CSF1R-mediated myeloid cell depletion shifts the ratio of motor cortical excitatory to inhibitory neurons in a multiple system atrophy model.

Motor cortical circuit functions depend on the coordinated fine-tuning of two functionally diverse neuronal populations: glutamatergic pyramidal neurons providing synaptic excitation and GABAergic interneurons adjusting the response of pyramidal neurons through synaptic inhibition. Microglia are brain resident macrophages which dynamically refine cortical circuits by monitoring perineuronal extracellular matrix and remodelling synapses. Previously, we showed that colony-stimulating factor 1 receptor (CSF1R)-mediated myeloid cell depletion extended the lifespan, but impaired motor functions of MBP29 mice, a mouse model for multiple system atrophy. In order to better understand the mechanisms underlying these motor deficits we characterized the microglial involvement in the cortical balance of GABAergic interneurons and glutamatergic pyramidal neurons in 4-months-old MBP29 mice following CSF1R inhibition for 12 weeks. Lack of myeloid cells resulted in a decreased number of COUP TF1 interacting protein 2-positive (CTIP2+) layer V pyramidal neurons, however in a proportional increase of calretinin-positive GABAergic interneurons in MBP29 mice. While myeloid cell depletion did not alter the expression of important presynaptic and postsynaptic proteins, the loss of cortical perineuronal net area was attenuated by CSF1R inhibition in MBP29 mice. These cortical changes may restrict synaptic plasticity and potentially modify parvalbumin-positive perisomatic input. Collectively, this study suggests, that the lack of myeloid cells shifts the neuronal balance toward an increased inhibitory connectivity in the motor cortex of MBP29 mice thereby potentially deteriorating motor functions.

Distinct forebrain regions define a dichotomous astrocytic profile in multiple system atrophy.

The growing recognition of a dichotomous role of astrocytes in neurodegenerative processes has heightened the need for unraveling distinct astrocytic subtypes in neurological disorders. In multiple system atrophy (MSA), a rare, rapidly progressing atypical Parkinsonian disease characterized by increased astrocyte reactivity. However the specific contribution of astrocyte subtypes to neuropathology remains elusive. Hence, we first set out to profile glial fibrillary acidic protein levels in astrocytes across the human post mortem motor cortex, putamen, and substantia nigra of MSA patients and observed an overall profound astrocytic response. Matching the post mortem human findings, a similar astrocytic phenotype was present in a transgenic MSA mouse model. Notably, MSA mice exhibited a decreased expression of the glutamate transporter 1 and glutamate aspartate transporter in the basal ganglia, but not the motor cortex. We developed an optimized astrocyte isolation protocol based on magnetic-activated cell sorting via ATPase Na+/K+ transporting subunit beta 2 and profiled the transcriptomic landscape of striatal and cortical astrocytes in transgenic MSA mice. The gene expression profile of astrocytes in the motor cortex displayed an anti-inflammatory signature with increased oligodendroglial and pro-myelinogenic expression pattern. In contrast, striatal astrocytes were defined by elevated pro-inflammatory transcripts accompanied by dysregulated genes involved in homeostatic functions for lipid and calcium metabolism. These findings provide new insights into a region-dependent, dichotomous astrocytic response-potentially beneficial in the cortex and harmful in the striatum-in MSA suggesting a differential role of astrocytes in MSA-related neurodegenerative processes.

Smoking Modifies the Genetic Risk for Early-Onset Periodontitis.

