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The use of flow cytometry to enumerate microorganisms is gaining traction over the traditional plate count technique on the basis of superior accuracy, precision and time-to-result. Here, we assessed the suitability of live/dead flow cytometry for the enumeration of mixed populations of probiotic bacteria (L. acidophilus, L. paracasei, L. plantarum, L. salivarius, B. lactis and B. bifidum) whilst comparing outcomes with plate counting. Using a novel gating strategy designed specifically for the enumeration of mixed populations, the application of flow cytometry resulted in the detection of higher numbers of viable bacteria with a greater level of repeatability than plate counting (RSD of 6.82 and 13.14% respectively). Across all multi-species blends tested, viable cell input was more accurately recovered by flow cytometry (101.8 ± 6.95%) than plate counts (81.37 ± 16.03%). However, when certain probiotic mixtures contained preparations with high numbers of non-viable cells in their total population, flow cytometry had the potential for overestimation of the viable population. Nevertheless, the comparative plate counts of these mixtures were low and variable, thus supporting the use of flow cytometry for the enumeration of viable bacteria in mixed populations.

Atherosclerosis, an inflammatory disorder of the vasculature and the underlying cause of cardiovascular disease, is responsible for one in three global deaths. Consumption of active food ingredients such as omega-3 polyunsaturated fatty acids, flavanols and phytosterols has many beneficial effects on cardiovascular disease. However, their combined actions on the risk factors for atherosclerosis remains poorly understood. We have previously shown that a formulation containing each of these active components at physiologically relevant doses modulated several monocyte/macrophage processes associated with atherosclerosis in vitro, including inhibition of cytokine-induced pro-inflammatory gene expression, chemokine-driven monocyte migration, expression of M1 phenotype markers, and promotion of cholesterol efflux. The objectives of the present study were to investigate whether the protective actions of the formulation extended in vivo and to delineate the potential underlying mechanisms. The formulation produced several favourable changes, including higher plasma levels of HDL and reduced levels of macrophages and myeloid-derived suppressor cells in the bone marrow. The mRNA expression of liver-X-receptor-α, peroxisome proliferator-activated receptor-γ and superoxide dismutase-1 was induced in the liver and that of interferon-γ and the chemokine (C–X–C motif) ligand 1 decreased, thereby suggesting the potential mechanisms for many beneficial effects. Other changes were also observed such as increased plasma levels of triglycerides and lipid peroxidation that may reflect potential activation of brown fat. This study provides new insights into the protective actions and the potential underlying mechanisms of the formulation in vivo, particularly in relation to risk factors together with changes in systemic inflammation and hepatic lipid alterations associated with atherosclerosis and metabolic syndrome, and supports further assessments in human trials., A combination of nutritionally active ingredients containing omega-3 polyunsaturated fatty acids, flavanols and phytosterols produce many beneficial effects on atherosclerosis-associated risk factors and gene expression in mice fed a high fat diet.

This 9-month randomised, parallel, double-blind, single-centre, placebo-controlled study (PROBE, ISRCTN18030882) assessed the impact of probiotic supplementation on bodyweight. Seventy overweight Bulgarian participants aged 45–65 years with BMI 25–29.9 kg/m2 received a daily dose of the Lab4P probiotic comprising lactobacilli and bifidobacteria (50 billion cfu/day). Participants maintained their normal diet and lifestyle over the duration of the study. The primary outcome was change from baseline in body weight and secondary outcomes included changes in waist circumference, hip circumference and blood pressure. A significant between group decrease in body weight (3.16 kg, 95% CI 3.94, 2.38, p p p

The anti-inflammatory and cholesterol lowering capabilities of probiotic bacteria highlight them as potential prophylactics against chronic inflammatory diseases, particularly cardiovascular disease. Previous studies in silico, in vitro, and in vivo suggest that the Lab4 probiotic consortium may harbour such capabilities and in the current study, we assessed plasma levels of cytokines/chemokines, short chain fatty acids and lipids and faecal levels of bile acids in a subpopulation of healthy Wistar rats included in 90-day repeat dose oral toxicity study. In the rats receiving Lab4, circulating levels of pro-inflammatory interleukin-6, tumour necrosis factor-α and keratinocyte chemoattractant/growth regulated oncogene were significantly lower compared to the control group demonstrating a systemic anti-inflammatory effect. These changes occurred alongside significant reductions in plasma low density lipoprotein cholesterol and increases in faecal bile acid excretion implying the ability to lower circulating cholesterol via the deconjugation of intestinal bile acids. Correlative analysis identified significant associations between plasma tumour necrosis factor-α and the plasma total cholesterol:high density lipoprotein cholesterol ratio and faecal levels of bifidobacteria in the Lab4 rats. Together, these data highlight Lab4 supplementation as a holistic approach to CVD prevention and encourages further studies in humans.

