The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

Time-Course In Vivo Trafficking Pattern and Effector Function of Donor-Specific Regulatory T Cells in Response to an Allograft.

Background: Experimental data have suggested that adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs), capable of controlling immune responses to specifi c auto- or alloantigens, could be used as a therapeutic strategy to promote specifi c tolerance in T-cell mediated diseases and in organ transplantation (Tx). However, before advocating the application of immunotherapy with Tregs in Tx, we need to improve our understanding of their in vivo homeostasis, traffi cking pattern and effector function in response to alloantigens. Methods : Donor-antigen specifi c murine Tregs were generated and characterized in vitro following our described protocols. Using an adoptive transfer and skin allotransplantation model, we have analyzed the in vivo expansion and homing of fl uorescent-labeled effector T cells (Teff) and Tregs, at different time-points after Tx, using fl ow-cytometry as well as fl uorescence microscopy techniques. Results: Tregs expressed CD62L, CCR7 and CD103 allowing their homing into lymphoid and non-lymphoid tissues (gut, skin) after intravenous injection. While hyporesponsive to TCR stimulation in vitro, transferred Tregs survived, migrated to secondary lymphoid organs and preferentially expanded within the allograft draining lymph nodes. Furthermore, Foxp3+ cells could be detected inside the allograft as early as day 3-5 after Tx. At a much later time-point (day 60 after Tx), graft-infi ltrating Foxp3+ cells were also detectable in tolerant recipients. When transferred alone, CD4+CD25- Teff cells expanded within secondary lymphoid organs and infi ltrated the allograft by day 3-5 after Tx. The co-transfer of Tregs limited the expansion of alloreactive Teff cells as well as their recruitment into the allograft. The promotion of graft survival observed in the presence of Tregs was in part mediated by the inhibition of the production of effector cytokines by CD4+CD25- T cells. Conclusion: Taken together, our results suggest that the suppression of allograft rejection and the induction of Tx tolerance are in part dependant on the alloantigendriven homing and expansion of Tregs. Thus, the appropriate localization of Tregs may be critical for their suppressive function in vivo.

A hormonal regulatory module that provides flexibility to tropic responses.

Plants orient their growth depending on directional stimuli such as light and gravity, in a process known as tropic response. Tropisms result from asymmetrical accumulation of auxin across the responding organ relative to the direction of the stimulus, which causes differential growth rates on both sides of the organ. Here, we show that gibberellins (GAs) attenuate the gravitropic reorientation of stimulated hypocotyls of dark-grown Arabidopsis (Arabidopsis thaliana) seedlings. We show that the modulation occurs through induction of the expression of the negative regulator of auxin signaling INDOLE-3-ACETIC ACID INDUCIBLE19/MASSUGU2. The biological significance of this regulatory mechanism involving GAs and auxin seems to be the maintenance of a high degree of flexibility in tropic responses. This notion is further supported by observations that GA-deficient seedlings showed a much lower variance in the response to gravity compared to wild-type seedlings and that the attenuation of gravitropism by GAs resulted in an increased phototropic response. This suggests that the interplay between auxin and GAs may be particularly important for plant orientation under competing tropic stimuli.

Ribonucleotide reductase genes of Bacillus prophages: a refuge to introns and intein coding sequences.

The ribonucleotide reductase gene tandem bnrdE/bnrdF in SPbeta-related prophages of different Bacillus spp. isolates presents different configurations of intervening sequences, comprising one to three of six non-homologous splicing elements. Insertion sites of group I introns and intein DNA are clustered in three relatively short segments encoding functionally important domains of the ribonucleotide reductase. Comparison of the bnrdE homologs reveals mutual exclusion of a group I intron and an intein coding sequence flanking the codon that specifies a conserved cysteine. In vivo splicing was demonstrated for all introns. However, for two of them a part of the mRNA precursor molecules remains unspliced. Intergenic bnrdE-bnrdF regions are unexpectedly long, comprising between 238 and 541 nt. The longest encodes a putative polypeptide related to HNH homing endonucleases.

An activation-independent role of transcription factors in insulator function.

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

Eokines: synthesis, storage and release from human eosinophils

Eosinophils are prominent inflammatory cells in asthma and other allergic disorders, as well as in helminthic parasite infections. Recently, eosinophils have been reported to synthesize and store a range of regulatory proteins within their secretory granules (eokines). Eokines comprise a group of cytokines, chemokines, and growth factors which are elaborated by eosinophils. These proteins, and the messages which encode them, appear to be identical to those produced by lymphocytes and other tissues. Interestingly, immunoreactivity to many of these eokines has been found to co-localize to the eosinophil´s secretory granules. In this review, we have discussed the repertoire of 18 eokines so far identified in eosinophils, and focused on four of these, namely, interleukin-2 (IL-2), IL-4, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES. These four eokines co-localize to the crystalloid granules in eosinophils, as shown in studies using subcellular fractionation and immunogold labeling in electron microscopy. During stimulation by physiological triggers, for example, with serum-coated particles, eosinophils release these mediators into the surrounding supernatant. In addition, eokines are likely to be synthesized within eosinophils rather than taken up by endocytosis, as show in detection of mRNA for each of these proteins using in situ hybridization, RT-PCR, and in the case of RANTES, in situ RT-PCR. Eokines synthesis and release from eosinophils challenges the commonly held notion that these cells act downstream of key elements in immune system, and indicate that they may instead belong to the afferent arm of immunity.

