages. Concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8 and IL-10 were determined using a cytometric bead array kit. PCA at 0.31, 1.25 and 20 µM and PGA at 5 and 20 µM decreased the concentration of IL-6 in the monocyte cultures, but there were no effects on TNF-α, IL-1ß, IL-8 and IL-10 and there were no effects of the other compounds. In the macrophage cultures, PGA at 20 µM decreased the concentrations of IL-6 and IL-10, but there was no effect on TNF-α, IL-1ß and IL-8 and there were no effects of the other compounds. In conclusion, while the effects of PGA were only observed at the higher, supraphysiological concentration and are thus considered of limited physiological relevance overall, the anti-inflammatory properties of PCA were observed at both the lower, physiologically relevant, and the higher concentrations; however, effects were modest and limited to IL-6 and monocytes. These preliminary data suggest potential for physiologically attainable PCA concentrations to modulate IL-6 production by monocytes.

Farnesoid X Receptor and Liver X Receptor Ligands Initiate Formation of Coated Platelets.

OBJECTIVES: The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. APPROACH AND RESULTS: We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbß3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. CONCLUSIONS: We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbß3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo.

Impact of phenolic-rich olive leaf extract on blood pressure, plasma lipids and inflammatory markers: a randomised controlled trial.

PURPOSE: Dietary polyphenols have been demonstrated to favourably modify a number of cardiovascular risk markers such as blood pressure (BP), endothelial function and plasma lipids. We conducted a randomised, double-blind, controlled, crossover trial to investigate the effects of a phenolic-rich olive leaf extract (OLE) on BP and a number of associated vascular and metabolic measures. METHODS: A total of 60 pre-hypertensive [systolic blood pressure (SBP): 121-140 mmHg; diastolic blood pressure (DBP): 81-90 mmHg] males [mean age 45 (±SD 12.7 years, BMI 26.7 (±3.21) kg/m2] consumed either OLE (136 mg oleuropein; 6 mg hydroxytyrosol) or a polyphenol-free control daily for 6 weeks before switching to the alternate arm after a 4-week washout. RESULTS: Daytime [-3.95 (±SD 11.48) mmHg, p = 0.027] and 24-h SBP [-3.33 (±SD 10.81) mmHg, p = 0.045] and daytime and 24-h DBP [-3.00 (±SD 8.54) mmHg, p = 0.025; -2.42 (±SD 7.61) mmHg, p = 0.039] were all significantly lower following OLE intake, relative to the control. Reductions in plasma total cholesterol [-0.32 (±SD 0.70) mmol/L, p = 0.002], LDL cholesterol [-0.19 (±SD 0.56) mmol/L, p = 0.017] and triglycerides [-0.18 (±SD 0.48), p = 0.008] were also induced by OLE compared to control, whilst a reduction in interleukin-8 [-0.63 (±SD 1.13) pg/ml; p = 0.026] was also detected. Other markers of inflammation, vascular function and glucose metabolism were not affected. CONCLUSION: Our data support previous research, suggesting that OLE intake engenders hypotensive and lipid-lowering effects in vivo.

Gastrointestinal modifications and bioavailability of brown seaweed phlorotannins and effects on inflammatory markers.

Brown seaweeds such as Ascophyllum nodosum are a rich source of phlorotannins (oligomers and polymers of phloroglucinol units), a class of polyphenols that are unique to Phaeophyceae. At present, there is no information on the bioavailability of seaweed polyphenols and limited evidence on their bioactivity in vivo. Consequently, we investigated the gastrointestinal modifications in vitro of seaweed phlorotannins from A. nodosum and their bioavailability and effect on inflammatory markers in healthy participants. In vitro, some phlorotannin oligomers were identified after digestion and colonic fermentation. In addition, seven metabolites corresponding to in vitro-absorbed metabolites were identified. Urine and plasma samples contained a variety of metabolites attributed to both unconjugated and conjugated metabolites (glucuronides and/or sulphates). In both urine and plasma, the majority of the metabolites were found in samples collected at late time points (6-24 h), suggesting colonic metabolism of high-molecular-weight phlorotannins, with three phlorotannin oligomers (hydroxytrifuhalol A, 7-hydroxyeckol, C-O-C dimer of phloroglucinol) identified in urine samples. A significant increase of the cytokine IL-8 was also observed. Our study shows for the first time that seaweed phlorotannins are metabolised and absorbed, predominantly in the large intestine, and there is a large inter-individual variation in their metabolic profile. Three phlorotannin oligomers present in the capsule are excreted in urine. Our study is the first investigation of the metabolism and bioavailability of seaweed phlorotannins and the role of colonic biotransformation. In addition, IL-8 is a possible target for phlorotannin bioactivity.

