Topical equol preparation improves structural and molecular skin parameters.

OBJECTIVE: Equol has been shown to improve skin health and regeneration, due to its antioxidative, phytoestrogenic and epigenetic characteristics. The effects of a topical intervention on skin structure, telomere length and epigenetic markers in skin cells were analysed. METHODS: Sixty-four participants were divided in four groups and three of them treated topically with the following: emulsion with Equol powder (Isoflavandiol-E-55-RS®); emulsion with microencapsulated Equol (Vesisorb® Isoflavandiol-E-55-RS®) and an emulsion with lecithin (Vesisorb® placebo). A control group of 6 volunteers did not receive any intervention. The active compound was a 0.5% equol-racemate. For 58 participants, all samples were collected. Structural analysis, molecular analysis and questionnaires were performed at the start of the study and after 8 weeks of intervention, twice a day. Structural skin parameters were analysed by Visioscan® VC 98 and Cutometer® dual MPA 580. Molecular analyses from epidermal cells collected by skin stripping of the forehead included telomere length and LINE-1 methylation, following DNA extraction, bisulfite conversion and qPCR as well as high-resolution melting curve analysis. Effects of nutrition and lifestyle habits were evaluated with a standardized food and lifestyle questionnaire. RESULTS AND DISCUSSION: The surface analysis showed significant improvements in skin roughness, skin texture and skin smoothness after both interventions. Cutometer® dual MPA 580 measurement revealed improvement of skin firmness and elasticity parameters for both preparations. A decrease in mean LINE-1 methylation (n.s.) and telomere length (sign. P < 0.05) was observed in the sample group with age. In the treated groups, significantly longer telomeres were observed after intervention. Whether changes in telomere length reflect changes in the regulation of telomerase, epigenetic interactions or turnover of keratinocytes needs further research. Stability and availability of preparations in skin seems to be high as not many significant differences in the activity of pure or encapsulated substances were seen. CONCLUSION: The results of this study indicate that equol has beneficial effects on structural as well as molecular skin parameters and encourages further investigations to decipher the epigenetic regulation of skin ageing and interactions of equol.

Faecalibacterium prausnitzii phylotypes in type two diabetic, obese, and lean control subjects.

Faecalibacterium prausnitzii is one of the main butyrate producers in the healthy human gut. Information on its genetic diversity is lacking, although two genetic phylotypes have been differentiated. In the present study, F. prausnitzii phylotypes were examined in faeces of obese and type two diabetes with similar eating behaviour compared to a lean control group. The purpose of the study was to analyse if an excessive butyrate production induced by different F. prausnitzii phylotypes discriminates between obese developing type two diabetes or not. The faecal samples were analysed for the total abundance of F. prausnitzii 16S rRNA copies, fragment lengths polymorphism, high resolution melt curve analysis (HRM) and the butyryl-CoA:acetate CoA-transferase gene copies and melt curve variances. The diabetic group was found to differ significantly from the lean control group in the results of qPCR, butyryl-CoA:acetyate CoA-transferase gene melt curve, and HRM. F. prausnitzii phylotypes differed in obese with and without developed diabetes type two. Different phylotypes of F. prausnitzii may lead to differences in the inflammatory genesis in the host. F. prausnitzii phylotypes may have an influence on developing type two diabetes and might also act as starting points for prevention and therapy of obesity associated disease.

Gut microbiota composition correlates with changes in body fat content due to weight loss.