In a double-blind, randomised, parallel-group, placebo-controlled study, healthy school children aged 3-10 years received a probiotic based supplement daily for 6 months to assess the impact on the incidence and duration of upper respiratory tract infection (URTI) symptoms. The intervention comprised Lab4 probiotic (Lactobacillus acidophilus CUL21 and CUL60, Bifidobacterium bifidum CUL20 and Bifidobacterium animalis subsp. lactis CUL34) at 12.5 billion cfu/day plus 50 mg vitamin C or a matching placebo. 171 children were included in the analysis (85 in placebo and 86 in active group). Incidence of coughing was 16% (P=0.0300) significantly lower in the children receiving the active intervention compared to the placebo. No significant differences in the incidence rate of other URTI symptoms were observed. There was significantly lower risk of experiencing five different URTI related symptoms in one day favouring the active group (Risk ratio: 0.31, 95% confidence interval: 0.12, 0.81, P=0.0163). Absenteeism from school and the use of antibiotics was also significantly reduced for those in the active group (-16%, P=0.0060 and -27%, P=0.0203, respectively). Our findings indicate that six months daily supplementation with the Lab4 probiotic and vitamin C combination reduces the incidence of coughing, absenteeism and antibiotic usage in 3 to 10 year old children.

Gut microbiome manipulation to alter the gut-lung axis may potentially protect humans against respiratory infections, and clinical trials of probiotics show promise in this regard in healthy adults and children. However, comparable studies are lacking in overweight/obese people, who have increased risks in particular of viral upper respiratory tract infections (URTI). This Addendum further analyses our recent placebo-controlled trial of probiotics in overweight/obese people (focused initially on weight loss) to investigate the impact of probiotics upon the occurrence of URTI symptoms. As well as undergoing loss of weight and improvement in certain metabolic parameters, study participants taking probiotics experienced a 27% reduction in URTI symptoms versus control, with those ≥45 years or BMI ≥30 kg/m2 experiencing greater reductions. This symptom reduction is apparent within 2 weeks of probiotic use. Gut microbiome diversity remained stable throughout the study in probiotic-treated participants. Our data provide support for further trials to assess the potential role of probiotics in preventing viral URTI (and possibly also COVID-19), particularly in overweight/obese people.

We read with interest Rodriguez and colleagues’ study, using microbiota transfer from obese stool donors into inulin-treated hum-ob mice, to define a gut microbiome signature predicting response to prebiotic.1 However, the impact of other microbiome-based interventions (and particularly probiotics) on weight loss in humans is highly-variable between individuals.2 We were interested as to whether baseline gut microbiota, or aspects of host physiology, may predict weight loss during probiotic studies. In our recent double-blind study (ISRCTN12562026), overweight/obese adults were randomised to either 6 months of Lab4P probiotic (containing lactobacilli and bifidobacteria) or placebo.3 A higher proportion of participants receiving probiotic lost weight compared with those receiving placebo, and the extent of weight loss in the probiotic arm was greater than the placebo group.3 We subsequently performed metataxonome and metabonome analysis on stool samples from study participants (using 16S rRNA gene sequencing and liquid chromatography-mass spectrometry bile acid profiling, respectively, applying established protocols4 5). We observed no difference in stool microbiota alpha-diversity at baseline between participants losing weight during the study versus those who did not (figure 1A); furthermore, no differences were observed in microbiota composition between groups at any taxonomic level. Conversely, on stool bile acid profiling, …

In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years old) with BMI 25–34.9 kg/m2 received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, p 2, p p p p p = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (−2.5%, p p = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, p

Atherosclerosis and its complications are responsible for one in three global deaths. Nutraceuticals show promise in the prevention and treatment of atherosclerosis but require an indepth understanding of the mechanisms underlying their actions. A previous study showed that the omega-6 fatty acid, dihomo-γ-linolenic acid (DGLA), attenuated atherosclerosis in the apolipoprotein E deficient mouse model system. However, the mechanisms underlying such protective effects of DGLA are poorly understood and were therefore investigated. We show that DGLA attenuates chemokine-driven monocytic migration together with foam cell formation and the expression of key pro-atherogenic genes induced by three pro-inflammatory cytokines in human macrophages. The effect of DGLA on interferon-γ signaling was mediated via inhibition of signal transducer and activator of transcription-1 phosphorylation on serine 727. In relation to anti-foam cell action, DGLA inhibits modified LDL uptake by both macropinocytosis and receptor-mediated endocytosis, the latter by reduction in expression of two key scavenger receptors (SR-A and CD36), and stimulates cholesterol efflux from foam cells. DGLA also improves macrophage mitochondrial bioenergetic profile by decreasing proton leak. Gamma-linolenic acid and prostaglandin E1, upstream precursor and key metabolite respectively of DGLA, also acted in an anti-atherogenic manner. The actions of DGLA extended to other key atherosclerosis-associated cell types with attenuation of endothelial cell proliferation and migration of smooth muscle cells in response to platelet-derived growth factor. This study provides novel insights into the molecular mechanisms underlying the anti-atherogenic actions of DGLA and supports further assessments on its protective effects on plaque regression in vivo and in human trials., Highlights • Dihomo-γ-linolenic acid (DGLA) attenuates atherosclerosis in a mouse model system. • The mechanisms underlying anti-atherogenic actions of DGLA are poorly understood. • DGLA inhibited atherogenic processes in three key cell types in this disease. • Mechanisms underlying such protective actions of DGLA were identified. • Studies inform on the beneficial anti-atherogenic actions of DGLA.