Periodontitis has low-prevalence, highly severe disease manifestations with an early onset and rapid progression. The diagnosis is based on severe destruction of the alveolar bone in adolescents and young adults. Genetic susceptibility variants and smoking are well-established risk factors, but their interactions in modifying disease susceptibility have not been studied. We aimed to identify genetic risk variants of early-onset periodontitis that unmask their effects on tobacco smoke exposure. To this end, we analyzed 79,780,573 common variants in 741 northwest Europeans diagnosed to have >30% bone loss at >2 teeth before 35 y of age, using imputed genotypes of the OmniExpress BeadChip. Never versus ever smokers were compared in a logistic regression analysis via a case-only approach. To explore the effect of tobacco smoke on the expression of the G×S-associated genes, cultures of primary gingival fibroblasts (n = 9) were exposed to cigarette smoke extract, and transcripts were quantified by reverse transcription polymerase chain reaction. We identified 16 loci for which our analysis suggested an association with G×S increased disease risk (P < 5 × 10-5). Nine loci had previously been reported to be associated with spirometric measures of pulmonary function by an earlier G×S genome-wide association study. Genome-wide significant cis expression quantitative trait loci were reported for G×S-associated single-nucleotide polymorphisms at ST8SIA1 and SOST, indicating a causal role of these genes in tobacco-related etiopathology. Notably, SOST is a negative regulator of bone growth, and ST8SIA1 has a role in tissue remodeling. Cigarette smoke extract significantly altered the expression of 2 associated genes: SSH1 (P = 5 × 10-07), which is required for NF-κB activation and innate immune responses to bacterial invasion, and ST8SIA1 (P = 0.0048). We conclude that the genetic predisposition to early-onset periodontitis is in part triggered by smoking and that tobacco smoke directly affects the expression of genes involved in bone homeostasis, tissue repair, and immune response.

Administration of systemic antibiotics during non-surgical periodontal therapy-a consensus report.

AIM: The aim of this meta-review was to evaluate whether there is a meaningful clinical benefit regarding the use of systemic adjunctive antibiotics in the treatment of patients with periodontitis. Additionally, a consensus regarding possible recommendations for future administration of antibiotics should be reached. METHODS: A structured literature search was performed by two independent investigators focusing on systematic reviews (SR) covering adjunctive systemic antibiosis during non-surgical periodontal therapy. Additionally, recent randomized clinical trials (RCT, July 2015 to July 2017) were searched systematically to update the latest SR. Results were summarized and discussed in a plenary to reach a consensus. RESULTS: Mostly, systematic reviews and RCTs showed a significant positive effect of adjunctive systematic antibiosis compared to controls. These positive effects gain clinical relevance in patients with severe periodontal disease aged 55 years and younger. CONCLUSION: Systemic antibiotics as an adjunct to non-surgical periodontal therapy should be sensibly administered and restrictively used. Only certain groups of periodontitis patients show a significant and clinically relevant benefit after intake of systemic antibiosis during periodontal therapy. CLINICAL RELEVANCE: Avoiding antibiotic resistance and possible side effects on the human microbiome should be a focus of dentists and physicians. Thus, a sensible administration of antibiotics is mandatory. This manuscript suggests guidelines for a reasonable use.

[Angiographic Results After Coronary Artery Bypass Grafting].

The main place among cardiovascular diseases takes coronary heart disease. The number of coronary artery bypass surgery increases every year. The large number of coronary artery bypass grafting (CABG) performed worldwide. The need for assessment of grafts patency is enormous. OBJECTIVE: We are performed analyze of graft patency results, after CABG surgery in our clinic. MATERIALS AND METHODS: This paper presents the results of a retrospective analysis of angiographic graft patency data depends of TIMI flow, Syntax score, diameter and degree of vascular lesions, as well as the surgery type. RESULTS: According angiographic data of 142 patients, we found that 74 (52.1 %) had no graft dysfunction. In the 68 (47.9 %) patients had various types graft dysfunction which is 3.0 % of the total number of operated patients in our center for coronary heart disease. 31 (46 %) patients were operated under Off pump, 19 (28 %) - On pump and 18 (26 %) - in a parallel bypass technic. According to our data parameters such as Syntax score, the diameter of the vessel and the percentage of lesion, its did not significantly affect the grafts patency in terms of up to 12,7±6,5 months. Preoperative coronary blood flow (assessed by TIMI scale) the significantly affects the grafts patency. CONCLUSIONS: In the graft patency for perioperative period and follow-up, significantly affected preoperative coronary blood flow assessed by TIMI. The results of beating heart (off pump and using a parallel IR) and On pump surgery similar in the immediate postoperative period. But there is tend to increase graft dysfunction in up to 30 months in patients after off pump surgery. We don't found relation between Syntax score, the diameter of the coronary arteries, the percentage of stenosis and graft patency after 12 months follow-up.

Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines.

