Development and validation of a ready to use cryo-EROD assay for the standardized screening of dioxins and dioxin-like compounds in foodstuffs.

Recent European regulations have indicated the need for new bioanalytical screening methods capable of monitoring dioxin and dioxin-like compounds in foodstuffs and environmental samples, cost-effectively and with a quicker turnaround. Cryo-cells of the hepatic H4IIE line preserved in 96-well plates were exposed to sample extracts prepared from various foodstuffs and analysed for their content of dioxins and dioxin-like compounds by means of the 7-Ethoxyresorufin-O-Deethylase (EROD)-assay in two laboratories. Assay data were compared between both laboratories and results from instrumental analysis used as a confirmatory method. Additionally, cut-off values for the different studied matrices were derived. The current European regulation regarding methods of analysis for the control of foodstuffs was applied with the aim of determining the feasibility of the cryo-methodology. Results obtained in both laboratories were in congruence with the required validation parameters of the Commission Regulation (EU) No 2017/644. Cut-off values should be established matrix-dependent to reduce the rate of false compliant results and to keep the rate of false non-compliant results under control. In summary, the ready-to-use cryo-assay method for the bioanalytical screening of foodstuffs in control laboratories without cell-culture facilities has successfully proven to be accurate, far quicker and more cost effective than current methods.

Impact of perfluorooctanesulfonate and perfluorooctanoic acid on human peripheral leukocytes.

Perfluorinated compounds (PFCs), such as perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA), are xenobiotics that can be detected worldwide in the environment, wildlife, and humans. So far, the immunotoxicity of PFCs has only been investigated in rodents, but not in humans. In this study, we explore the impact of PFOS and PFOA on selected functions of human leukocytes in vitro. PFOS induced a significant decrease of natural killer-cell activity and reduced the release of the pro-inflammatory cytokine TNF-α following lipopolysaccharide (LPS)-stimulation. Furthermore, the plasma PFOS concentrations (2.09-8.98 ng/ml) found in our study subjects correlated positively with the LPS-stimulated IL-6 release. PFOA augmented significantly calcitriol-induced monocytic differentiation of the HL-60 cell line. Additionally, there was a significant linear relationship between LPS-stimulated TNF-α and IL-6 release, and the plasma PFOA (1.20-6.92ng/ml) concentrations of the study subjects. In conclusion, the investigated PFCs affect human immune cells mainly with regard to natural killer-cell cytotoxicity and the pro-inflammatory cytokine release by stimulated macrophages.

Bolus ingestion but not regular consumption of native or dealcoholized red wine modulates selected immunological functions of leukocytes in healthy volunteers.

BACKGROUND/AIMS: Red wine (RW) consumption reduces the risk for coronary heart disease (CHD). Since immunological mechanisms involved in CHD were modulated by RW polyphenols in vitro, we investigated whether consumption of native or dealcoholized RW (DRW) affects selected immunological parameters in healthy adults ex vivo. METHODS: Twenty-seven nonsmokers were randomized to consume a single dose of 200 ml RW, 175 ml DRW or 200 ml water (controls). The same doses of RW (n = 24) and DRW (n = 25) were ingested daily for 6 weeks in addition to the subjects' usual diet. Controls (n = 25) did not receive any study drink. Blood was drawn before/90/360 min after supplementation or before and after 6-week intervention, respectively, to determine T cell apoptosis, phagocytosis and burst of neutrophils and monocytes. RESULTS: T cell apoptosis decreased after 360 min in group DRW [median (interquartile range); t(0): 71 (68; 75)% vs. t(360): 65 (64; 67)%; p = 0.008], but also in controls [t(0): 71 (65; 76)% vs. t(360): 64 (60; 65)%; p = 0.038] and both changes were different from group RW (DRW, p = 0.006; control, p = 0.024). Burst-positive monocytes increased after bolus ingestion of DRW [t(0): 27 (14; 69)% vs. t(360): 47 (29; 74)%; p = 0.012], and this change was different from controls (p = 0.008). Immunological changes related to daily consumption of RW or DRW did not occur. CONCLUSIONS: Other mechanisms than a modulation of phagocytosis, burst or T cell apoptosis by RW polyphenols, ingested either with or without alcohol, might explain the protective effects of RW against CHD observed in epidemiological studies.

