International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Dietary factors and low-grade inflammation in relation to overweight and obesity.

Low-grade inflammation is a characteristic of the obese state, and adipose tissue releases many inflammatory mediators. The source of these mediators within adipose tissue is not clear, but infiltrating macrophages seem to be especially important, although adipocytes themselves play a role. Obese people have higher circulating concentrations of many inflammatory markers than lean people do, and these are believed to play a role in causing insulin resistance and other metabolic disturbances. Blood concentrations of inflammatory markers are lowered following weight loss. In the hours following the consumption of a meal, there is an elevation in the concentrations of inflammatory mediators in the bloodstream, which is exaggerated in obese subjects and in type 2 diabetics. Both high-glucose and high-fat meals may induce postprandial inflammation, and this is exaggerated by a high meal content of advanced glycation end products (AGE) and partly ablated by inclusion of certain antioxidants or antioxidant-containing foods within the meal. Healthy eating patterns are associated with lower circulating concentrations of inflammatory markers. Among the components of a healthy diet, whole grains, vegetables and fruits, and fish are all associated with lower inflammation. AGE are associated with enhanced oxidative stress and inflammation. SFA and trans-MUFA are pro-inflammatory, while PUFA, especially long-chain n-3 PUFA, are anti-inflammatory. Hyperglycaemia induces both postprandial and chronic low-grade inflammation. Vitamin C, vitamin E and carotenoids decrease the circulating concentrations of inflammatory markers. Potential mechanisms are described and research gaps, which limit our understanding of the interaction between diet and postprandial and chronic low-grade inflammation, are identified.

Glycolaldehyde-modified ß-lactoglobulin AGEs are unable to stimulate inflammatory signaling pathways in RAGE-expressing human cell lines.

SCOPE: Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. METHODS AND RESULTS: In our study, we used glycolaldehyde-modified ß-lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X-114 (TX-114). Affinity column-purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde-modified AGEs purified by extraction with TX-114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin-like activity was demonstrated in the detergent phase after extraction with TX-114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. CONCLUSION: Our results demonstrate that glycolaldehyde-modified AGEs are unable to induce inflammatory signaling in receptor for AGE-expressing cells. The observed cell-activating activity can be ascribed to an endotoxin-like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.

Protection of dystrophic muscle cells with polyphenols from green tea correlates with improved glutathione balance and increased expression of 67LR, a receptor for (-)-epigallocatechin gallate.

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by the absence of the protein dystrophin. Because oxidative stress contributes to the pathogenesis of DMD, we investigated if a green tea polyphenol blend (GTP) and its major polyphenol (-)-epigallocatechin gallate (EGCg), could protect muscle cell primary cultures from oxidative damage induced by hydrogen peroxide (H(2)O(2)) in the widely used mdx mouse model. On-line fluorimetric measurements using an H(2)O(2)-sensitive probe indicated that GTP and EGCg scavenged peroxide in a concentration-dependent manner. A 48 h exposure to EGCg increased glutathione content but did not alter the expression of proteins involved in membrane stabilization and repair. Pretreatment of dystrophic cultures with GTP or EGCg 48 h before exposure to H(2)O(2) improved cell survival. Normal cultures were protected by GTP but not by EGCg. 67LR, a receptor for EGCg, was seven times more abundant in dystrophic compared with normal cultures. Altogether our results demonstrate that GTP and EGCg protect muscle cells by scavenging H(2)O(2) and by improving the glutathione balance. In addition, the higher levels of 67LR in dystrophic muscle cells compared with normal ones likely contribute to EGCg-mediated survival.

Analysis of advanced glycation endproducts in dairy products by isotope dilution liquid chromatography-electrospray tandem mass spectrometry. The particular case of carboxymethyllysine.