ENaC and its regulatory proteins as drug targets for blood pressure control.

Hypertension is a serious medical problem affecting millions of people worldwide. A key protein regulating blood pressure is the Epithelial Na(+) Channel (ENaC). In accord, loss of function mutations in ENaC (PHA1) cause hypotension, whereas gain of function mutations (Liddle syndrome) result in hypertension. The region mutated in Liddle syndrome, called the PY motif (L/PPxY), serves as a binding site for the ubiquitin ligase Nedd4-2, a C2-WW-Hect E3 ubiquitin ligase. Nedd4-2 binds the ENaC-PY motif via it WW domains, ubiquitylates the channel and targets it for endocytosis, a process impaired in Liddle syndrome due to poor binding of the channel to Nedd4-2. This leads to accumulation of active channels at the cell surface and increased Na(+) (and fluid) absorption in the distal nephron, resulting in elevated blood volume and blood pressure. Compounds that destabilize cell surface ENaC, or enhance Nedd4-2 activity in the kidney, could potentially serve as drug targets for hypertension. In addition, recent discoveries of regulation of activation of ENaC by proteases such as furin, prostasin and elastase, which cleave the extracellular domain of this channel leading to it activation, as well as the identification of inhibitors that block the activity of these proteases, provide further avenues for drug targeting of ENaC and the control of blood pressure.

Progesterone/RANKL is a major regulatory axis in the human breast.

Estrogens and progesterones are major drivers of breast development but also promote carcinogenesis in this organ. Yet, their respective roles and the mechanisms underlying their action in the human breast are unclear. Receptor activator of nuclear factor κB ligand (RANKL) has been identified as a pivotal paracrine mediator of progesterone function in mouse mammary gland development and mammary carcinogenesis. Whether the factor has the same role in humans is of clinical interest because an inhibitor for RANKL, denosumab, is already used for the treatment of bone disease and might benefit breast cancer patients. We show that progesterone receptor (PR) signaling failed to induce RANKL in PR(+) breast cancer cell lines and in dissociated, cultured breast epithelial cells. In clinical specimens from healthy donors and intact breast tissue microstructures, hormone response was maintained and RANKL expression was under progesterone control, which increased RNA stability. RANKL was sufficient to trigger cell proliferation and was required for progesterone-induced proliferation. The findings were validated in vivo where RANKL protein expression in the breast epithelium correlated with serum progesterone levels and the protein was expressed in a subset of luminal cells that express PR. Thus, important hormonal control mechanisms are conserved across species, making RANKL a potential target in breast cancer treatment and prevention.

Policy Credibility and Delegation to Independent Regulatory Agencies: A Comparative Empirical Analysis

Independent regulatory agencies are one of the main institutional features of the 'rising regulatory state' in Western Europe. Governments are increasingly willing to abandon their regulatory competencies and to delegate them to specialized institutions that are at least partially beyond their control. This article examines the empirical consistency of one particular explanation of this phenomenon, namely the credibility hypothesis, claiming that governments delegate powers so as to enhance the credibility of their policies. Three observable implications are derived from the general hypothesis, linking credibility and delegation to veto players, complexity and interdependence. An independence index is developed to measure agency independence, which is then used in a multivariate analysis where the impact of credibility concerns on delegation is tested. The analysis relies on an original data set comprising independence scores for thirty-three regulators. Results show that the credibility hypothesis can explain a good deal of the variation in delegation. The economic nature of regulation is a strong determinant of agency independence, but is mediated by national institutions in the form of veto players.

Synchronous versus asynchronous modeling of gene regulatory networks.

MOTIVATION: In silico modeling of gene regulatory networks has gained some momentum recently due to increased interest in analyzing the dynamics of biological systems. This has been further facilitated by the increasing availability of experimental data on gene-gene, protein-protein and gene-protein interactions. The two dynamical properties that are often experimentally testable are perturbations and stable steady states. Although a lot of work has been done on the identification of steady states, not much work has been reported on in silico modeling of cellular differentiation processes. RESULTS: In this manuscript, we provide algorithms based on reduced ordered binary decision diagrams (ROBDDs) for Boolean modeling of gene regulatory networks. Algorithms for synchronous and asynchronous transition models have been proposed and their corresponding computational properties have been analyzed. These algorithms allow users to compute cyclic attractors of large networks that are currently not feasible using existing software. Hereby we provide a framework to analyze the effect of multiple gene perturbation protocols, and their effect on cell differentiation processes. These algorithms were validated on the T-helper model showing the correct steady state identification and Th1-Th2 cellular differentiation process. AVAILABILITY: The software binaries for Windows and Linux platforms can be downloaded from http://si2.epfl.ch/~garg/genysis.html.