Scope Previous studies showed that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (Lab4P) reduced diet-induced weight gain and plasma cholesterol levels in C57BL/6J mice fed a high fat diet (HFD). The effect of Lab4P on atherosclerosis is not known and was therefore investigated. Methods and results Atherosclerosis-associated parameters were analyzed in LDL receptor deficient mice fed HFD for 12 weeks alone or supplemented with Lab4P. Lab4P increased plasma HDL and triglyceride levels and decreased LDL/VLDL levels. Lab4P also reduced plaque burden and content of lipids and macrophages, indicative of dampened inflammation, and increased smooth muscle cell content, a marker of plaque stabilization. Atherosclerosis arrays showed that Lab4P altered the liver expression of 19 key disease-associated genes. Lab4P also decreased the frequency of macrophages and T-cells in the bone marrow. In vitro assays using conditioned media from probiotic bacteria demonstrated attenuation of several atherosclerosis-associated processes in vitro such as chemokine-driven monocytic migration, proliferation of monocytes and macrophages, foam cell formation and associated changes in expression of key genes, and proliferation and migration of vascular smooth muscle cells. Conclusion This study provides new insights into the anti-atherogenic actions of Lab4P together with the underlying mechanisms and supports further assessments in human trials. This article is protected by copyright. All rights reserved.

Neurodegeneration has been linked to changes in the gut microbiota and this study compares the neuroprotective capability of two bacterial consortia, known as Lab4 and Lab4b, using the established SH-SY5Y neuronal cell model. Firstly, varying total antioxidant capacities (TAC) were identified in the intact cells from each consortia and their secreted metabolites, referred to as conditioned media (CM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Crystal Violet (CV) assays of cell viability revealed that Lab4 CM and Lab4b CM could induce similar levels of proliferation in SH-SY5Y cells and, despite divergent TAC, possessed a comparable ability to protect undifferentiated and retinoic acid-differentiated cells from the cytotoxic actions of rotenone and undifferentiated cells from the cytotoxic actions of 1-methyl-4-phenylpyridinium iodide (MPP+). Lab4 CM and Lab4b CM also had the ability to attenuate rotenone-induced apoptosis and necrosis with Lab4b inducing the greater effect. Both consortia showed an analogous ability to attenuate intracellular reactive oxygen species accumulation in SH-SY5Y cells although the differential upregulation of genes encoding glutathione reductase and superoxide dismutase by Lab4 CM and Lab4b CM, respectively, implicates the involvement of consortia-specific antioxidative mechanisms of action. This study implicates Lab4 and Lab4b as potential neuroprotective agents and justifies their inclusion in further in vivo studies.

The transcription of the ATP-binding cassette transporter A1 (ABCA1) gene, which plays a key anti-atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) in the LXRs-mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have, therefore, investigated a potential role for PKC in the action of LXR/RXR agonist 22-(R)-hydroxycholesterol (22-(R)-HC)/9-cis-retinoic acid (9cRA) in THP-1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein-1 (AP-1) DNA binding activity in macrophages treated with 22-(R)-HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype-specific inhibitors. These studies, therefore, reveal a potentially important role for PKC in the action of 22-(R)-HC and 9cRA in human macrophages.

