Association of a common TLR-6 polymorphism with coronary artery disease – implications for healthy ageing?

Background

The pro-inflammatory status of the elderly triggers most of the age-related diseases such as cancer and atherosclerosis. Atherosclerosis, the leading cause world wide of morbidity and death, is an inflammatory disease influenced by life-style and genetic host factors. Stimuli such as oxLDL or microbial ligands have been proposed to trigger inflammation leading to atherosclerosis. It has recently been shown that oxLDL activates immune cells via the Toll-like receptor (TLR) 4/6 complex. Several common single nucleotide polymorphisms (SNPs) of the TLR system have been associated with atherosclerosis. To investigate the role of TLR-6 we analyzed the association of the TLR-6 SNP Pro249Ser with atherogenesis.

Results

Genotyping of two independent groups with CAD, as well as of healthy controls revealed a significant association of the homozygous genotype with a reduced risk for atherosclerosis (odds ratio: 0.69, 95% CI 0.51-0.95, P?=?0.02). In addition, we found a trend towards an association with the risk of restenosis after transluminal coronary angioplasty (odds ratio: 0.53, 95% CI 0.24-1.16, P?=?0.12). In addition, first evidence is presented that the frequency of this protective genotype increases in a healthy population with age. Taken together, our results define a role for TLR-6 and its genetic variations in modulating the inflammatory response leading to atherosclerosis.

Conclusions

These results may lead to a better risk stratification, and potentially to an improved prophylactic treatment of high-risk populations. Furthermore, the protective effect of this polymorphism may lead to an increase of this genotype in the healthy elderly and may therefore be a novel genetic marker for the well-being during aging.

     

The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS,Cigarette Smoke or Allergen Challenge

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4^−/− and Mrp4^+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4^−/− and Mrp4^+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4^+/+ or Mrp4^−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

Neonatal environment exerts a sustained influence on the development of the intestinal microbiota and metabolic phenotype

The postnatal environment, including factors such as weaning and acquisition of the gut microbiota, has been causally linked to the development of later immunological diseases such as allergy and autoimmunity, and has also been associated with a predisposition to metabolic disorders. We show that the very early-life environment influences the development of both the gut microbiota and host metabolic phenotype in a porcine model of human infants. Farm piglets were nursed by their mothers for 1 day, before removal to highly controlled, individual isolators where they received formula milk until weaning at 21 days. The experiment was repeated, to create two batches, which differed only in minor environmental fluctuations during the first day. At day 1 after birth, metabolic profiling of serum by ¹H nuclear magnetic resonance spectroscopy demonstrated significant, systemic, inter-batch variation which persisted until weaning. However, the urinary metabolic profiles demonstrated that significant inter-batch effects on 3-hydroxyisovalerate, trimethylamine-N-oxide and mannitol persisted beyond weaning to at least 35 days. Batch effects were linked to significant differences in the composition of colonic microbiota at 35 days, determined by 16 S pyrosequencing. Different weaning diets modulated both the microbiota and metabolic phenotype independently of the persistent batch effects. We demonstrate that the environment during the first day of life influences development of the microbiota and metabolic phenotype and thus should be taken into account when interrogating experimental outcomes. In addition, we suggest that intervention at this early time could provide ‘metabolic rescue'' for at-risk infants who have undergone aberrant patterns of initial intestinal colonisation.     

On the Evolutionary History of Uleiella chilensis,a Smut Fungus Parasite of Araucaria araucana in South America: Uleiellales ord. nov. in Ustilaginomycetes