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na2S or cysteine-HCl generates the required anaerobic environment by releasing metabolites similar to those produced when protein is fermented, possibly inducing a dysbiosis. An experiment was conducted to study the impact of two reducing agents on results yielded by such in vitro fermentation models. Protein (soybean proteins, casein) and carbohydrate (potato starch, cellulose) ingredients were fermented in vitro by bacteria isolated from fresh feces obtained from three sows in three carbonate-based incubation media differing in reducing agent: (i) Na2S, (ii) cysteine-HCl and (iii) control with a mere saturation with CO2 and devoid of reducing agent. The gas production during fermentation was recorded over 72 h. Short-chain fatty acids (SCFA) production after 24 and 72 h and microbial composition of the fermentation broth after 24 h were compared between ingredients and between reducing agents. The fermentation residues after 24 h were also evaluated in terms of cytotoxicity using Caco-2 cell monolayers. Results showed that the effect of the ingredient induced higher differences than the reducing agent. Among the latter, cysteine-HCl induced the strongest differences compared with the control, whereas Na2S was similar to the control for most parameters. For all ingredients, final gas produced per g of substrate was similar (P>0.10) for the three reducing agents whereas the maximum rate of gas production (R max) was reduced (P0.10) after 24 h of fermentation with Na2S and in the control without reducing agent. Molar ratios of branched chain-fatty acids were higher (P<0.05) for protein (36.5% and 9.7% for casein and soybean proteins, respectively) than for carbohydrate (<4%) ingredients. Only fermentation residues of casein showed a possible cytotoxic effect regardless of the reducing agent (P<0.05). Concerning the microbial composition of the fermentation broth, most significant differences in phyla and in genera ascribable to the reducing agent were found with potato starch and casein. In conclusion, saturating the incubation media with CO2 seems sufficient to generate a suitable anaerobic environment for intestinal microbes and the use of a reducing agent can be omitted.

Influence of operating conditions on nitrous oxide formation during nitritation and nitrification.

Nitrous oxide (N2O), a strong greenhouse gas, can be produced by ammonium-oxidizing bacteria (AOB) as a by-product of ammonium oxidation and can potentially be formed in all types of nitrification processes. However, partial nitritation has been reported to cause significantly higher N2O emissions than complete nitrification. In the study presented here, the mechanisms and factors that drive N2O formation by AOB were investigated with respect to different operational strategies to achieve nitrite accumulation base on combined evaluation of oxygen uptake rate (OUR) and N2O formation rate. On the one hand, N2O formation during partial nitritation and nitrification in a continuously stirred tank reactor (CSTR) with continuous aerobic conditions was observed. On the other hand, the effect of intermittent aeration on N2O formation during nitrification was investigated. The presence of nitrite, the extend of sludge-specific ammonium loading, low oxygen concentration, and transition from aerobic to anoxic conditions significantly increased N2O formation in this reactor independently from each other, indicating that different formation pathways, supposedly via nitrite or hydroxylamine, were active.

Potential of an in vitro toolbox combined with exposure data as a first step for the risk assessment of dietary chemical contaminants.

In vitro risk assessment of dietary contaminants has become a priority in human food safety. This paper proposes an in vitro approach associating different complementary tools in an original toolbox and aims to improve the assessment of the toxicological impact of dietary contaminants at realistic human exposure levels, with a special focus on the intestinal compartment. The system is based on the use of four complementary cellular tools, namely stress gene induction in transgenic strains of Escherichia coli, modulation of the activity of key biotransformation enzymes (cytochrome P-450 (CYP) 1A1 and 3A4) in a human intestinal cell line, and activation of aryl hydrocarbon receptor (AhR) and oestrogenic receptor (ER)-dependent genes in agonistic and antagonistic assays with luciferase reporter cells. It was applied to four chosen model molecules: ochratoxin A (OTA) and deoxynivalenol (DON), two common food-borne mycotoxins, and imazalil (IMA) and benomyl (BEN), two fungicides widely occurring in foodstuffs. All these assays were performed at or around a realistic intestinal concentration, determined through a deterministic approach based on the calculation of a theoretical maximum daily intake (TMDI). Using the four model molecules, it is clearly highlighted that induction of CYP1A1 activity and inhibition of CYP3A4 activity occurred in Caco-2 cells at a realistic intestinal concentration of IMA. Furthermore, some bacterial stress genes were induced in a range of realistic concentrations, following exposure to DON and IMA. In addition, BEN clearly provoked an ER agonistic activity in a human oestrogen sensitive reporter cell line. All these results are in accordance with the literature, suggesting that the in vitro toolbox constitutes an interesting approach in order to obtain a first 'fingerprint' of dietary contaminants at realistic human exposure for further risk assessment.