Objective: The objective of this study was to identify the immunomodulatory potential of two consortia of lactic acid bacteria, “Lab4” and “Lab4b”, using human macrophages as an in vitro model system. Methods: THP-1 monocyte-derived macrophages were exposed to metabolites of Lab4 or Lab4b. RT-qPCR was performed on macrophages to determine the expression of M1 pro-inflammatory (IL-1β, IL-18 and CD80) or M2 anti-inflammatory (CD206) marker mRNA in addition to IL-1β protein and inflammasome (NLRP3, Caspase-1, NLRP1, NLRC4 and AIM2) mRNA expression. Bacterial (LPS and ATP) and viral (Poly I:C) challenge were stimulated to determine the potential of these consortia to regulate IL-1β protein, inflammasome mRNA expression, and antiviral IL-12 mRNA expression and protein under inflammatory conditions. The ability of these consortia to modulate macrophage phagocytosis of E. coliwas also assessed. Results:Results: Lab4 and Lab4b metabolites promoted an M1 phenotype in macrophages in vitro by increasing mRNA expression of IL-1β, IL-18, and CD80 and reducing mRNA expression of CD206. Induction of IL-1β protein suggested involvement of the inflammasome. mRNA expression of NLRP3, Caspase-1, NLRP1 and AIM2 was induced by Lab4 and mRNA expression of NLRP3 and Caspase-1 was induced by Lab4b suggesting different potential modes of action of the two consortia. Lab4 and Lab4b metabolites, in combination with this LPS and ATP challenge, enhanced IL-1β mRNA and protein expression further, accompanied by different mRNA expression profiles of inflammasome genes by the two consortia. Lab4 and Lab4b also induced expression of mRNA and protein of the antiviral response gene, IL-12. In combination with Poly I:C challenge, Lab4 induced IL-12 protein further, while Lab4b induced IL-12p25/IL-12p40 mRNA further highlighting potential differences. Both consortia were also able to induce phagocytosis of E. coli particles. Conclusion: Data generated from this study suggest the potential for organism-dependent control of the immunoregulatory response seen in human macrophages.

Hypercholesterolaemia is a major risk factor for cardiovascular disease and it has been found that some probiotic bacteria possess cholesterol-lowering capabilities. In this study, the ability of the Lab4 probiotic consortium to hydrolyse bile salts, assimilate cholesterol and regulate cholesterol transport by polarised Caco-2 enterocytes was demonstrated. Furthermore, in wild-type C57BL/6J mice fed a high fat diet, 2-weeks supplementation with Lab4 probiotic consortium plus Lactobacillusplantarum CUL66 resulted in significant reductions in plasma total cholesterol levels and suppression of diet-induced weight gain. No changes in plasma levels of very low-density lipoprotein/low-density lipoprotein, high-density lipoprotein, triglycerides, cytokines or bile acids were observed. Increased amounts of total and unconjugated bile acids in the faeces of the probiotic-fed mice, together with modulation of hepatic small heterodimer partner and cholesterol-7α-hydroxylase mRNA expression, implicates bile salt hydrolase activity as a potential mechanism of action. In summary, this study demonstrates the cholesterol-lowering efficacy of short-term feeding of the Lab4 probiotic consortium plus L. plantarum CUL66 in wild-type mice and supports further assessment in human trials.

Background:\ud This pilot study investigates the efficacy of a probiotic consortium (Lab4) in combination with vitamin C on the prevention of respiratory tract infections in children attending preschool facilities.\ud \ud Subjects/methods:\ud In a double-blind, randomised, placebo-controlled pilot study with children aged 3–6 years, 57 received 1.25 × 1010 colony-forming units of Lactobacillus acidophilus CUL21 (NCIMB 30156), Lactobacillus acidophilus CUL60 (NCIMB 30157), Bifidobacterium bifidum CUL20 (NCIMB 30153) and Bifidobacterium animalis subsp. lactis CUL34 (NCIMB 30172) plus 50 mg vitamin C or a placebo daily for 6 months.\ud \ud Results:\ud Significant reductions in the incidence rate of upper respiratory tract infection (URTI; 33%, P=0.002), the number of days with URTI symptoms (mean difference: −21.0, 95% confidence interval (CI):−35.9, −6.0, P=0.006) and the incidence rate of absence from preschool (30%, P=0.007) were observed in the active group compared with the placebo. The number of days of use of antibiotics, painkillers, cough medicine or nasal sprays was lower in the active group and reached significance for use of cough medicine (mean difference: −6.6, 95% CI: −12.9, −0.3, P=0.040). No significant differences were observed in the incidence rate ratio or duration of lower respiratory tract infection or in the levels of plasma cytokines, salivary immunoglobulin A or urinary metabolites.\ud \ud Conclusions:\ud Supplementation with a probiotic/vitamin C combination may be beneficial in the prevention and management of URTIs.

