Persistence of fermented food bacteria in the oral cavity of rats after one week of consumption.

Fermented foods constitute a wide source of exogenous lactic acid bacteria (LAB) which could modify the endogenous microbiota of the consumer's digestive tract. This assumption depends on the ability of LAB to persist, at least transiently, in the oral cavity. The aim of this study was to evaluate the persistence of 3 LAB species, Streptococcus thermophilus, Lactococcus lactis, and Lacticaseibacillus paracasei in the oral cavity of rats by feeding them with a daily portion of model cheese containing the three bacteria for one week. Saliva samples were collected 16 h and one week after the last daily cheese intake. Their cheese bacteria contents were quantified by qPCR. When 16 h after cheese consumption, cheese bacteria were detected in the oral cavity of about ⅓ of the rats ranging from 10-3% to 10-1% of the total bacteria. One week after the last cheese intake, 1/8 LAB remains in the oral cavity. This study is a proof of concept showing that LAB can remain in the oral cavity several days after fermented food consumption with variability depending on individuals.

CRISPR/Cas9-mediated knockout of Abcd1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the Abcd1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.

A microglial cell model for acyl-CoA oxidase 1 deficiency.

Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) ß-oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H2O2, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1ß and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal ß-oxidation defect on oxidative stress, inflammation and cellular functions.

Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol.

BACKGROUND AND AIMS: Oxysterols are assumed to play important roles in age-related macular degeneration, a major cause of blindness. So we characterized the cytotoxic, oxidative, inflammatory, and angiogenic activities of oxysterols (7ß-hydroxycholesterol (7ß-OH), 7-ketocholesterol (7KC), 25-hydroxycholesterol (25-OH)) in human retinal ARPE-19 cells, and evaluated the protective effects of resveratrol (Rsv: 1 µM), a polyphenol from red wine. METHODS: ARPE-19 cells were treated with 7ß-OH, 7KC, or 25-OH (5-40 µg/mL; 24-48 h) without or with Rsv. Cell viability was determined using trypan blue and the MTT assay. Cell death was characterized by electron microscopy and in situ detection of activated caspases with fluorochrome-labeled inhibitors of caspases. Reactive oxygen species (ROS) production was measured with hydroethidine. ELISA methods and a cytometric bead assay were used to quantify cytokines involved in inflammation (IL-8, IL-1ß, IL-6, IL-10, IL-12p70, TNF-α, MCP-1) and VEGF. RESULTS: 7ß-OH and 7KC triggered a caspase-independent cell death process associated with the presence of multilamellar cytoplasmic structures evocating phospholipidosis, increased ROS production, and IL-8 secretion. 7ß-OH enhanced VEGF secretion. No cytotoxic effects were identified with 25-OH, which highly stimulated ROS production, MCP-1, and VEGF secretion. With oxysterols, no IL-10, TNF-α, and IL-12p70 secretion were detected. 25-OH induced IL-8 secretion through the MEK/ERK½ signaling pathway, and Rsv showed cytoprotective activities and inhibited VEGF secretion. CONCLUSION: 7ß-OH, 7KC, and 25-OH have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells. As Rsv has some protective effects against oxysterol-induced cell death and VEGF secretion it could be valuable in ARMD treatment.

The biokinetics and radiotoxicology of curium: a comparison with americium.

The human and animal data on the biokinetics of (242)Cm and (244)Cm are reviewed and shown to be very similar to those for (241)Am. Liver and skeleton are the main organs of deposition and the retention of curium in the skeleton is very prolonged in all the species examined. Retention of both curium and americium in the liver appears to be species-dependent, being relatively rapidly removed from the liver of rats, and probably humans, but being tenaciously retained in dogs and some other species. The radiotoxicity of curium is also reviewed and it is shown that, as with (241)Am, lung and bone tumour induction are the major hazards from inhaled and systemically deposited (244)Cm. The use of chelating agents for the treatment of accidental contamination of the human body with (242,244)Cm is also discussed.

TIARA: treatment initiatives after radiological accidents.

This paper describes the objectives, and reviews the progress, of the European project 'Treatment Initiatives After Radiological Accidents' (TIARA). TIARA forms part of the 'Preparatory Action for Security Research' (PASR) launched by the European Commission in 2004. The Preparatory Action is intended to reach preliminary conclusions on the needs for the security of EU citizens. It prepared a comprehensive Security Research Programme as part of the Commission's Seventh Framework Programme proposal, which was adopted in 2006 and launched in 2007. The principal purpose of TIARA is to constitute a European network that will participate in facilitating the management of a crisis in the event of the malevolent dispersal of radionuclides into the public environment.

Development of a database: DACTARI for a radiotoxic element ranking methodology.