Screening, identification and quantification of glucosinolates in black radish (Raphanus sativus L. niger) based dietary supplements using liquid chromatography coupled with a photodiode array and liquid chromatography-mass spectrometry.

The glucosinolate profile of black radish (Raphanus sativus L. niger) based dietary supplements has been investigated by HPLC-PDA, LC-ESI-MS/MS and LC-APCI-MS/MS systems. Optimization of the MS/MS parameters and LC conditions was performed using sinigrin reference standard and rapeseed certified reference material (BC190) respectively. An LC-ESI-MS/MS system was used to detect (screen) and identify the naturally occurring intact glucosinolates (GLs). The intact GLs identified were then desulfated and quantified on an HPLC-PDA system as desulfo-glucosinolates (DS-GLs). Prior to quantification, the DS-GLs were identified using an APCI-MS/MS. The HPLC-PDA method performance criteria were evaluated using glucotropaeolin potassium salt. The validated method was applied for the analysis of six dietary supplements. In total, six glucosinolates were identified and quantified in the dietary supplements; glucoraphasatin (0.2-0.48 mg/g), glucosisaustricin (0.37-0.91 mg/g), glucoraphenin (0.84-1.27 mg/g), glucoputrajivin (0.14-0.28 mg/g), glucosisymbrin (0.70-0.99 mg/g) and gluconasturtiin (0.06-0.12 mg/g). Glucoraphenin was the most abundant glucosinolate in all samples.

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells.

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 µg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 µg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 µg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.

Determination of the nitrous oxide emission potential of deammonification under anoxic conditions.

Various studies have been performed to determine nitrous oxide (N2O) emissions from conventional biological nitrogen removal processes in wastewater treatment like nitrification and denitrification in the main stream. However, with respect to the overall emissions of a wastewater treatment plant, part-stream treatment for high-strength wastewater (e.g., sludge liquor) is also expected to hold a significant emission potential because of high concentrations and extreme boundary conditions. This paper presents results from a laboratory-scale study on nitrous oxide production by biomass from a deammonification process (nitritation + anammox) under anoxic conditions. It was discovered that N2O formation results from incomplete endogenous denitrification rather than anammox and is dependent on substrate availability. Based on direct measurements of the dissolved N2O concentrations in a sequencing batch reactor, the dynamic behavior of N2O production is characterized in more detail. The results show that, during anoxic conditions, the N2O emission potential of deammonification is significantly lower than from conventional denitrification.

In vitro identification of targeting ligands of human M cells by phage display.

To improve transport of vaccine-loaded nanoparticles, the phage display technology was used to identify novel lead peptides targeting human M cells. Using an in vitro model of the human follicle-associated epithelium (FAE) which contains both Caco-2 and M cells, a T7 phage display library was screened for its ability either to bind the apical cell surface of or to undergo transcytosis across Caco-2 cells or FAE. The selection for transcytosis across both enterocytes and FAE identified three different peptide sequences (CTGKSC, PAVLG and LRVG) with high frequency. CTGKSC and LRVG sequences enhanced phage transport across M-like cells. When polymeric nanoparticles were grafted with the sequences CTGKSC and LRVG, their transport by FAE was significantly enhanced. These peptides could therefore be used to enhance the transport of vaccine-loaded nanoparticles across the intestinal mucosal barrier.

Adjuvant extension of chemotherapy after neoadjuvant therapy may not improve outcome in early-stage breast cancer.