Liver X receptors (LXRs) belong to the nuclear receptor superfamily of ligand-dependent transcription factors. LXRs are activated by oxysterols, metabolites of cholesterol, and therefore act as intracellular sensors of this lipid. There are two LXR genes (α and β) that display distinct tissue/cell expression profiles. LXRs interact with regulatory sequences in target genes as heterodimers with retinoid X receptor. Such direct targets of LXR actions include important genes implicated in the control of lipid homeostasis, particularly reverse cholesterol transport. In addition, LXRs attenuate the transcription of genes associated with the inflammatory response indirectly by transrepression. In this review, we describe recent evidence that both highlights the key roles of LXRs in atherosclerosis and inflammation and provides novel insights into the mechanisms underlying their actions. In addition, we discuss the major limitations of LXRs as therapeutic targets for the treatment of atherosclerosis and how these are being addressed.

Cardiovascular disease results in more deaths globally than any other ailment. A major contributing factor to its pathology is atherosclerosis; an inflammatory disorder characterized by the development of fibrotic plaques within the arterial walls. Key to the progression of atherosclerosis are macrophages that contribute to plaque development by transforming into lipid-loaded foam cells upon internalization of modified lipoproteins. Accumulation of such foam cells in the arterial wall initiates the formation of fatty streaks that subsequently develop into advanced plaques that are prone to rupture. Clearly, macrophage lipid metabolism and foam cell biology represent a key avenue of research during the ongoing search for novel therapeutic targets that can be used in the clinical intervention of atherosclerosis. In this article, we aim to summarize the current status of research on macrophages, lipid metabolism and gene expression in relation to atherogenesis and both current and potential future therapies.

Atherosclerosis is an inflammatory disorder of the vasculature that is orchestrated by the action of cytokines. Macrophages play a prominent role in all stages of this disease, including foam cell formation, production of reactive oxygen species, modulation of the inflammatory response and the regulation of the stability of atherosclerotic plaques. The role of the matrix metalloproteinase family in the control of plaque stability is well established. A disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) family has been implicated in several diseases and the expression of ADAMTS-4 in macrophages of atherosclerotic lesions has suggested a potential role for this protease in atherosclerosis. However, the action of cytokines on the expression of ADAMTS-4 in macrophages is poorly understood. We have investigated here the effect of transforming growth factor-β (TGF-β) on ADAMTS-4 expression in macrophages along with the regulatory mechanisms underlying its actions. Consistent with the anti-atherogenic role of TGF-β, this cytokine decreased the expression of ADAMTS-4 mRNA and protein in human macrophages. Transient transfection assays showed that the −100 to +10 promoter region contained the minimal TGF-β response elements. Small-interfering RNA-mediated knockdown revealed a critical role for Smads, p38 mitogen-activated protein kinase and c-Jun in the action of TGF-β on ADAMTS-4 mRNA expression. These studies show for the first time that TGF-β inhibits the expression of ADAMTS-4 in human macrophages and identifies the signalling pathways underlying this response. The inhibition of macrophage ADAMTS-4 expression is likely to contribute to the anti-atherogenic, plaque stabilisation action of TGF-β.

The proinflammatory cytokine IFN-γ is a master regulator of atherosclerosis and mediates its cellular actions mainly through STAT1. Unfortunately, the impact of other IFN-γ inducible pathways on STAT1 activation and the regulation of downstream responses associated with atherosclerosis in human macrophages are poorly understood and were therefore investigated. In this study, we demonstrate that the IFN-γ–mediated phosphorylation of STAT1 on Ser727, crucial for its maximal activity, was attenuated in human macrophages by pharmacological inhibition of ERK. In these cells, IFN-γ induced changes in the expression of several key genes implicated in atherosclerosis, such as MCP-1, through an ERK-dependent mechanism. Additionally, the IFN-γ–induced activity of STAT1-responsive promoters was attenuated by transfection of dominant-negative forms of ERK and other key components of this pathway. Furthermore, the IFN-γ–induced uptake of acetylated and oxidized low-density lipoprotein by human macrophages was attenuated by pharmacological inhibition or RNA interference–mediated knockdown of ERK. These studies suggest a critical role for ERK signaling in the IFN-γ–mediated changes in macrophage cholesterol homeostasis and gene expression during atherosclerosis.

Hypercholesterolemia drives the development of cardiovascular disease, the leading cause of mortality in western society. Supplementation with probiotics that interfere with cholesterol metabolism may provide a contribution to disease prevention. Lactobacillus plantarum CUL66 (NCIMB 30280) has been assessed in vitro for its ability to impact cholesterol absorption. L. plantarum CUL66 tested positive for bile salt hydrolase activity and the ability to assimilate cholesterol from culture media. RT-qPCR analysis showed that the bacterium significantly decreased the expression of Niemann-Pick C1-like 1 and ATP-binding cassette transporter-1 in polarised Caco-2 cells after 6 h exposure. Conversely, the expression of ATP-binding cassette sub-family G member (ABCG)-5 and ABCG-8, and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased. Using a radiolabelled assay, we also observed significant reductions in the uptake and basolateral efflux of cholesterol by Caco-2 cells exposed to L. plantarum CUL66. This in vitro study identified L. plantarum CUL66 as a cholesterol lowering bacteria by highlighting its ability to beneficially regulate multiple in vitro events associated with intestinal cholesterol metabolism and provides evidence of efficacy for its inclusion in future in vivo studies.