The evolutionary history, divergence times and phylogenetic relationships of Uleiella chilensis (Ustilaginomycotina, smut fungi) associated with Araucaria araucana were analysed. DNA sequences from multiple gene regions and morphology were analysed and compared to other members of the Basidiomycota to determine the phylogenetic placement of smut fungi on gymnosperms. Divergence time estimates indicate that the majority of smut fungal orders diversified during the Triassic–Jurassic period. However, the origin and relationships of several orders remain uncertain. The most recent common ancestor between Uleiella chilensis and Violaceomyces palustris has been dated to the Lower Cretaceous. Comparisons of divergence time estimates between smut fungi and host plants lead to the hypothesis that the early Ustilaginomycotina had a saprobic lifestyle. As there are only two extant species of Araucaria in South America, each hosting a unique Uleiella species, we suggest that either coevolution or a host shift followed by allopatric speciation are the most likely explanations for the current geographic restriction of Uleiella and its low diversity. Phylogenetic and age estimation analyses, ecology, the unusual life-cycle and the peculiar combination of septal and haustorial characteristics support Uleiella chilensis as a distinct lineage among the Ustilaginomycotina. Here, we describe a new ustilaginomycetous order, the Uleiellales to accommodate Uleiella. Within the Ustilaginomycetes, Uleiellales are sister taxon to the Violaceomycetales.     

Methicillin-Resistant Staphylococcus aureus in Saarland,Germany: The Long-Term Care Facility Study

BackgroundMultiresistant organisms pose a threat for patients and care recipients. Control interventions need to be tailored to region, the type of institution considered, and risk factors. The German state of Saarland is ideally suited to study colonisation epidemiology throughout its various health and care institutions. After conclusion of a large admission prevalence study in acute care hospitals, we now performed a methicillin-resistant Staphylococcus aureus (MRSA) point prevalence study in Saarland long term care facilities (LTCF), allowing for a direct comparison with respect of MRSA prevalence and associated risk factors between these two institutional types located within a confined region.ConclusionAs already known from previous studies, colonisation with MRSA is common in LTCF residents even in an area with relatively low MRSA prevalence. This found prevalence can now be related to the acute care admission prevalence (2.2%) as well as to the admission prevalence in acute care geriatric departments (7.6%). The common clonal attribution (spa type) of MRSA isolates prevalent in the LTCF population as well as in the acute care admission population points towards a close relationship between both types of institutions. However, the ostensible absence of risk factors such as “previous hospitalisation” in conjunction with newly identified factors such as “multiple decolonisation cycles” refers to MRSA colonisation risks independent of contact with acute care facilities. Overall, this large LTCF point prevalence study allows data-based, region-tailored decisions on MRSA screening policies and provides a basis for additional preventative measures.     

A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application

Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl₂, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.     

Eco‐evolutionary dynamics in a coevolving host–virus system

Eco‐evolutionary dynamics have been shown to be important for understanding population and community stability and their adaptive potential. However, coevolution in the framework of eco‐evolutionary theory has not been addressed directly. Combining experiments with an algal host and its viral parasite, and mathematical model analyses we show eco‐evolutionary dynamics in antagonistic coevolving populations. The interaction between antagonists initially resulted in arms race dynamics (ARD) with selective sweeps, causing oscillating host–virus population dynamics. However, ARD ended and populations stabilised after the evolution of a general resistant host, whereas a trade‐off between host resistance and growth then maintained host diversity over time (trade‐off driven dynamics). Most importantly, our study shows that the interaction between ecology and evolution had important consequences for the predictability of the mode and tempo of adaptive change and for the stability and adaptive potential of populations.     

Photocatalytic antibacterial effects on TiO2-anatase upon UV-A and UV-A/VIS threshold irradiation

Photocatalysis mediated by the anatase modification of titanium dioxide (TiO₂) has shown antibacterial effects in medical applications. The aim of this study was to investigate the possibility of expanding the excitation wavelengths for photocatalytic antibacterial effects from ultraviolet (UV) into the visible light range. After deposition of salivary pellicle and adhesion of Streptococcus gordonii on anatase, different irradiation protocols were applied to induce photocatalysis: ultraviolet A (UV-A) > 320 nm; ultraviolet/visible (UV-A/VIS) light > 380 nm and > 390 nm; and VIS light 400–410 nm. A quartz crystal microbalance with dissipation (QCM-D) tests and microscopic examination were used to observe the photoinduced antibacterial effects. Salivary pellicle could be photocatalytically decomposed under all irradiation protocols. In contrast, effective photocatalytic attack of bacteria could be observed by UV-A as well as by UV-A/VIS at 380 nm < λ < 390 nm only. Wavelengths above 380 nm show promise for in situ therapeutic antifouling applications.