Genetics, lifestyle, and dietary habits contribute to metabolic syndrome, but also an altered gut microbiota has been identified. Based on this knowledge it is suggested that host bacterial composition tends to change in response to dietary factors and weight loss. The aim of this study was to identify bacteria affecting host metabolism in obesity during weight loss and to correlate them with changes of the body composition obtained from bioelectrical impedance analysis (BIA). We recruited obese individuals receiving a dietary intervention according DACH (German, Austrian, and Swiss Society of Nutrition) reference values and guidelines for 'prevention and therapy of obesity' of DAG e.V., DDG, DGE e.V., and DGEM e.V. over three months. Faecal microbiota and BIA measurements were conducted at three time points, before, during, and after the intervention. Gut microbiota was analysed on the basis of 16S rDNA with quantitative real time PCR. Additionally, a food frequency questionnaire with questions to nutritional behaviour, lifestyle, and physical activity was administered before intervention. After weight reduction, obese individuals showed a significant increase of total bacterial abundance. The ratio of Firmicutes/Bacteroidetes significantly decreased during intervention. Lactobacilli significantly increased between the first and the second time point. These differences also correlated with differences in weight percentage. During the intervention period Clostridium cluster IV increased significantly between the second and the third time point. In contrast Clostridium cluster XIVa showed a decreased abundance. The dominant butyrate producer, Faecalibacterium prausnitzii, significantly increased as did the abundance of the butyryl-CoA: acetate CoA-transferase gene. Archaea and Akkermansia were significantly more prevalent after weight reduction. Our results show a clear difference in the gut bacterial composition before and after dietary intervention with a rapid change in gut microbial composition after a few weeks, but also indicate that a major shift requires long term dietary treatment.

Therapeutic perspectives of epigenetically active nutrients.

Many nutrients are known for a wide range of activities in prevention and alleviation of various diseases. Recently, their potential role in regulating human health through effects on epigenetics has become evident, although specific mechanisms are still unclear. Thus, nutriepigenetics/nutriepigenomics has emerged as a new and promising field in current epigenetics research in the past few years. In particular, polyphenols, as part of the central dynamic interaction between the genome and the environment with specificity at physiological concentrations, are well known to affect mechanisms underlying human health. This review summarizes the effects of dietary compounds on epigenetic mechanisms in the regulation of gene expression including expression of enzymes and other molecules responsible for drug absorption, distribution, metabolism and excretion in cancer, metabolic syndrome, neurodegenerative disorders and hormonal dysfunction.

Impact of a probiotic, inulin, or their combination on the piglets' microbiota at different intestinal locations.

Natural feed additives are used to maintain health and to promote performance of pigs without antibiotics. Effects of a probiotic, inulin, and their combination (synbiotic), on the microbial diversity and composition at different intestinal locations were analysed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and 16S rRNA gene pyrosequencing. Bacterial diversity assessed by DGGE and/or pyrosequencing was increased by inulin in all three gut locations and by the synbiotic in the caecum and colon. In contrast, the probiotic did only affect the microbiota diversity in the ileum. Shifts in the DGGE microbiota profiles of the caecum and colon were detected for the pro- and synbiotic fed animals, whereas inulin profiles were more similar to the ones of the control. 16S rRNA gene pyrosequencing revealed that all three additives could reduce Escherichia species in each gut location, indicating a potential beneficial effect on the gut microbiota. An increase of relative abundance of Clostridiaceae in the large intestine was found in the inulin group and of Enterococcaceae in the ileum of probiotic fed pigs. Furthermore, real-time PCR results showed that the probiotic and synbiotic increased bifidobacterial numbers in the ileum, which was supported by sequencing results. The probiotic and inulin, to different extents, changed the diversity, relative abundance of phylotypes, and community profiles of the porcine microbiota. However, alterations of the bacterial community were not uniformly between gut locations, demonstrating that functionality of feed additives is site specific. Therefore, gut sampling from various locations is crucial when investigations aim to identify the composition of a healthy gut microbiota after its manipulation through feed additives.

Microbiota and epigenetic regulation of inflammatory mediators in type 2 diabetes and obesity.