Introduction Atherosclerosis is the underlying cause of cardiovascular disease that leads to more global mortalities each year than any other ailment. Consumption of active food ingredients such as phytosterols, omega-3 polyunsaturated fatty acids and flavanols are known to impart beneficial effects on cardiovascular disease although the combined actions of such agents in atherosclerosis is poorly understood. The aim of this study was to screen a nutritional supplement containing each of these active components for its anti-atherosclerotic effect on macrophages in vitro. Results The supplement attenuated the expression of intercellular adhesion molecule-1 and macrophage chemoattractant protein-1 in human and murine macrophages at physiologically relevant doses. The migratory capacity of human monocytes was also hindered, possibly mediated by eicosapentaenoic acid and catechin, while the ability of foam cells to efflux cholesterol was improved. The polarisation of murine macrophages towards a pro-inflammatory phenotype was also attenuated by the supplement. Conclusion The formulation was able to hinder multiple key steps of atherosclerosis development in vitro by inhibiting monocyte recruitment, foam cell formation and macrophage polarisation towards an inflammatory phenotype. This is the first time a combination these ingredients has been shown to elicit such effects and supports its further study in preclinical in vivo models.

The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-kappaB recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation.

The history of (and perhaps the lessons to be learned from) peritoneal CA125 investigation presents no better example of the diffi culties in assigning the status of a ‘marker’ predictive of membrane dysfunction to factors measured in dialysis effl uent. In 1995, Visser et al. [3] were the fi rst to suggest that CA125 could be used as ‘a bulk marker of mesothelial cell mass in stable PD patients’. This was suggested based largely on in vitro and ex vivo approaches that suggested a mesothelial-cell-specifi c location (i.e. it is not expressed by leucocytes). Their data also suggested passive secretion of the antigen and a relationship to the number of mesothelial cells in culture. These data on the ‘regulation’ of secretion, however, were at odds with both pre-existing and subsequently produced data suggestive of induction of CA125 by pro-infl ammatory cytokines [4, 5] . In the current issue, Br e borowicz et al. fan the fl ames of this debate further by suggesting, based exclusively on in vitro data, that CA125 production by peritoneal mesothelial cells neither correlates with, nor is regulated by pro-infl ammatory cytokines [6] . The evidence ascribing CA125 as a clinical marker of mesothelial cell or membrane integrity was apparently strengthened by the studies of Ho-dac Panekeet et al. [7] who presented cross-sectional data suggesting a relationIdentifying easily measurable markers that are of prognostic value in predicting membrane failure in peritoneal dialysis (PD) patients remains one of the major challenges. Facilitating this endeavour has always been the ease of sampling of drained peritoneal effl uent within which, with the development of ever more sensitive measurement techniques, any number of mediators can be quantitated. Local production of these ‘factors’ (i.e. of peritoneal membrane or infi ltrating leucocyte origin) would then theoretically, at least, allow changes to be correlated with clinical outcomes and thereby their usefulness as indicators of altered membrane status. So far then there is an attractiveness and inescapable logic to this type of approach that would predict that after almost 20 years of investigation (since the fi rst measurements of prostaglandins in peritoneal effl uent [1, 2] ) there would now be suffi cient data on a number of markers predictive of clinical and membrane-associated changes in these patients. Unfortunately, however, despite signifi cant scientifi c endeavour (and as we will discuss a number of long-term clinical studies) we are really little nearer to identifying our holy grail. Published online: April 7, 2005

The objective of this study was to examine the effect of daily probiotic supplementation upon the immune profile of healthy participants by the assessment of ex vivo cytokine production. Twenty healthy adult volunteers received a multi-strain probiotic supplement consisting of two strains of Lactobacillus acidophilus (CUL60 and CUL21), Bifidobacterium lactis (CUL34) and Bifidobacterium bifidum (CUL20) and fructooligosaccharide for 12 weeks. Blood samples were collected at baseline, 6 and 12 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured ex vivo in the presence or absence of lipopolysaccharide and cytokine production was assessed. Postintervention, a significant decrease in the production of interleukin-6 and interleukin-1β was apparent when PBMCs were incubated in the presence of lipopolysaccharide, whilst a significant increase in IL-10 and transforning growth factor-β production was seen when the cells were incubated without an additional stimulus. This preliminary study demonstrates the potential of a multi-strain probiotic supplement to alter the immune response as demonstrated by changes in ex vivo cytokine production. Such results demonstrate the potential benefit of probiotic supplementation for healthy individuals and warrants further investigation.