A fully validated multiple-transition recording isotope dilution liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of N(epsilon)-carboxymethyllysine (CML) and lysine in dairy products is described. Internal standards were [N-1',2'-(13)C(2)]CML and [1,2,3,4,5,6-(13)C(6)-2,6-(15)N(2)]lysine, and the method was validated by evaluating the selectivity, linearity, precision (repeatability and reproducibility) and trueness, using both powder and liquid products. For liquid dairy products, the repeatability and reproducibility was 2.79% and 11.0%, while 4.85% and 4.92% were determined for powder dairy products, respectively. The trueness of the method ranged from -9.6% to -3.6% for powder and from -0.99% to 6.8% for liquid dairy products. The limit of detection for CML was estimated to be 8 ng CML per mg protein while the limit of quantification was 27 ng CML per mg protein. The method encompasses a proteolytic cleavage mediated by enzymatic digestion to reach a complete release of the amino acids prior to a sample cleanup based on solid phase extraction, and followed by LC-MS/MS analysis of CML and lysine residues. To ensure a suitable performance of the enzymatic digestion, CML measurements were compared to values obtained with an acid hydrolysis-mediated proteolysis. Finally, the method was employed for the analysis of CML in various dairy products. The values compare well to the data available in the literature when similar methods were used, even if some discrepancies were observed upon comparison with the results obtained by other techniques such as enzyme-linked immunosorbent assay and GC-MS.

Supplementation with oral probiotic bacteria protects human cutaneous immune homeostasis after UV exposure-double blind, randomized, placebo controlled clinical trial.

There is now strong evidence that probiotic bacteria can regulate inflammatory immune responses. Here, we analyzed whether oral supplementation with the probiotic bacterial strain Lactobacillus johnsonii (La1) could interfere with skin immune status following UV exposure. A randomized, double-blind, placebo controlled clinical trial was conducted with 54 healthy volunteers receiving either La1 or placebo, during six weeks prior to solar-simulated UV irradiation. Blister roofs and skin biopsies were recovered 1, 4 and 10 days after UV exposure from un-irradiated and irradiated skin and used for immunohistochemical analysis and mixed epidermal cell lymphocyte reaction (MECLR), respectively. La1 supplementation did not prevent the UV-induced phenotypic maturation of Langerhans cells (LCs) or the decrease in MECLR in irradiated skin samples, one day post-irradiation. On day 4, MECLR was still decreased in the placebo group, with a parallel reduction in the CD1a LC marker in irradiated epidermis. In contrast, the allostimulatory capacity of epidermal cells was totally recovered in the La1 group correlating with the normalization of CD1a expression within the epidermis. For the first time, the results provide evidence that ingested probiotic bacteria accelerate the recovery of skin immune homeostasis after UV-induced immunosuppression.

Dicarbonyls stimulate cellular protection systems in primary rat hepatocytes and show anti-inflammatory properties.

Advanced glycation endproducts (AGEs) and their precursor dicarbonyls are generally perceived as having adverse health effects. They are also considered to be initiators and promoters of disease and aging. However, proof for a causal relationship is lacking. On the other hand, it is known that AGEs and melanoidins possess beneficial properties, such as antioxidant and metal-chelating activities. Furthermore, some AGEs may stimulate the cellular detoxification system, generally known as the phase II drug metabolizing system. We show here that several reactive dicarbonyl intermediates have the capability to stimulate the cellular phase II detoxification systems in both a reporter cell line and primary rat hepatocytes. In addition, we demonstrate that dicarbonyls can attenuate the inflammatory signaling induced by tumor necrosis factor-alpha in a reporter cell system.

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products.

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

N(epsilon)-carboxymethyllysine-modified proteins are unable to bind to RAGE and activate an inflammatory response.

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.

A comparative study of proteolysis methods for the measurement of 3-nitrotyrosine residues: enzymatic digestion versus hydrochloric acid-mediated hydrolysis.

A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC-MS(/MS) or LC-MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.

Supplementation with oral probiotic bacteria maintains cutaneous immune homeostasis after UV exposure.