Effects of increased wholegrain consumption on immune and inflammatory markers in healthy low habitual wholegrain consumers.

PURPOSE: Wholegrain (WG) consumption is associated with reduced risk of cardiovascular disease, but clinical data on inflammation and immune function is either conflicting or limited. The objective of this study was to assess the impact of increasing WG consumption to at least 80 g/day on markers of inflammation and glucose metabolism and on phenotypic and functional aspects of the immune system, in healthy, middle-aged adults with low habitual WG intake. METHODS: Subjects consumed a diet high in WG (>80 g/day) or low in WG (<16 g/day, refined grain diet) in a crossover study, with 6-week intervention periods, separated by a 4-week washout. Adherence to the dietary regimes was achieved by dietary advice and provision of a range of food products, with compliance verified by analysis of plasma alkylresorcinols (ARs). RESULTS: On the WG intervention, WG consumption reached 168 g/day (P < 0.001), accompanied by an increase in plasma ARs (P < 0.001) and fibre intake (P < 0.001), without affecting other aspects of dietary intake. On the WG arm, there were trends for lower ex vivo activation of CD4(+) T cells and circulating concentrations of IL-10, C-reactive protein, C-peptide, insulin and plasminogen activator inhibitor-1. The percentage of CD4(+) central memory T cells and circulating levels of adipsin tended to increase during the WG intervention. CONCLUSIONS: Despite the dramatic increase in WG consumption, there were no effects on phenotypic or functional immune parameters, markers of inflammation or metabolic markers.

Effect of a synbiotic on the response to seasonal influenza vaccination is strongly influenced by degree of immunosenescence.

BACKGROUND: Ageing increases risk of respiratory infections and impairs the response to influenza vaccination. Pre- and probiotics offer an opportunity to modulate anti-viral defenses and the response to vaccination via alteration of the gut microbiota. This study investigated the effect of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52,486, combined with a prebiotic, gluco-oligosaccharide (B. longum + Gl-OS), on the response to seasonal influenza vaccination in young and older subjects in a double-blind, randomized controlled trial, taking into account the influence of immunosenescence markers at baseline. RESULTS: Vaccination resulted in a significant increase in total antibody titres, vaccine-specific IgA, IgM and IgG and seroprotection to all three subunits of the vaccine in both young and older subjects, and in general, the increases in young subjects were greater. There was little effect of the synbiotic, although it tended to reduce seroconversion to the Brisbane subunit of the vaccine and the vaccine-specific IgG response in older subjects. Immunological characterization revealed that older subjects randomized to the synbiotic had a significantly higher number of senescent (CD28(-)CD57(+)) helper T cells at baseline compared with those randomized to the placebo, and they also had significantly higher plasma levels of anti-CMV IgG and a greater tendency for CMV seropositivity. Moreover, higher numbers of CD28(-)CD57(+) helper T cells were associated with failure to seroconvert to Brisbane, strongly suggesting that the subjects randomized to the synbiotic were already at a significant disadvantage in terms of likely ability to respond to the vaccine compared with those randomized to the placebo. CONCLUSIONS: Ageing was associated with marked impairment of the antibody response to influenza vaccination in older subjects and the synbiotic failed to reverse this impairment. However, the older subjects randomized to the synbiotic were at a significant disadvantage due to a greater degree of immunosenscence at baseline compared with those randomized to the placebo. Thus, baseline differences in immunosenescence between the randomized groups are likely to have influenced the outcome of the intervention, highlighting the need for detailed immunological characterization of subjects prior to interventions. TRIAL REGISTRATION: Clinicaltrials.gov NCT01066377.

Increased whole grain consumption does not affect blood biochemistry, body composition, or gut microbiology in healthy, low-habitual whole grain consumers.