Highlights • Macrophages can internalise LDL through scavenger receptor-independent mechanisms. • Macropinocytosis has been shown to contribute significantly to foam cell formation. • Cytokines such as TGF-β, IL-33, IFN-γ and IL-17A can modulate macropinocytosis. • TGF-β mediated inhibition of macropinocytosis is a Smad-2/-3-independent process. • Macropinocytosis is a promising target for therapeutic intervention of atherosclerosis., A key event during the formation of lipid-rich foam cells during the progression of atherosclerosis is the uptake of modified low-density lipoproteins (LDL) by macrophages in response to atherogenic mediators in the arterial intima. In addition to scavenger receptor-dependent uptake of LDL, macropinocytosis is known to facilitate the uptake of LDL through the constitutive and passive internalization of large quantities of extracellular solute. In this study we confirm the ability of macropinocytosis to facilitate the uptake of modified LDL by human macrophages and show its modulation by TGF-β, IFN-γ, IL-17A and IL-33. Furthermore we show that the TGF-β-mediated inhibition of macropinocytosis is a Smad-2/-3-independent process.

The ubiquitous mammalian extracellular matrix glycosaminoglycan hyaluronan (HA) plays a pivotal role in the regulation of cell phenotype in fibrosis and scarring. Transforming growth factor-beta 1 (TGF-β1) and interleukin-1 beta (IL-1β) up-regulate hyaluronan synthase (HAS) 1 and HAS2 in dermal fibroblasts and renal proximal tubular epithelial cells, and subsequent HA synthesis regulates cell phenotype. In the present study, we investigated the mechanism of HAS1 transcriptional up-regulation in response to these cytokines. We used 5'-rapid amplification of cDNA ends analysis to identify the 5' end of HAS1 transcripts, resulting in an increase of 26 nucleotides to the HAS1 exon 1 sequence of reference sequence NM_001523. Constitutive luciferase activity of upstream DNA sequences was shown in luciferase reporter assays, but our reporter vector signals were refractory to the addition of TGF-β1 and IL-1β. Using siRNAs to knockdown transcription factor mRNAs, we showed that TGF-β1 up-regulation of HAS1 transcription was mediated via Smad3 but not Smad2, while HAS1 induction by IL-1β was Sp3, not Sp1, dependent.

Cardiovascular disease is the biggest killer globally and the principal contributing factor to the pathology is atherosclerosis; a chronic, inflammatory disorder characterized by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. Macrophages are fundamental to the immune response directed to the site of inflammation and their normal, protective function is harnessed, detrimentally, in atherosclerosis. Macrophages contribute to plaque development by internalizing native and modified lipoproteins to convert them into cholesterol-rich foam cells. Foam cells not only help to bridge the innate and adaptive immune response to atherosclerosis but also accumulate to create fatty streaks, which help shape the architecture of advanced plaques. Foam cell formation involves the disruption of normal macrophage cholesterol metabolism, which is governed by a homeostatic mechanism that controls the uptake, intracellular metabolism, and efflux of cholesterol. It has emerged over the last 20 years that an array of cytokines, including interferon-γ, transforming growth factor-β1, interleukin-1β, and interleukin-10, are able to manipulate these processes. Foam cell targeting, anti-inflammatory therapies, such as agonists of nuclear receptors and statins, are known to regulate the actions of pro- and anti-atherogenic cytokines indirectly of their primary pharmacological function. A clear understanding of macrophage foam cell biology will hopefully enable novel foam cell targeting therapies to be developed for use in the clinical intervention of atherosclerosis. Abbreviations ABCA-1/G-1, ATP-binding cassette transporter A-1/G-1; ACAT-1, Acyl-CoA:cholesterol acyltransferase-1; AcLDL, acetylated LDL; ADRP, adipocyte differentiation related protein; ApoE/A–I, apolipoprotein E/A–I; BMMs, bone-marrow derived macrophages; CD, cluster of differentiation; CHOP, C/EBP-homologous protein; CPT-1, carnitine palmitoyl transferase-1; CRP, C-reactive protein; CXCL, chemokine (C–X–C motif) ligand; CVD, cardiovasculardisease; DR3, Death Receptor 3; ECM, extracellular matrix; FAs, non-esterified fatty acids; HDL, high-density lipoprotein; HL, hepatic lipase; HMDMs, human monocyte-derived macrophages; HMG CoA, 3-hydroxy-3-methylglutaryl-CoA; ICAM1, intercellular adhesion molecule-1; IDL, intermediate density lipoprotein; IFN, interferon; IL, interleukin; IMT, intima-media thickness; JNK2, c-Jun N-terminal kinase 2; LIGHT, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells; LDL, low density lipoprotein; LDLr, low density lipoprotein receptor; LPL, lipoprotein lipase; LXR, liver X receptor; MAPK, mitogen activated protein kinase; M1, classically activated macrophage; M2, alternatively activated macrophage; MCP-1, monocyte chemoattractant protein-1; M-CSF, macrophage-colony stimulating factor; MMP, matrix metalloproteinase; MPMs, murine peritoneal macrophages; NPC, Niemann Pick type C; NCEH, neutral cholesteryl ester hydrolase; oxLDL, oxidized LDL; PPAR, peroxisome proliferator-activated receptor; sIFN-γR, soluble interferon-γ receptor; SOCS3, suppressor of cytokine signaling 3; sPLA2, secretory phospholipase A2; SR, scavenger receptor; SR-PSOX, scavenger receptor for phosphatidylserine and oxidized LDL; Th, T-helper; TGF-β1, transforming growth factor-β1; TL1A, TNF-like protein 1A; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TNFSF, tumor necrosis factor superfamily; TWEAK, TNF-like weak inducer of apoptosis; UPR, unfolded protein response; VCAM-1, vascular cell adhesion molecule-1; VLDL, very low-density lipoprotein; VSMC, vascular smooth muscle cell.