INTRODUCTION: Neoadjuvant chemotherapy (NAC) is equivalent to adjuvant therapy (AdC) in terms of survival and disease-free interval. Many institutions add AdC after NAC and surgery. However, such extended chemotherapy (ExC) is not evidence based. Study aim was to investigate if ExC improved disease-free (DFS) and overall survival (OS). PATIENTS AND METHODS: From 1998 to 2006 356 consecutive patients received NAC (45 pts), AdC (221 pts) or ExC (90 pts). We analysed these 3 groups to determine effects of ExC and to identify patients who might benefit. NAC consisted in 93% of 3-6 cycles of epirubicin+docetaxel, AdC comprised EC+/-taxanes in 72%. Median age in the NAC, AdC, and ExC-groups was 54, 56 and 52 years with follow-up of 30, 57, and 55 months. RESULTS: After NAC, 35% achieved downstaging and 10% pathologic complete remission. Surprisingly ExC seemed to result in reduction of 5-year DFS: compared to 85% and 82% after NAC and AdC, DFS was 61% after ExC (p=0.001). OS was not significantly affected (79, 91, and 78% after NAC, AdC and ExC, p=0.13). In multivariate analysis after correction for age, menopausal status, stage, grading, hormone receptors, her2-status, radiotherapy and surgery, ExC seemed to adversely affect DFS (HR 2.15, p=0.008), loco-regional and distant recurrence-rates (HR 3.0, p=0.03 and HR 2.0, p=0.02). DISCUSSION: In this single-center analysis ExC could not show advantages in terms of DFS and OS. Because multivariate analyses of retrospective data cannot account for all potential biases, these data require confirmation in randomized clinical trials. Until then, extended chemotherapy should be considered carefully. As in previous studies, no differences were found between NAC and AdC groups.

Determination and evaluation of the phosphorus load of an artificial shallow lake.

Enhanced eutrophication of lakes due to high nutrient loads from anthropogenic sources has become a worldwide problem. Dying ecosystems and limitation of uses are the consequences. In Bochum, Germany, Lake Umminger is an integral part of a recreation area, but also receives high nutrient loads from the local sewer system, as could be shown with the help of water and nutrient balances. Mass algae growth, the dying of fish and production of digestion gas implied a demand to rehabilitate the lake. Primarily, the urgency and sanitation potential as well as the applicability of external and internal enhancement measures had to be evaluated. The trophic classification needed was based upon the German guideline for the classification of the water quality of natural lakes according to trophic criteria, mainly using Vollenweider's eutrophication model. This paper focuses on a description and analysis of the problems that arose during the application of this model to Lake Umminger, stating that shallow, artificial lakes cannot be evaluated correctly with the existing methods. Although some suggestions for further improvement are given, the development of new evaluation criteria was not in the scope of the study presented.

Study of the gastrointestinal biotransformation of zearalenone in a Caco-2 cell culture system with liquid chromatographic methods.

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the detection of zearalenone (ZON), alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) in in vitro biological samples. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of ZON, alpha-ZOL, beta-ZOL, alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL) in in vitro biological samples. Zearalanone (ZAN) was used as internal standard in both methods. The limit of detection/limit of quantitation (LOD/LOQ) values for ZON, alpha-ZOL and beta-ZOL were 2/7, 2/7 and 4/13 microg l(-1), respectively, for the HPLC-FLD method. For the LC-MS/MS method LOD/LOQ values for ZON, alpha-ZOL, beta-ZOL, alpha-ZAL and beta-ZAL were 6/20, 5/17, 4/14, 9/30 and 6/19 microg l(-1), respectively. Within-day and between-day precision were less then 11 and 14%, respectively for the HPLC-FLD method, and both less then 20% for the LC-MS/MS method. The recovery of ZON and its metabolites ranged between 73 and 89% for the HPLC-FLD method and between 69 and 112% for the LC-MS/MS method. The methods were used for the detection of the compounds in in vitro biological samples, obtained with human intestinal Caco-2 cells culture experiments. The 8-days post-confluent Caco-2 cells were treated with ZON or a mixture of ZON and imazalil (IMA). After an incubation time of 24 h the samples were analysed with the HPLC-FLD method. Neither ZON nor its derivatives were detected in the samples. The disappearance of ZON could possibly point out the formation of phase II metabolites like glucuronide conjugates. Therefore, samples were pretreated with beta-glucuronidase before LC-MS/MS analysis. The LC-MS/MS results showed that ZON, alpha-ZOL and beta-ZOL could only be detected in the beta-glucuronidase pretreated samples. This confirmed the formation of glucuronide conjugates and the hydroxylation of ZON during the incubation with Caco-2 cells.