BACKGROUND: Whole-grain (WG) foods have been suggested to reduce the risk of cardiovascular disease, but studies are inconsistent and effects on cardiovascular risk markers are not clear. OBJECTIVE: The objective of this study was to assess the impact of increasing WG consumption to at least 80 g/d on overall dietary intake, body composition, blood pressure (BP), blood lipids, blood glucose, gastrointestinal microbiology, and gastrointestinal symptoms in healthy, middle-aged adults with habitual WG intake <24 g/d. METHODS: Eligible subjects [12 men, 21 women, aged 40-65 y, body mass index (BMI): 20-35 kg/m(2)] were identified through use of food frequency questionnaires and subsequently completed 3-day food diaries (3DFDs) to confirm habitual WG consumption. Subjects consumed diets high in WG (>80 g/d) or low in WG [<16 g/d, refined-grain (RG) diet] in a crossover study with 6-wk intervention periods separated by a 4-wk washout. Adherence was achieved by specific dietary advice and provision of a range of cereal food products. The 3DFDs, diet compliance diaries, and plasma alkylresorcinols were used to verify compliance. RESULTS: During the WG intervention, consumption increased from 28 g/d to 168 g/d (P < 0.001), accompanied by an increase in plasma alkylresorcinols (P < 0.001) and total fiber intake (P < 0.001), without any effect on energy or other macronutrients. Although there were no effects on studied variables, there were trends toward increased 24-h fecal weight (P = 0.08) and reduction in body weight (P = 0.10) and BMI (P = 0.08) during the WG intervention compared with the RG period. CONCLUSION: A combination of dietary advice and provision of commercially available food items enabled subjects with a low-moderate habitual consumption of WG to substantially increase their WG intake, but there was little effect on blood biochemical markers, body composition, BP, fecal measurements, or gut microbiology. This trial was registered at www.controlled-trials.com as ISRCTN36521837.

Secoiridoids delivered as olive leaf extract induce acute improvements in human vascular function and reduction of an inflammatory cytokine: a randomised, double-blind, placebo-controlled, cross-over trial.

The leaves of the olive plant (Olea europaea) are rich in polyphenols, of which oleuropein and hydroxytyrosol (HT) are most characteristic. Such polyphenols have been demonstrated to favourably modify a variety of cardiovascular risk factors. The aim of the present intervention was to investigate the influence of olive leaf extract (OLE) on vascular function and inflammation in a postprandial setting and to link physiological outcomes with absorbed phenolics. A randomised, double-blind, placebo-controlled, cross-over, acute intervention trial was conducted with eighteen healthy volunteers (nine male, nine female), who consumed either OLE (51 mg oleuropein; 10 mg HT), or a matched control (separated by a 4-week wash out) on a single occasion. Vascular function was measured by digital volume pulse (DVP), while blood collected at baseline, 1, 3 and 6 h was cultured for 24 h in the presence of lipopolysaccharide in order to investigate effects on cytokine production. Urine was analysed for phenolic metabolites by HPLC. DVP-stiffness index and ex vivo IL-8 production were significantly reduced (P< 0.05) after consumption of OLE compared to the control. These effects were accompanied by the excretion of several phenolic metabolites, namely HT and oleuropein derivatives, which peaked in urine after 8-24 h. The present study provides the first evidence that OLE positively modulates vascular function and IL-8 production in vivo, adding to growing evidence that olive phenolics could be beneficial for health.

Biological significance of short-chain fatty acid metabolism by the intestinal microbiome.

PURPOSE OF REVIEW: Evidence suggests that short-chain fatty acids (SCFAs) derived from microbial metabolism in the gut play a central role in host homeostasis. The present review describes the current understanding and physiological implications of SCFAs derived from microbial metabolism of nondigestible carbohydrates. RECENT FINDINGS: Recent studies indicate a role for SCFAs, in particular propionate and butyrate, in metabolic and inflammatory disorders such as obesity, diabetes and inflammatory bowel diseases, through the activation of specific G-protein-coupled receptors and modification of transcription factors. Established prebiotics, such as fructooligosaccharides and galactooligosaccharides, which support the growth of Bifidobacteria, mainly mediate acetate production. Thus, recent identification of prebiotics which are able to stimulate the production of propionate and butyrate by benign saccharolytic populations in the colon is of interest. SUMMARY: Manipulation of saccharolytic fermentation by prebiotic substrates is beginning to provide information on structure-function relationships relating to the production of SCFAs, which have multiple roles in host homeostasis.

Development of a food-exchange model to replace saturated fat with MUFAs and n-6 PUFAs in adults at moderate cardiovascular risk.