There is evidence that long chain n-3 PUFA (such as from fish oils) provide atheroprotection through, in part, changes in macrophage function although it has not been fully determined whether these n-3 PUFA target cellular mechanisms that control macrophage foam cell formation. Therefore, we investigated whether the n-3 PUFA, EPA and DHA, modulate modified low-density lipoprotein (LDL) uptake by human macrophages. The uptake of fluorophore labeled acetylated LDL (AcLDL) and/or oxidized LDL (OxLDL) by THP-1 macrophages and primary human monocyte-derived macrophages were measured by flow cytometry following co-incubation with EPA or DHA in vitro. DHA inhibited both AcLDL and OxLDL uptake in human macrophages whilst EPA reduced AcLDL and increased OxLDL uptake. These effects were only partly explained by changes in the mRNA and protein expression of key scavenger receptors, such as CD36 and scavenger receptor-A, in these cells suggesting the involvement of a scavenger receptor-independent mechanism. EPA and DHA inhibited macropinocytosis, as measured by Lucifer Yellow uptake, in human macrophages and attenuated the expression of Syndecan-4, which has been implicated in the uptake of other modified forms of LDL. EPA and DHA reduced modified LDL uptake by human macrophages through a mechanism that is in part scavenger receptor-independent and may involve inhibition of macropinocytosis and Syndecan-4 expression. This suggests that both EPA and DHA are capable of regulating macrophage foam cell formation and adds to the evidence describing an atheroprotective role for n-3 PUFA, implicating them as potential therapeutic agents for the treatment of clinical atherosclerosis.

The anti-atherogenic cytokine, TGF-β, plays a key role during macrophage foam cell formation by modulating the expression of key genes involved in the control of cholesterol homeostasis. Unfortunately, the molecular mechanisms underlying these actions of TGF-β remain poorly understood. In this study we examine the effect of TGF-β on macrophage cholesterol homeostasis and delineate the role of Smads-2 and ‐3 during this process. Western blot analysis showed that TGF-β induces a rapid phosphorylation-dependent activation of Smad-2 and ‐3 in THP-1 and primary human monocyte-derived macrophages. Small interfering RNA-mediated knockdown of Smad-2/3 expression showed that the TGF-β-mediated regulation of key genes implicated in the uptake of modified low density lipoproteins and the efflux of cholesterol from foam cells was Smad-dependent. Additionally, through the use of virally delivered Smad-2 and/or Smad-3 short hairpin RNA, we demonstrate that TGF-β inhibits the uptake of modified LDL by macrophages through a Smad-dependent mechanism and that the TGF-β-mediated regulation of CD36, lipoprotein lipase and scavenger receptor-A gene expression was dependent on Smad-2. These studies reveal a crucial role for Smad signaling, particularly Smad-2, in the inhibition of foam cell formation by TGF-β through the regulation of expression of key genes involved in the control of macrophage cholesterol homeostasis., Highlights ► Anti-atherogenic cytokine TGF-β inhibits macrophage foam cell formation. ► The role of Smads in the control of macrophage cholesterol homeostasis was studied. ► Smads were found to play a key role in the TGF-β-mediated uptake of modified LDL. ► A dominant role of Smad2 was identified in the regulation of gene expression. ► The TGF-β-Smad axis may represent a powerful anti-foam cell therapeutic target.