Skeletal deformations in medaka (Oryzias latipes) visualized by synchrotron radiation micro-computer tomography (SRmicroCT).

Synchrotron radiation micro-computer tomography (SRmicroCT) offers the possibility to investigate biomineralized structures in high detail. Two animals of adult medaka fish (Oryzias latipes) were analyzed by SRmicroCT with a resolution of 6.55 microm: the wild-type animal was normally developed whereas the second animal showed an idiopathic deformation of the cranial and axial skeleton. These deformations could be followed on the macro- and on the microscale (i.e., on the level of the individual ribs and fin bones). Our study clearly demonstrates that SRmicroCT is an excellent technique to study alterations in the skeletal structure of fish in detail.

Inflammatory cytokines induce protein tyrosine nitration in rat astrocytes.

Protein tyrosine nitration may be relevant for the pathogenesis of hepatic encephalopathy (HE). Infections, sepsis, and trauma precipitate HE episodes. Recently, serum levels of tumor necrosis factor (TNF)-alpha were shown to correlate with severity of HE in chronic liver failure. Here the effects of inflammatory cytokines on protein tyrosine nitration in cultured rat astrocytes and rat brain in vivo were studied. In cultured rat astrocytes TNF-alpha (50 pg/ml-10 ng/ml) within 6h increased protein tyrosine nitration. TNF-alpha-induced tyrosine nitration was related to an increased formation of reactive oxygen and nitrogen intermediates, which was downstream from a NMDA-receptor-dependent increase of intracellular [Ca(2+)](i) and nNOS-catalyzed NO production. Astroglial tyrosine nitration was also elevated in brains of rats receiving a non-lethal injection of lipopolysaccharide, as indicated by colocalization of nitrotyrosine immunoreactivity with glial fibrillary acidic protein and glutamine synthetase, and by identification of the glutamine synthetase among the tyrosine-nitrated proteins. It is concluded that reactive oxygen and nitrogen intermediates as well as protein tyrosine nitration by inflammatory cytokines may alter astrocyte function in an NMDA-receptor-, Ca(2+)-, and NOS-dependent fashion. This may be relevant for the pathogenesis of HE and other conditions involving cytokine exposure the brain.

Dynamic expression of sparc precedes formation of skeletal elements in the Medaka (Oryzias latipes).

Sparc is a secreted calcium-binding glycoprotein that regulates mineralization of bone tissues in mammals. In other vertebrates, its function remains largely unclear. Here, we describe the isolation, genomic organization and expression of the sparc gene in the teleost Medaka (Oryzias latipes), an established vertebrate model for developmental studies. During earliest stages of Medaka embryogenesis, sparc is expressed in the sclerotome compartment of the somites that gives rise to precursor cells of the axial skeleton. Importantly, in this area its expression precedes that of twist-1, which is a crucial regulator of osteoblast formation. Dynamic expression is also found in the floor plate of the neural tube and the notochord. Both structures are passed by migrating skeletal precursors shortly before they differentiate and form the vertebrae. In general, sparc is expressed before the formation and mineralization of bone elements and expression of bone markers like collagen type 1a in the fins and axial skeleton of Medaka embryos. It is also expressed in several non-skeletal tissues of embryos and adult fish, suggesting possible other functions not related to bone mineralization. Taken together, the Medaka sparc gene represents an excellent marker for early sclerotome development. Its restricted and highly dynamic expression suggests a novel function during migration of sclerotome cells and their differentiation into early vertebrae. This marker thus allows the analysis of early skeletal development and formation of extracellular bone matrix in this vertebrate model.

Single and repeated moderate consumption of native or dealcoholized red wine show different effects on antioxidant parameters in blood and DNA strand breaks in peripheral leukocytes in healthy volunteers: a randomized controlled trial (ISRCTN68505294).