Metabolic syndrome is associated with alterations in the structure of the gut microbiota leading to low-grade inflammatory responses. An increased penetration of the impaired gut membrane by bacterial components is believed to induce this inflammation, possibly involving epigenetic alteration of inflammatory molecules such as Toll-like receptors (TLRs). We evaluated changes of the gut microbiota and epigenetic DNA methylation of TLR2 and TLR4 in three groups of subjects: type 2 diabetics under glucagon-like peptide-1 agonist therapy, obese individuals without established insulin resistance, and a lean control group. Clostridium cluster IV, Clostridium cluster XIVa, lactic acid bacteria, Faecalibacterium prausnitzii and Bacteroidetes abundances were analysed by PCR and 454 high-throughput sequencing. The epigenetic methylation in the regulatory region of TLR4 and TLR2 was analysed using bisulfite conversion and pyrosequencing. We observed a significantly higher ratio of Firmicutes/ Bacteroidetes in type 2 diabetics compared to lean controls and obese. Major differences were shown in lactic acid bacteria, with the highest abundance in type 2 diabetics, followed by obese and lean participants. In comparison, F. prausnitzii was least abundant in type 2 diabetics, and most abundant in lean controls. Methylation analysis of four CpGs in the first exon of TLR4 showed significantly lower methylation in obese individuals, but no significant difference between type 2 diabetics and lean controls. Methylation of seven CpGs in the promoter region of TLR2 was significantly lower in type 2 diabetics compared to obese subjects and lean controls. The methylation levels of both TLRs were significantly correlated with body mass index. Our data suggest that changes in gut microbiota and thus cell wall components are involved in the epigenetic regulation of inflammatory reactions. An improved diet targeted to induce gut microbial balance and in the following even epigenetic changes of pro-inflammatory genes may be effective in the prevention of metabolic syndrome.

Regulation of TLR4, p38 MAPkinase, IκB and miRNAs by inactivated strains of lactobacilli in human dendritic cells.

Strain specific properties of probiotics in providing supportive health effects in the immune system and the gastrointestinal tract have been widely investigated in vivo and in vitro. However, the underlying responsible mechanism is poorly described. By unravelling the probiotic-induced responses in a complex network of interacting signalling pathways, we investigated the effect of heat-inactivated Lactobacillus rhamnosus GG (LGG) and Lactobacillus delbrueckii subsp. bulgaricus (L.del) on the expression of TLR4 and signalling factors such as p38 MAPK and I?B at transcription level in human monocyte-derived dendritic cells (DCs). Our findings demonstrated that even inactivated probiotic strains can affect TLR4 expression in a down-regulatory direction as with lipopolysaccharides after 12 hours. LGG significantly down-regulated expression of p38 while I?B expression was significantly reduced in L.del-treated DCs. Moreover, we found these Lactobacillus strains could even modify the immune response at post-transcriptional level by modifying miRNAs expression. Based on our results LGG induced a significant down-regulatory effect on miR-146a expression which is known as a novel fine negative regulator of immune response targeting NFκB. On the other hand, miR-155 was up-regulated by LGG which is consistent with down-regulation of p38 and in LGG-treated DCs. These findings provide genetic and epigenetic explanations for the responsible underlying mechanisms by which probiotics influence immune response by targeting DCs.

Culture free DGGE and cloning based monitoring of changes in bacterial communities of salad due to processing.

To assess the possibilities of a culture-independent monitoring of bacterial communities in the food chain, samples of salad from farming sites as well as corresponding, processed products in stores were analysed. The bacterial DNA was extracted using a modified soil extraction protocol. Amplification of 16S rDNA was carried out using primers specific for eubacteria and enterobacteriaceae. Fingerprints of 200/370 bp respectively were obtained by denaturing gradient gel electrophoresis (DGGE) analysis following PCR and nested PCR amplification. In parallel to DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. DGGE analysis indicated a high diversity of bacterial communities in salad samples. Fingerprints indicated clearly reduced diversity of bacterial communities in processed samples from markets compared to field-grown salads. Surprisingly, primers pointed out in literature as specific for enterobacteriaceae did amplify pseudomonadeceae as well. Therefore, the more specific primers fD2 and rP1 were used subsequently in this study to amplify specific members of the family enterobacteriaceae. A total of 11 different 16S rDNA sequences were obtained and subjected to sequencing and phylogenetic affiliation. Sequences derived from the eubacterial clone library from organically farmed salad were affiliated to the family microbacteriaceae and pseudomonadaceae. In addition, a potential new genus within the family of enterobacteriaceae was detected. Furthermore, a sequence showing 98.9% similarity to Pseudomonas libaniensis (fluorescence subgroup) was found in a processed salad sample but not in the corresponding field samples. This species is generally known as an opportunistic pathogen. Whereas molecular based monitoring of bacterial communities in food still may need more experience and standardisation to detect specific bacteria present, the monitoring strategy presented in this paper, combining DGGE analysis with the construction of clone libraries, is an attractive method for culture-independent monitoring of changes of bacterial communities in the food chain.