The recommendation to reduce saturated fatty acid (SFA) consumption to ≤10% of total energy (%TE) is a key public health target aimed at lowering cardiovascular disease (CVD) risk. Replacement of SFA with unsaturated fats may provide greater benefit than replacement with carbohydrates, yet the optimal type of fat is unclear. The aim of the DIVAS (Dietary Intervention and Vascular Function) study was to develop a flexible food-exchange model to investigate the effects of substituting SFAs with monounsaturated fatty acids (MUFAs) or n-6 (ω-6) polyunsaturated fatty acids (PUFAs) on CVD risk factors. In this parallel study, UK adults aged 21-60 y with moderate CVD risk (50% greater than the population mean) were identified using a risk assessment tool (n = 195; 56% females). Three 16-wk isoenergetic diets of specific fatty acid (FA) composition (%TE SFA:%TE MUFA:%TE n-6 PUFA) were designed using spreads, oils, dairy products, and snacks as follows: 1) SFA-rich diet (17:11:4; n = 65); 2) MUFA-rich diet (9:19:4; n = 64); and 3) n-6 PUFA-rich diet (9:13:10; n = 66). Each diet provided 36%TE total fat. Dietary targets were broadly met for all intervention groups, reaching 17.6 ± 0.4%TE SFA, 18.5 ± 0.3%TE MUFA, and 10.4 ± 0.3%TE n-6 PUFA in the respective diets, with significant overall diet effects for the changes in SFAs, MUFAs, and n-6 PUFAs between groups (P < 0.001). There were no differences in the changes of total fat, protein, carbohydrate, and alcohol intake or anthropometric measures between groups. Plasma phospholipid FA composition showed changes from baseline in the proportions of total SFAs, MUFAs, and n-6 PUFAs for each diet group, with the changes in SFAs and MUFAs differing between the groups (P < 0.001). In conclusion, successful implementation of the food-exchange model broadly achieved the dietary target intakes for the exchange of SFAs with MUFAs or n-6 PUFAs with minimal disruption to the overall diet in a free-living population. This trial was registered at clinicaltrials.gov as NCT01478958.

Effect of salmon consumption during pregnancy on maternal and infant faecal microbiota, secretory IgA and calprotectin.

The gut microbiota plays an important role in the development of the immune and gastrointestinal systems of infants. In the present study, we investigated whether increased salmon consumption during pregnancy, maternal weight gain during pregnancy or mode of infant feeding alter the markers of gut immune defence and inflammation. Women (n 123) who rarely ate oily fish were randomly assigned to continue consuming their habitual diet or to consume two 150 g portions of farmed salmon per week from 20 weeks of pregnancy to delivery. Faecal samples were collected from the mothers (n 75) at 38 weeks of gestation and from their infants (n 38) on days 7, 14, 28 and 84 post-partum. Fluorescence in situ hybridisation was used to determine faecal microbiota composition and ELISA to measure faecal secretory IgA (sIgA) and calprotectin concentrations. There was no effect of salmon consumption on maternal faecal microbiota or on maternal or infant faecal sIgA and calprotectin concentrations. The degree of weight gain influenced maternal faecal microbiota, and the mode of infant feeding influenced infant faecal microbiota. Faecal samples collected from infants in the salmon group tended to have lower bacterial counts of the Atopobium cluster compared with those collected from infants in the control group (P=0·097). This difference was significant in the formula-fed infants (P< 0·05), but not in the exclusively breast-fed infants. In conclusion, the impact of oily fish consumption during pregnancy on maternal and infant gut microbiota composition is limited, but significant differences are associated with maternal weight gain during pregnancy and mode of infant feeding.

Xylo-oligosaccharides alone or in synbiotic combination with Bifidobacterium animalis subsp. lactis induce bifidogenesis and modulate markers of immune function in healthy adults: a double-blind, placebo-controlled, randomised, factorial cross-over study.

Prebiotics, probiotics and synbiotics are dietary ingredients with the potential to influence health and mucosal and systemic immune function by altering the composition of the gut microbiota. In the present study, a candidate prebiotic (xylo-oligosaccharide, XOS, 8 g/d), probiotic (Bifidobacterium animalis subsp. lactis Bi-07, 109 colony-forming units (CFU)/d) or synbiotic (8 g XOS+109 CFU Bi-07/d) was given to healthy adults (25-65 years) for 21 d. The aim was to identify the effect of the supplements on bowel habits, self-reported mood, composition of the gut microbiota, blood lipid concentrations and immune function. XOS supplementation increased mean bowel movements per d (P= 0·009), but did not alter the symptoms of bloating, abdominal pain or flatulence or the incidence of any reported adverse events compared with maltodextrin supplementation. XOS supplementation significantly increased participant-reported vitality (P= 0·003) and happiness (P= 0·034). Lowest reported use of analgesics was observed during the XOS+Bi-07 supplementation period (P= 0·004). XOS supplementation significantly increased faecal bifidobacterial counts (P= 0·008) and fasting plasma HDL concentrations (P= 0·005). Bi-07 supplementation significantly increased faecal B. lactis content (P= 0·007), lowered lipopolysaccharide-stimulated IL-4 secretion in whole-blood cultures (P= 0·035) and salivary IgA content (P= 0·040) and increased IL-6 secretion (P= 0·009). XOS supplementation resulted in lower expression of CD16/56 on natural killer T cells (P= 0·027) and lower IL-10 secretion (P= 0·049), while XOS and Bi-07 supplementation reduced the expression of CD19 on B cells (XOS × Bi-07, P= 0·009). The present study demonstrates that XOS induce bifidogenesis, improve aspects of the plasma lipid profile and modulate the markers of immune function in healthy adults. The provision of XOS+Bi-07 as a synbiotic may confer further benefits due to the discrete effects of Bi-07 on the gut microbiota and markers of immune function.