BACKGROUND: Red wine (RW) is rich in antioxidant polyphenols that might protect from oxidative stress related diseases, such as cardiovascular disease and cancer. Antioxidant effects after single ingestion of RW or dealcoholized RW (DRW) have been observed in several studies, but results after regular consumption are contradictory. Thus, we examined if single or repeated consumption of moderate amounts of RW or DRW exert antioxidant activity in vivo. METHODS: Total phenolic content and concentration of other antioxidants in plasma/serum, total antioxidant capacity (TEAC) in plasma as well as DNA strand breaks in peripheral leukocytes were measured in healthy non-smokers A) before, 90 and 360 min after ingestion of one glass of RW, DRW or water; B) before and after consumption of one glass of RW or DRW daily for 6 weeks. DNA strand breaks (SB) were determined by single cell gel electrophoresis (Comet Assay) in untreated cells and after induction of oxidative stress ex vivo with H2O2 (300 microM, 20 min). RESULTS: Both RW and DRW transiently increased total phenolic content in plasma after single consumption, but only RW lead to a sustained increase if consumed regularly. Plasma antioxidant capacity was not affected by single or regular consumption of RW or DRW. Effects of RW and DRW on DNA SB were conflicting. DNA strand breaks in untreated cells increased after a single dose of RW and DRW, whereas H2O2 induced SB were reduced after DRW. In contrast, regular RW consumption reduced SB in untreated cells but did not affect H2O2 induced SB. CONCLUSION: The results suggest that consumption of both RW and DRW leads to an accumulation of phenolic compounds in plasma without increasing plasma antioxidant capacity. Red wine and DRW seem to affect the occurrence of DNA strand breaks, but this cannot be referred to antioxidant effects.

Antigen-specific targeting and elimination of EBV-transformed B cells by allergen toxins.

BACKGROUND: With the exception of antigen-specific immunotherapy, current treatments for atopic diseases provide only symptomatic relief. Because of the increasing incidence of such diseases, the development of novel strategies and concepts for the treatment of allergies is urgently needed. OBJECTIVE: Here we present a new approach for the treatment of atopic diseases. The strategy is comparable to the application of immunotoxins in cancer therapy, in which a cytotoxic peptide is coupled to a cancer cell-specific antibody fragment or ligand. In the case of so-called allergen toxins (ATs), the target cell-specific moiety is an allergen or allergen-derived fragment, which should be bound only by allergen-reactive cells. After receptor-mediated internalization, allergen-specific cells are killed, and the allergic pathogenesis is interrupted. METHODS: Proof of the AT principle was shown by using a human ex vivo system in which EBV was used to transform human B cells specific for the timothy grass pollen allergen Phl p 5b. The AT is composed of the major B-cell and T-cell epitopes of the Phl p 5b (P5) allergen fused to a truncated form of the highly toxic Pseudomonas aeruginosa exotoxin A (ETA'). RESULTS: Allergen-specific and nonspecific B cells were challenged with P5-ETA', but only the Phl p 5b-reactive B cells showed selective binding and cytotoxicity. CONCLUSION: This approach represents an initial step toward a novel therapeutic strategy in the treatment of atopic diseases.

Modulation of endothelial and smooth muscle function by bed rest and hypoenergetic, low-fat nutrition.

Prolonged microgravity alters the regulation of the peripheral vasculature. The influence of reduced food intake, as often observed in astronauts, on vascular function is unclear. In a randomized, four-phase, crossover study, the effect of simulated microgravity (13 days of bed rest), energetic restriction (-25%, fat reduced), and their combination on endothelium-dependent and -independent vasodilation was compared with ambulatory control conditions. Using venous occlusion plethysmography, cumulative intra-arterial dose-response curves to endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasodilators were constructed in 10 healthy male volunteers before and on day 13 of each of the four intervention periods. Bed rest combined with normoenergetic nutrition impaired the dose-response to acetylcholine (ANOVA, P = 0.004) but not to sodium nitroprusside, whereas hypoenergetic diet under ambulatory conditions improved responses to acetylcholine (P = 0.044) and sodium nitroprusside (P < 0.001). When bed rest was combined with hypoenergetic diet, acetylcholine responses did not change. Similarly, under control conditions, no change was observed. Individual changes in the total cholesterol-to-HDL ratio were correlated with changes in endothelial and vascular smooth muscle relaxation. In conclusion, short-term bed rest impairs endothelium-dependent arterial relaxation in humans. A hypoenergetic, low-fat diet modulates serum lipids, improves endothelium-dependent and -independent relaxation, and may antagonize the unfavorable effects of simulated microgravity on endothelial function.

A proinflammatory role for Fas in joints of mice with collagen-induced arthritis.

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death - Fas (CD95)/FasL - in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor alpha (TNF-alpha) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-alpha and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.