Activation, stimulation and uptake of bacterial ghosts in antigen presenting cells.

Bacterial ghosts have been shown to be an innovative system to prepare vaccines of various bacteria with all features of the intact bacterial cell envelopes, especially all antigenic epitopes, but also to target recombinant proteins inserted in the cell envelopes of the ghost preparations to specific antigen presenting cells. To investigate the activation of the antigen presenting cell by bacterial ghosts in more detail we studied the uptake of bacterial ghosts in dendritic porcine cells and RAW macrophages and the induction of inflammatory mediators or mediators directing the immune response in THP-1 human macrophage cell line. The synthesis of inflammatory macrophage mediators such as TNFalpha in the THP1 cell line was stimulated by a hundred-fold higher dose of ghosts from Vibrio cholerae than the corresponding LPS using ELISA-analysis. These results confirm in vivo experiments indicating no toxic effects of ghosts in rabbits even after intravenous administration in doses stimulating significant humoral responses. We were also able to see a significant activation of IL-12 indicated by the analysis of IL-12(p70) synthesis and IL-12(p40) mRNA accumulation. This interleukine is of special importance in the activation of cellular TH1 immune responses. A rapid uptake of bacterial ghosts in macrophages within 10-30 min could be confirmed by electron microscopy. As antigen presentation is especially effective in porcine dendritic cells (DC) and even a low capacity of antigen uptake is sufficient for an induction of immune responses we investigated uptake and activation of bacterial ghosts by DC. DC are known to be phagocytic in specific immature stages. We found a significant uptake of bacterial ghosts from Actinobacillus pleuropneumoniae (App) and V. cholerae conjugated with FITC (fluorescinisothiocyanate) within 2 h. These data suggest that bacterial ghosts effectively stimulate monocytes and macrophages for the induction of TH1 directed immune responses and dendritic cells treated with bacterial ghosts may serve as a promising vehicle for active immunization and immunotherapy in situ.

Substantial equivalence of antinutrients and inherent plant toxins in genetically modified novel foods.

For a safety evaluation of foodstuff derived from genetically modified crops, the concept of the substantial equivalence of modified organisms with their parental lines is used following an environmental safety evaluation. To assess the potential pleiotropic effect of genetic modifications on constituents of modified crops data from US and EC documents were investigated with regard to inherent plant toxins and antinutrients. Analysed were documents of rape (glucosinolates, phytate), maize (phytate), tomato (tomatine, solanine, chaconine, lectins, oxalate), potato (solanine, chaconine, protease-inhibitors, phenols) and soybean (protease-inhibitors, lectins, isoflavones, phytate). In several documents used for notifications no declarations even on essential inherent plant toxins and antinutrients could be found, for instance data on phytate in modified maize were provided only in one of four documents. Significant variations in the contents of these compounds in parental and modified plants especially due to environmental influences were observed: drought stress, for example, was made responsible for significantly increased glucosinolate levels of up to 72.6micromol/g meal in modified and parental rape plants in field trials compared to recommended standard concentrations of less than 30micromol/g. Taking into account these wide natural variations generally the concentrations of inherent plant toxins and antinutrients in modified products were in the range of the concentrations in parental organisms. The results presented indicate that the concept of the substantial equivalence is useful for the risk assessment of genetically modified organisms (GMOs) used for novel foods but possible environmental influences on constituents of modified crops need more attention. Consistent guidelines, specifying data of relevant compounds which have to be provided for notification documents of specific organisms have to be established. Because of the importance of inherent plant toxins and antinutrients on nutritional safety, also coherent databases of standard parental lines and clear criteria for mandatory declarations are necessary.

Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines.

Gram-negative bacterial ghosts produced by controlled expression of the plasmid-encoded lysis gene E offers a promising approach in non-living vaccine technology. Bacterial cell wall complex and hence the antigenic determinants of the living cells are not affected by denaturation due to cell killing. However, the endotoxin content of the Gram-negative cell wall has been discussed as a potential problem for this kind of whole cell or envelope vaccines. Here we show that bacterial ghosts prepared from Escherichia coli O26:B6 and Salmonella typhimurium C5 induce dose-dependent antibody responses against bacterial cells or their corresponding lipopolysaccharides (LPS) in doses 25 ng kg-1 when administered intravenously to rabbits in a standard immunization protocol. No differences between the immune responses of the rabbits were observed when comparing equivalent doses of bacterial ghosts and antibiotic-treated whole cells. The results indicate that the bacterial ghosts exhibit all the antigenic properties of the living cells. No significant fever responses in rabbits have been recorded in doses of < 250 ng kg-1 E. coli O26:B6 ghosts and up to doses of 250 ng kg-1 S. typhimurium C5 ghosts when applying test methods recommended by the US pharmacopoeia. These findings correlate with cell culture experiments where doses 100 ng ml-1 of bacterial ghosts were needed for the release of tumour necrosis factor alpha (TNF alpha) and prostaglandin E2 (PGE2) from RAW mouse macrophage cultures. Free LPS of Salmonella abortus equi commonly used as a LPS-standard, however, stimulated TNF alpha and PGE2 synthesis of RAW cells in doses of 1 ng ml-1. The endotoxic activity of our bacterial preparations analysed by a standard limulus amoebocyte lysate and 2-keto-3-deoxyoctonate assay correlated with the capacity to stimulate the release of PGE2 and TNF alpha in RAW mouse macrophage cultures and the endotoxic responses in rabbits. It can be concluded that these in vitro systems can be used as easy predictive test systems for preparations of bacterial vaccines, particularly for bacterial ghosts.

Alterations of pharmacokinetic properties of gentamicin, penicillin G and two cephalosporins in septicaemic rabbits.

The pharmacokinetics of gentamicin, penicillin G, latamoxef and CPW 86-363, a novel third generation cephalosporin, were studied in healthy and septicaemic rabbits. Elevation of body temperature in infected animals was paralleled by statistically significant decreases in serum drug levels during the early stages of the distribution phase for penicillin G, latamoxef and CPW 86-363 whereas gentamicin showed increased serum drug levels during the early period. No significant differences were seen in tissue fluid levels (STIF) or normal and septicaemic rabbits for the four antibiotics used. Haemodynamic alterations and an increased permeability of blood vessel walls are presumed to contribute to changes in distribution properties of various drugs during experimental septicaemia. The qualitative differences among the antibiotics tested seem to be related to their physico-chemical characteristics.

A technique for repeated sampling of pure cerebrospinal fluid from the conscious rabbit.

For the study of drug pharmacokinetics' a reliable method for repeated sampling of uncontaminated cerebrospinal fluid (CSF) from a conscious rabbit was established. The subdural space was punctured between a spinous processes of the lumbar vertebrae. A catheter was inserted through a cannula and pushed up to the region of the cisterna magna. After removing the cannula, the distal end of the catheter was sealed and placed subcutaneously until the onset of the experiments. Ten days after the insertion, samples of clear and colorless CSF up to 1 ml could be withdrawn.