Dosimetric impact studies aim at evaluating potential radiological effects of chronic or acute releases from nuclear facilities. A methodology for ranking radionuclides (RN) in terms of their health-related impact on the human population was first developed at CEA with specific criteria for each RN that could be applied to a variety of situations. It is based, in particular, on applying physico-chemical criteria to the complete RN inventory (present in the release or in the source term) and on applying norms related to radiation protection and chemical toxicology. The initial step consisted in identifying and collecting data necessary to apply the methodology, with reference to a previous database of long-lived radionuclides (LLRN, with half-lives ranging from 30 to 10(14) y) containing 95 radionuclides. The initial results have allowed us to identify missing data and revealed the need to complete the study for both toxic and radiotoxic aspects. This led us to the next step, developing a specific database, DAtabase for Chemical Toxicity and Radiotoxicity Assessment of RadIonuclides (DACTARI), to collect data on chemical toxicity and radiotoxicity, including acute or chronic toxicity, the chemical form of the compounds, the contamination route (ingestion, inhalation), lethal doses, target organs, intestinal and maternal-foetal transfer, drinking water guidelines and the mutagenic and carcinogenic properties.

Treatment of accidental intakes of plutonium and americium: guidance notes.

The scientific basis for the treatment of the contamination of the human body by plutonium, americium and other actinides is reviewed. Guidance Notes are presented for the assistance of physicians and others who may be called upon to treat workers or members of the public who may become contaminated internally with inhaled plutonium nitrate, plutonium tributyl phosphate, americium nitrate or americium oxide.

Study of uranium transfer across the blood-brain barrier.

Uranium is a heavy metal which, following accidental exposure, may potentially be deposited in human tissues and target organs, the kidneys and bones. A few published studies have described the distribution of this element after chronic exposure and one of them has demonstrated an accumulation in the brain. In the present study, using inductively coupled plasma mass spectrometry (ICP-MS) for the quantification of uranium, uranium transfer across the blood-brain barrier (BBB) has been assessed using the in situ brain perfusion technique in the rat. For this purpose, a physiological buffered bicarbonate saline at pH 7.4 containing natural uranium at a given concentration was perfused. After checking the integrity of the BBB during the perfusion, the background measurement of uranium in control rats without uranium in the perfusate was determined. The quantity of uranium in the exposed rat hemisphere, which appeared to be significantly higher than that in the control rats, was measured. Finally, the possible transfer of the perfused uranium not only in the vascular space but also in the brain parenchyma is discussed.

Uncertainties on the committed equivalent dose to the thyroid as a function of age for different iodine isotopes.

This study compares uncertainties of equivalent doses after internal contamination by 125I, 129I or 131I. Uncertainties were calculated using reported distributions of physiological parameters and Monte Carlo simulation. In adults, uncertainties increase from 131I to 125I and 129I with 1% of the population receiving 3.9, 4.0 and 7.2 times the median dose for the respective isotopes. In newborns, these values were 7.5, 12.3 and 19.0 for 131I, 125I and 129I respectively. The ratio of the beta dose delivered to the epithelium versus a homogeneous distributed dose was estimated for different iodine concentrations in colloid, epithelium and interstitium. In adults, for 131I, about 40% of the beta dose was delivered to the epithelial cells, whereas this fraction varied depending on the concentration for 125I and 129I, i.e. 20-30% at a relative epithelial concentration of 20% and 7-14% at a concentration of 3%. Small variations were observed depending on age.

Biokinetics of radionuclides and treatment of accidental intakes.

This paper describes the objectives and reviews the progress of EULEP Working Party 5, convened under the auspices of the European Union's Fifth Framework Programme, to 'cluster' two EU-supported contracts, Biokinetics and Dosimetry of Internal Contamination (BIODOS (EU Contract FIS5-1999-00214)) and Radionuclide Biokinetics Database (EULEP) (RBDATA-EULEP (Concerted Action Contract FIS5-1999-00218), and two non-EU funded projects, Biokinetics of Radionuclides in Human Volunteers (RNHV (non-EU funded project)) and Treatment of Accidental Intakes of Radionuclides (TAIR (part funded by EULEP)).

Kinetics of radiation-induced apoptosis in the cerebellum of 14-day-old rats after acute or during continuous exposure.

We have studied, by histological methods, cytological progression, frequency and distribution of apoptosis in the external granular layer of the cerebellum after whole-body irradiation of 14-day-old rats by gamma-rays from 60 Co. After acute exposure to 0.25, 0.5, 1.5 and 3 Gy (18 cGy/min), the duration of the apoptotic process gradually increased with dose from 6-9 h after 0.25 Gy, to > 24 h after 3 Gy. Up to 1 Gy, maximal frequency was found 6 h after exposures, and at this postirradiation time a linear increase in apoptosis with dose was observed. No effect of dose-rate on apoptosis induction could be demonstrated 6 h after delivering 1 Gy at dose-rates from 2.2 to 18 cGy/min. Continuous irradiation at 1.8 cGy/h induced a gradual increase of apoptosis that remained at a plateau value of about 3% from 15 to 29 h (controls 0.12%, SD = 0.07) and then gradually decreased to 1% at 53 h. At this time the mitotic index was similar to that measured in controls. Apoptosis occurring 3 h after acute irradiation, confined to proliferative cells, was only observed for doses of 1.5 and 3 Gy.