Dietary n-3 polyunsaturated fatty acids alter the number, fatty acid profile and coagulatory activity of circulating and platelet-derived extracellular vesicles: a randomized, controlled crossover trial.

BACKGROUND: Extracellular vesicles (EVs) are proposed to play a role in the development of cardiovascular diseases (CVDs) and are considered emerging markers of CVDs. n-3 PUFAs are abundant in oily fish and fish oil and are reported to reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFAs on the generation and function of EVs. OBJECTIVES: We aimed to investigate the effects of fish oil supplementation on the number, generation, and function of EVs in subjects with moderate risk of CVDs. METHODS: A total of 40 participants with moderate risk of CVDs were supplemented with capsules containing either fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12 wk in a randomized, double-blind, placebo-controlled crossover intervention study. The effects of fish oil supplementation on conventional CVD and thrombogenic risk markers were measured, along with the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs). PDEV proteome profiles were evaluated, and their impact on coagulation was assessed using assays including fibrin clot formation, thrombin generation, fibrinolysis, and ex vivo thrombus formation. RESULTS: n-3 PUFAs decreased the numbers of circulating EVs by 27%, doubled their n-3 PUFA content, and reduced their capacity to support thrombin generation by >20% in subjects at moderate risk of CVDs. EVs derived from n-3 PUFA-enriched platelets in vitro also resulted in lower thrombin generation, but did not alter thrombus formation in a whole blood ex vivo assay. CONCLUSIONS: Dietary n-3 PUFAs alter the number, composition, and function of EVs, reducing their coagulatory activity. This study provides clear evidence that EVs support thrombin generation and that this EV-dependent thrombin generation is reduced by n-3 PUFAs, which has implications for prevention and treatment of thrombosis. CLINICAL TRIAL REGISTRY: This trial was registered at clinicaltrials.gov as NCT03203512.

Premature impairment of methylation pathway and cardiac metabolic dysfunction in fa/fa obese Zucker rats.

Increasing evidence suggests that obesity is a chronic inflammatory disease, in which adipose tissue is involved in a network of endocrine signals to modulate energy homeostasis. These oxidative-inflammatory pathways, which are associated with cardiovascular complications, are also observed during the aging process. In this study, we investigated the interaction between aging and the development of obesity in a hyperphagic rat model. Metabolic profiles of the liver, white adipose tissue (WAT) and heart from young and adult Zucker lean (fa/+) and obese (fa/fa) rats were characterized using a (1)H NMR-based metabonomics approach. We observed premature metabolic modifications in all studied organs in obese animals, some of which were comparable to those observed in adult lean animals. In the cardiac tissue, young obese rats displayed lower lactate and scyllo-inositol levels associated with higher creatine, choline and phosphocholine levels, indicating an early modulation of energy and membrane metabolism. An early alteration of the hepatic methylation and transsulfuration pathways in both groups of obese rats indicated that these pathways were affected before diabetic onset. These findings therefore support the hypothesis that obesity parallels some metabolic perturbations observed in the aging process and provides new insights into the metabolic modifications occurring in prediabetic state.

Modulation of vaccine response by concomitant probiotic administration.

Evidence suggests that probiotic bacteria modulate both innate and adaptive immunity in the host, and in some situations can result in reduced severity of common illnesses, such as acute rotavirus infection and respiratory infections. Responses to vaccination are increasingly being used to provide high quality information on the immunomodulatory effects of dietary components in humans. The present review focuses on the effect of probiotic administration upon vaccination response. The majority of studies investigating the impact of probiotics on responses to vaccination have been conducted in healthy adults, and at best they show modest effects of probiotics on serum or salivary IgA titres. Studies in infants and in elderly subjects are very limited, and it is too early to draw any firm conclusions regarding the potential for probiotics to act as adjuvants in vaccination. Although some studies are comparable in terms of duration of the intervention, age and characteristics of the subjects, most differ in terms of the probiotic selected. Further well designed, randomized, placebo-controlled studies are needed to understand fully the immunomodulatory properties of probiotics, whether the effects exerted are strain-dependent and age-dependent and their clinical relevance in enhancing immune protection following vaccination.