Probiotic bacteria have been shown to modulate the immune system of the gut and protect against infectious and inflammatory diseases of the gastro-intestinal tract. Ultraviolet radiation (UVR) is known to alter the cutaneous and systemic immune systems implicated in the development of skin tumors. In this study we investigated whether oral probiotics are able to modulate the immune system of the skin using hairless Skh:hr1 mice exposed to an acute dose of UVR. We show that nutritional supplementation with Lactobacillus johnsonii (La1) at 10(8) cfu/day for 10 days was able to protect against the UVR-induced suppression of contact hypersensitivity, the decreased epidermal Langerhans cell density and the increased IL-10 serum levels. In the absence of UV exposure, probiotic bacteria had no detectable effect on the immune system of the skin, thus acting only to re-establish skin homeostasis. In conclusion, our data demonstrate that ingested probiotic bacteria can maintain cutaneous immune capacity after UV exposure.

Green tea extract and its major polyphenol (-)-epigallocatechin gallate improve muscle function in a mouse model for Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a frequent muscular disorder caused by mutations in the gene encoding dystrophin, a cytoskeletal protein that contributes to the stabilization of muscle fiber membrane during muscle activity. Affected individuals show progressive muscle wasting that generally causes death by age 30. In this study, the dystrophic mdx(5Cv) mouse model was used to investigate the effects of green tea extract, its major component (-)-epigallocatechin gallate, and pentoxifylline on dystrophic muscle quality and function. Three-week-old mdx(5Cv) mice were fed for either 1 or 5 wk a control chow or a chow containing the test substances. Histological examination showed a delay in necrosis of the extensor digitorum longus muscle in treated mice. Mechanical properties of triceps surae muscles were recorded while the mice were under deep anesthesia. Phasic and tetanic tensions of treated mice were increased, reaching values close to those of normal mice. The phasic-to-tetanic tension ratio was corrected. Finally, muscles from treated mice exhibited 30-50% more residual force in a fatigue assay. These results demonstrate that diet supplementation of dystrophic mdx(5Cv) mice with green tea extract or (-)-epigallocatechin gallate protected muscle against the first massive wave of necrosis and stimulated muscle adaptation toward a stronger and more resistant phenotype.

Cyclosporin A generates superoxide in smooth muscle cells.

Cyclosporin A (CsA) generates superoxide in smooth muscle cells. Our earlier studies have demonstrated that the increase in the vasopressin type 1 receptor induced in vascular smooth muscle cells in the presence of CsA is probably due to superoxide (Krauskopf et al., J Biol Chem 278, 41685-41690, 2003). This increase in vasopressin receptor is likely at the base of increased vascular responsiveness to vasoconstrictor hormones and hypertension induced by CsA. Here, we demonstrate that CsA produces superoxide. In addition, our data show that superoxide generation does not originate from the major cellular superoxide generating systems NAD(P)H oxidase or xanthine oxidase. Our results suggest that the side effects of CsA could be diminished with the help of SOD mimetic drugs.

Role of superoxide as a signaling molecule.

Superoxide is known to affect vascular physiology in several ways and has also been recognized to contribute significantly to vascular physiopathology. Here we discuss the emerging role of superoxide as an essential signaling molecule in normal physiology.

Vasopressin type 1A receptor up-regulation by cyclosporin A in vascular smooth muscle cells is mediated by superoxide.

Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-L-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.

Pharmacological control of cellular calcium handling in dystrophic skeletal muscle.

Duchenne muscular dystrophy arises due to the lack of the cytoskeletal protein dystrophin. In Duchenne muscular dystrophy muscle, the lack of dystrophin is accompanied by alterations in the dystrophin-glycoprotein complex. We and others have found that the absence of dystrophin in cells of the Duchenne muscular dystrophy animal model, the mdx mouse, leads to elevated Ca(2+) influx and cytosolic Ca(2+) concentrations when exposed to stress. We have also shown that alpha-methylprednisolone, the only drug used successfully in the therapy of Duchenne muscular dystrophy, and creatine lowered cytosolic Ca(2+) levels in mdx myotubes. It is likely that chronic elevation of [Ca(2+)] in the cytosol in response to stress is an initiating event for apoptosis and/or necrosis in Duchenne muscular dystrophy or mdx muscle and that alterations in mitochondrial function and metabolism are involved. Other cellular signalling pathways (e.g. nitric oxide) might also be affected.