Dirk Haller

Critical review: vegetables and fruit in the prevention of chronic diseases

BackgroundVegetables and fruit provide a significant part of human nutrition, as they are important sources of nutrients, dietary fibre, and phytochemicals. However, it is uncertain whether the risk of certain chronic diseases can be reduced by increased consumption of vegetables or fruit by the general public, and what strength of evidence has to be allocated to such an association.MethodsTherefore, a comprehensive analysis of the studies available in the literature and the respective study results has been performed and evaluated regarding obesity, type 2 diabetes mellitus, hypertension, coronary heart disease (CHD), stroke, cancer, chronic inflammatory bowel disease (IBD), rheumatoid arthritis (RA), chronic obstructive pulmonary disease (COPD), asthma, osteoporosis, eye diseases, and dementia. For judgement, the strength of evidence for a risk association, the level of evidence, and the number of studies were considered, the quality of the studies and their estimated relevance based on study design and size.ResultsFor hypertension, CHD, and stroke, there is convincing evidence that increasing the consumption of vegetables and fruit reduces the risk of disease. There is probable evidence that the risk of cancer in general is inversely associated with the consumption of vegetables and fruit. In addition, there is possible evidence that an increased consumption of vegetables and fruit may prevent body weight gain. As overweight is the most important risk factor for type 2 diabetes mellitus, an increased consumption of vegetables and fruit therefore might indirectly reduces the incidence of type 2 diabetes mellitus. Independent of overweight, there is probable evidence that there is no influence of increased consumption on the risk of type 2 diabetes mellitus. There is possible evidence that increasing the consumption of vegetables and fruit lowers the risk of certain eye diseases, dementia and the risk of osteoporosis. Likewise, current data on asthma, COPD, and RA indicate that an increase in vegetable and fruit consumption may contribute to the prevention of these diseases. For IBD, glaucoma, and diabetic retinopathy, there was insufficient evidence regarding an association with the consumption of vegetables and fruit.ConclusionsThis critical review on the associations between the intake of vegetables and fruit and the risk of several chronic diseases shows that a high daily intake of these foods promotes health. Therefore, from a scientific point of view, national campaigns to increase vegetable and fruit consumption are justified. The promotion of vegetable and fruit consumption by nutrition and health policies is a preferable strategy to decrease the burden of several chronic diseases in Western societies.

Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures

BACKGROUND AND AIM Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis. Reactivity of IEC to bacterial signals may depend on interactions with immunocompetent cells. To address the question of whether non-pathogenic bacteria modify the immune response of the intestinal epithelium, we co-cultivated enterocyte-like CaCO-2 cells with human blood leucocytes in separate compartments of transwell cultures. METHODS CaCO-2/PBMC co-cultures were stimulated with non-pathogenic bacteria and enteropathogenic Escherichia coli. Expression of tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-8, monocyte chemoattracting protein 1 (MCP-1), and IL-10 was studied by enzyme linked immunosorbent assays (cytokine secretion) and by semiquantitative reverse transcription-polymerase chain reaction. RESULTS Challenge of CaCO-2 cells with non-pathogenic E coli andLactobacillus sakei induced expression of IL-8, MCP-1, IL-1β, and TNF-α mRNA in the presence of underlying leucocytes. Leucocyte sensitised CaCO-2 cells produced TNF-α and IL-1β whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells. CaCO-2 cells alone remained hyporesponsive to the bacterial challenge. Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mRΝA in leucocyte sensitised CaCO-2 cells. TNF-α was identified as one of the early mediators involved in cellular cross talk. In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenicE coli and non-pathogenic bacteria. CONCLUSION The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.

Safety assessment of probiotics for human use

The safety of probiotics is tied to their intended use, which includes consideration of potential vulnerability of the consumer or patient, dose and duration of consumption, and both the manner and frequency of administration. Unique to probiotics is that they are alive when administered, and unlike other food or drug ingredients, possess the potential for infectivity or in situ toxin production. Since numerous types of microbes are used as probiotics, safety is also intricately tied to the nature of the specific microbe being used. The presence of transferable antibiotic resistance genes, which comprises a theoretical risk of transfer to a less innocuous member of the gut microbial community, must also be considered. Genetic stability of the probiotic over time, deleterious metabolic activities, and the potential for pathogenicity or toxicogenicity must be assessed depending on the characteristics of the genus and species of the microbe being used. Immunological effects must be considered, especially in certain vulnerable populations, including infants with undeveloped immune function. A few reports about negative probiotic effects have surfaced, the significance of which would be better understood with more complete understanding of the mechanisms of probiotic interaction with the host and colonizing microbes. Use of readily available and low cost genomic sequencing technologies to assure the absence of genes of concern is advisable for candidate probiotic strains. The field of probiotic safety is characterized by the scarcity of studies specifically designed to assess safety on the one hand contrasted with the long history of safe use of many of these microbes in foods on the other hand.

High-fat diet alters gut microbiota physiology in mice

The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.