The impact of obesity-related SNP on appetite and energy intake.

An increasing number of studies have reported a heritable component for the regulation of energy intake and eating behaviour, although the individual polymorphisms and their 'effect size' are not fully elucidated. The aim of the present study was to examine the relationship between specific SNP and appetite responses and energy intake in overweight men. In a randomised cross-over trial, forty overweight men (age 32 (sd 09) years; BMI 27 (sd 2) kg/m2) attended four sessions 1 week apart and received three isoenergetic and isovolumetric servings of dairy snacks or water (control) in random order. Appetite ratings were determined using visual analogue scales and energy intake at an ad libitum lunch was assessed 90 min after the dairy snacks. Individuals were genotyped for SNP in the fat mass and obesity-associated (FTO), leptin (LEP), leptin receptor (LEPR) genes and a variant near the melanocortin-4 receptor (MC4R) locus. The postprandial fullness rating over the full experiment following intake of the different snacks was 17·2 % (P= 0·026) lower in A carriers compared with TT homozygotes for rs9939609 (FTO, dominant) and 18·6 % (P= 0·020) lower in G carriers compared with AA homozygotes for rs7799039 (LEP, dominant). These observations indicate that FTO and LEP polymorphisms are related to the variation in the feeling of fullness and may play a role in the regulation of food intake. Further studies are required to confirm these initial observations and investigate the 'penetrance' of these genotypes in additional population subgroups.

Immune factors and fatty acid composition in human milk from river/lake, coastal and inland regions of China.

Breast milk fatty acid composition may be affected by the maternal diet during gestation and lactation. The influence of dietary and breastmilk fatty acids on breast milk immune factors is poorly defined. We determined the fatty acid composition and immune factor concentrations of breast milk from women residing in river/lake, coastal and inland regions of China, which differ in their consumption of lean fish and oily fish. Breast milk samples were collected on days 3­5 (colostrum), 14 and 28 post-partum (PP) and analysed for soluble CD14 (sCD14), transforming growth factor (TGF)-b1, TGF-b2, secretory IgA (sIgA) and fatty acids. The fatty acid composition of breast milk differed between the regions and with time PP. The concentrations of all four immune factors in breast milk decreased over time, with sCD14, sIgA and TGF-b1 being highest in the colostrum in the river and lake region. Breast milk DHA and arachidonic acid (AA) were positively associated, and g-linolenic acid and EPA negatively associated, with the concentrations of each of the four immune factors. In conclusion, breast milk fatty acids and immune factors differ between the regions in China characterised by different patterns of fish consumption and change during the course of lactation. A higher breast milk DHA and AA concentration is associated with higher concentrations of immune factors in breast milk, suggesting a role for these fatty acids in promoting gastrointestinal and immune maturation of the infant.

Immunomodulatory effects of a probiotic drink containing Lactobacillus casei Shirota in healthy older volunteers.

PURPOSE: There is growing evidence that probiotics confer health benefits to the host by modulating immune function, especially in older people, where immunosenescence is a feature even of healthy ageing. The aim of this study was to investigate the effect of a probiotic drink containing Lactobacillus casei Shirota (LcS) on immune function in a healthy non-immunocompromised older population. METHODS: Thirty healthy old volunteers were recruited into a randomized placebo-controlled, single-blind crossover study. The volunteers were supplemented with the probiotic drink containing 1.3 × 10(10) CFU LcS or skimmed milk per day for 4 weeks, followed by 4 weeks of washout and were crossed over to the other treatment. Peripheral blood and saliva samples were collected at baseline and end of each treatment. RESULTS: Probiotic consumption was associated with a significant increase in natural killer (NK) cell activity relative to baseline and a significant decrease in the mean fluorescence intensity of CD25 expression in the resting T cells compared with placebo. Additionally, there was a trend towards an increased ratio of IL-10 to IL-12 relative to baseline after LcS intake. CONCLUSIONS: Consumption of a probiotic drink containing LcS improved NK cell activity and tended to produce a more anti-inflammatory cytokine profile in an older population.