Liquid chromatographic methods for biotransformation studies of ochratoxin A.

Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OTalpha), 10-hydroxy OTA (10-OHOTA), 4R-hydroxy OTA (4R-OHOTA) and the ethyl ester of OTA (OTC) in in vitro samples, obtained with Caco-2 cell culture experiments and in in vivo urine samples from sheep. A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed and validated for the detection of OTA and its metabolites OTalpha, 10-OHOTA, 4R-OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OTalpha, 4R-OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 microg/L, respectively, for the HPLC-FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 microg/L, respectively for the LC-MS/MS method. Within-day and between-day precision were both <12% for the HPLC-FLD method, and <10% for the LC-MS/MS method. The recovery of OTA and its metabolites ranged between 71 and 111% for the HPLC-FLD method and between 79 and 110% for the LC-MS/MS method. In the first experiment only OTA was added to the Caco-2 cells while in the second experiment 3-methylcholanthrene (3MC) was also present in the cell culture systems. Besides OTA, which was recovered in all the samples, an unknown compound was also observed in the second experiment. When 3MC was added, the results showed that the OTA concentration in the basolateral samples was decreased by 50%. The methods were also implemented for the analysis of urine samples of sheep, fed increasing amounts of OTA. With the HPLC-FLD method it could be concluded that the concentration of OTA and OTalpha increased according to ingested amounts of OTA, with OTalpha being the most abundant compound. The results obtained with the LC-MS/MS method confirmed these results.

Transport mechanisms of mmePEG750P(CL-co-TMC) polymeric micelles across the intestinal barrier.

Monomethylether poly(ethyleneglycol)(750)-poly(caprolactone-co-trimethylene carbonate) (mmePEG750)P(CL-co-TMC)) which spontaneously form micelles, can cross lipid bilayers via passive diffusion and demonstrate an oral bioavailability of 40% in rats. The aim of the current work was to study the transport mechanism(s) of drug-loaded mmePEG750P(CL-co-TMC) micelles across the intestinal barrier. The transport of radiolabelled polymer across Caco-2 cell monolayer was investigated by disrupting tight junctions and by inhibiting endocytosis. The polymer and drugs loaded in micelles independently crossed Caco-2 cell monolayers and did not use either the paracellular route or M-cells. The polymer did not affect P-gp pumps. This mechanistic study suggests that whereas drug-loaded micelles were absorbed by fluid-phase endocytosis, polymeric unimers diffused passively across the membrane concomitantly with micellar endocytosis.

Contaminants in organically and conventionally produced winter wheat (Triticum aestivum) in Belgium.

A database has been compiled with the levels of important contaminants (mycotoxins, heavy metals and pesticides) measured from 2002 to 2005 in winter wheat (Triticum aestivum) grown in Belgium according to the organic and conventional farming systems. Assuming no further change in contaminant levels during cereal processing and during the preparation of foodstuffs, conservative intakes are estimated for the consumers of cereal-based products such as flour, bread, breakfast cereals, dough and pastry. The results show that for the consumer of organic foodstuffs, estimated daily intakes are 0.56 microg deoxynivalenol (DON), 0.03 microg zearalenone (ZEA), 0.19 microg Cd, 0.28 microg Pb and 0.0006 microg Hg kg(-1) body weight, taking into account the average contaminant levels in unprocessed grains and the average cereal products consumptions in Belgium. For the consumers of conventional foodstuffs, the corresponding estimated daily intakes are 0.99 microg DON, 0.06 microg ZEA, 0.17 microg Cd, 0.12 microg Pb and 0.0007 microg Hg kg(-1) body weight. In addition, it appears that for the consumers of conventional products, intakes of some post-harvest insecticides have to be taken into account (0.11 microg chlorpyriphos-methyl, 0.2 microg dichlorvos and 0.24 microg pirimiphos-methyl kg(-1) bw). When expressed as a percentage of the tolerable/acceptable daily intake (TDI/ADI), it seems that the corresponding estimated (conservative) intakes are the highest for DON (56% for organic and 99% for conventional cereal products), ZEA (16% for organic and 32% for conventional cereal products), and Cd (19% for organic and 17% for conventional cereal products), all other estimated intakes of contaminants (including pesticides) being lower than 10% of the TDI/ADI.