Depletion of luminal iron alters the gut microbiota and prevents Crohn's disease-like ileitis

Background Iron replacement therapy is a common treatment in patients with anaemia and Crohns disease, but oral iron supplements are less tolerated. The pathogenesis of Crohns disease is attributed to intestinal bacteria and environmental factors that trigger disease in a genetically predisposed host. The aim of this study was to characterise the interrelationship between luminal iron sulfate, systemic iron, the gut microbiota and the development of chronic ileitis in a murine model of Crohns disease. Methods Wild type (WT) and heterozygous TNFΔARE/WT mice were fed with an iron sulfate containing or iron sulfate free diet in combination with intraperitoneal control injections or iron injections for 11 weeks. Results TNFΔARE/WT mice develop severe inflammation of the distal ileum but remained completely healthy when transferred to an iron sulfate free diet, even if iron was systemically repleted. Absence of luminal iron sulfate reduced cellular markers of endoplasmic reticulum (ER) stress responses and pro-apoptotic mechanisms in the ileal epithelium. Phenotype or reactivity of major effector intraepithelial CD8αβ+ T cells were not altered in the absence of luminal iron. Interestingly, ER stress mechanisms sensitised the small intestinal epithelial cell (IEC) line Mode-K to cytotoxic function of effector T cells from TNF∆ARE/WT mice. Pyrosequencing of 16S rRNA tags of the caecal microbiota revealed that depletion of luminal iron sulfate induced significant compositional alterations, while total microbial diversity (Shannons diversity index) and number of total operational taxonomic units were not affected. Conclusion This study showed that an iron sulfate free diet in combination with systemic iron repletion prevents the development of chronic ileitis in a murine model of Crohns disease. Luminal iron may directly affect IEC function or generate a pathological milieu in the intestine that triggers epithelial cell stress-associated apoptosis through changes in microbial homeostasis. These results suggest that oral replacement therapy with iron sulfate may trigger inflammatory processes associated with progression of Crohns disease-like ileitis.

Gene-environment interactions in chronic inflammatory disease

Chronic inflammatory diseases represent a major challenge for both clinical research and patient care, and evidence indicates that these disorders develop as a result of complex gene-environment interactions. Better understanding of their cause-and-effect relationship is the basis for emerging proposals for therapy and prevention.

Enterococcus faecalis metalloprotease compromises epithelial barrier and contributes to intestinal inflammation.

BACKGROUND & AIMS Matrix metalloproteases (MMPs) mediate pathogenesis of chronic intestinal inflammation. We characterized the role of the gelatinase (GelE), a metalloprotease from Enterococcus faecalis, in the development of colitis in mice. METHODS Germ-free, interleukin-10-deficient (IL-10(-/-)) mice were monoassociated with the colitogenic E faecalis strain OG1RF and isogenic, GelE-mutant strains. Barrier function was determined by measuring E-cadherin expression, transepithelial electrical resistance (TER), and translocation of permeability markers in colonic epithelial cells and colon segments from IL-10(-/-) and TNF(ΔARE/Wt) mice. GelE specificity was shown with the MMP inhibitor marimastat. RESULTS Histologic analysis (score 0-4) of E faecalis monoassociated IL-10(-/-) mice revealed a significant reduction in colonic tissue inflammation in the absence of bacteria-derived GelE. We identified cleavage sites for GelE in the sequence of recombinant mouse E-cadherin, indicating that it might be degraded by GelE. Experiments with Ussing chambers and purified GelE revealed the loss of barrier function and extracellular E-cadherin in mice susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) before inflammation developed. Colonic epithelial cells had reduced TER and increased translocation of permeability markers after stimulation with GelE from OG1RF or strains of E faecalis isolated from patients with Crohns disease and ulcerative colitis. CONCLUSIONS The metalloprotease GelE, produced by commensal strains of E faecalis, contributes to development of chronic intestinal inflammation in mice that are susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) by impairing epithelial barrier integrity.

PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.

Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence

Objectives Dysbiosis of the intestinal microbiota is associated with Crohns disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNFdeltaARE mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement. Design Heterozygous TNFdeltaARE mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis. Results GF-TNFdeltaARE mice were free of inflammation in the gut and antibiotic treatment of CONV-TNFdeltaARE mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNFdeltaARE mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNFdeltaARE recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNFdeltaARE mice with the human CD-related Escherichia coli LF82 did not induce ileitis. Conclusions We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.

A Consideration of Biomarkers to be Used for Evaluation of Inflammation in Human Nutritional Studies

To monitor inflammation in a meaningful way, the markers used must be valid: they must reflect the inflammatory process under study and they must be predictive of future health status. In 2009, the Nutrition and Immunity Task Force of the International Life Sciences Institute, European Branch, organized an expert group to attempt to identify robust and predictive markers, or patterns or clusters of markers, which can be used to assess inflammation in human nutrition studies in the general population. Inflammation is a normal process and there are a number of cells and mediators involved. These markers are involved in, or are produced as a result of, the inflammatory process irrespective of its trigger and its location and are common to all inflammatory situations. Currently, there is no consensus as to which markers of inflammation best represent low-grade inflammation or differentiate between acute and chronic inflammation or between the various phases of inflammatory responses. There are a number of modifying factors that affect the concentration of an inflammatory marker at a given time, including age, diet and body fatness, among others. Measuring the concentration of inflammatory markers in the bloodstream under basal conditions is probably less informative compared with data related to the concentration change in response to a challenge. A number of inflammatory challenges have been described. However, many of these challenges are poorly standardised. Patterns and clusters may be important as robust biomarkers of inflammation. Therefore, it is likely that a combination of multiple inflammatory markers and integrated readouts based upon kinetic analysis following defined challenges will be the most informative biomarker of inflammation.