Intake of ochratoxin A and deoxynivalenol through beer consumption in Belgium.

Estimations of ochratoxin A (OTA) and 4-deoxynivalenol (DON) exposure of the Belgian population through beer consumption were made using the results of the recent Belgian food survey and the compiled data set of OTA and DON levels in conventionally and organically produced beers in 2003-05. For the consumers of organic beers, the daily intake of OTA was 0.86 (in 2003), 1.76 (in 2004) and 0.72 (in 2005) ng kg(-1) body weight (bw), considering the mean beer consumption (0.638 litres) and the average level of OTA in 2003, 2004 and 2005, respectively. Using the 97.5th percentile of beer consumption (1.972 litres), the corresponding OTA daily intakes were 2.65, 5.44 and 2.24 ng kg(-1) bw, which are close or above the tolerable daily intake (TDI) of 5 ng kg(-1) bw. For the consumers of conventional beers, the OTA intakes were low: 0.23, 0.23 and 0.11 ng kg(-1) bw day(-1) for the average beer consumption, in 2003, 2004 and 2005 against 0.72, 0.73 and 0.34 ng kg(-1) bw day(-1) when the 97.5th percentile level was considered. As for the DON intake, the estimates were quite low for both conventional and organic beer consumers when the provisional maximum TDI (PMTDI) of 1 microg kg(-1) bw was considered. Average consumption of organic beer led to daily intakes of 0.05 and 0.04 microg DON kg(-1) bw in 2003 and 2004, respectively, whilst for conventional beer, daily intakes were 0.07 and 0.05 microg DON kg(-1) bw. At the 97.5th percentile level of beer consumption, daily intakes of 0.15 and 0.13 microg kg(-1) bw were obtained for organic beers against 0.23 and 0.17 microg kg(-1) bw for conventional ones. The results showed that beer could be an important contributor to OTA exposure in Belgium, even though a declining trend seems to be apparent during the last year of monitoring. Therefore, efforts should be devoted to maintain the OTA levels as low as reasonably achievable, especially for organic beer.

Short communication: Ability of cultured mammary epithelial cells in a bicameral system to secrete milk fat.

Mammary epithelial cells from lactating cows were cultured onto inserts coated with type I collagen. Every second day, the rates of fatty acid synthesis and secretion were determined by measuring the amount of [14C]-labeled sodium acetate incorporated into lipids over a 4-h period. The [14C]-containing lipids were identified by thin layer chromatography fractionation. In parallel, the integrity of the cell layer was evaluated by measurement of transepithelial electrical resistance. The integrity increased progressively to reach a maximum after 8 d of culture. Cells incorporated acetate into lipids; 1.34% of acetate was incorporated into lipids produced by freshly isolated cells. This percentage decreased to 0.5% after 2 d of culture. Moreover, this capacity decreased with the duration in culture; on d 8, the rate of incorporation dropped to about 3% of that on d 2. In the cell extracts, the [14C]-labeled lipids were mainly triglycerides, although the proportion of diglycerides and phospholipids progressively increased as a part of total newly synthesized lipids. The proportion of triglycerides decreased 0.66 times between d 2 and 8 when the proportion of diglycerides and phospholipids increased 1.33 and 2.18 times, respectively. About 28% of the newly synthesized lipids were secreted within 4 h of incubation. Around 65 to 85% of these labeled lipids were found in the apical compartment, suggesting a partially vectorial secretion. But 58 to 80% of labeled lipids found in the apical and basolateral medium were free fatty acids. Functional tight junctions and incorporation of labeled fatty acids into triglycerides are not compatible with an inferred status of complete dedifferentiation of the cell layer. Moreover, triglyceride secretion seems compromised, probably due to the lack of an appropriate cell environment and cell shape.