NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2−/− mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1−/− mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition

Quasifree (p, 2p) Reactions on Oxygen Isotopes: Observation of Isospin Independence of the Reduced Single-Particle Strength

Quasifree one-proton knockout reactions have been employed in inverse kinematics for a systematic study of the structure of stable and exotic oxygen isotopes at the R3B/LAND setup with incident beam energies in the range of 300-450 MeV/u. The oxygen isotopic chain offers a large variation of separation energies that allows for a quantitative understanding of single-particle strength with changing isospin asymmetry. Quasifree knockout reactions provide a complementary approach to intermediate-energy one-nucleon removal reactions. Inclusive cross sections for quasifree knockout reactions of the type OA(p,2p)NA-1 have been determined and compared to calculations based on the eikonal reaction theory. The reduction factors for the single-particle strength with respect to the independent-particle model were obtained and compared to state-of-the-art ab initio predictions. The results do not show any significant dependence on proton-neutron asymmetry

A Novel Role for IκBζ in the Regulation of IFNγ Production

IκBζ is a novel member of the IκB family of NFκB regulators, which modulates NFκB activity in the nucleus, rather than controlling its nuclear translocation. IκBζ is specifically induced by IL-1β and several TLR ligands and positively regulates NFκB-mediated transcription of genes such as IL-6 and NGAL as an NFκB binding co-factor. We recently reported that the IL-1 family cytokines, IL-1β and IL-18, strongly synergize with TNFα for IFNγ production in KG-1 cells, whereas the same cytokines alone have minimal effects on IFNγ production. Given the striking similarities between the IL-1R and IL-18R signaling pathways we hypothesized that a common signaling event or gene product downstream of these receptors is responsible for the observed synergy. We investigated IκBζ protein expression in KG-1 cells upon stimulation with IL-1β, IL-18 and TNFα. Our results demonstrated that IL-18, as well as IL-1β, induced moderate IκBζ expression in KG-1 cells. However, TNFα synergized with IL-1β and IL-18, whereas by itself it had a minimal effect on IκBζ expression. NFκB inhibition resulted in decreased IL-1β/IL-18/TNFα-stimulated IFNγ release. Moreover, silencing of IκBζ expression led to a specific decrease in IFNγ production. Overall, our data suggests that IκBζ positively regulates NFκB-mediated IFNγ production in KG-1 cells

Effective proton-neutron interaction near the drip line from unbound states in 25,26 F

Background: Odd-odd nuclei, around doubly closed shells, have been extensively used to study proton-neutron interactions. However, the evolution of these interactions as a function of the binding energy, ultimately when nuclei become unbound, is poorly known. The F26 nucleus, composed of a deeply bound π0d5/2 proton and an unbound ν0d3/2 neutron on top of an O24 core, is particularly adapted for this purpose. The coupling of this proton and neutron results in a Jπ=11+-41+ multiplet, whose energies must be determined to study the influence of the proximity of the continuum on the corresponding proton-neutron interaction. The Jπ=11+,21+,41+ bound states have been determined, and only a clear identification of the Jπ=31+ is missing. Purpose: We wish to complete the study of the Jπ=11+-41+ multiplet in F26, by studying the energy and width of the Jπ=31+ unbound state. The method was first validated by the study of unbound states in F25, for which resonances were already observed in a previous experiment. Method: Radioactive beams of Ne26 and Ne27, produced at about 440AMeV by the fragment separator at the GSI facility were used to populate unbound states in F25 and F26 via one-proton knockout reactions on a CH2 target, located at the object focal point of the R3B/LAND setup. The detection of emitted γ rays and neutrons, added to the reconstruction of the momentum vector of the A-1 nuclei, allowed the determination of the energy of three unbound states in F25 and two in F26. Results: Based on its width and decay properties, the first unbound state in F25, at the relative energy of 49(9) keV, is proposed to be a Jπ=1/2- arising from a p1/2 proton-hole state. In F26, the first resonance at 323(33) keV is proposed to be the Jπ=31+ member of the Jπ=11+-41+ multiplet. Energies of observed states in F25,26 have been compared to calculations using the independent-particle shell model, a phenomenological shell model, and the ab initio valence-space in-medium similarity renormalization group method. Conclusions: The deduced effective proton-neutron interaction is weakened by about 30-40% in comparison to the models, pointing to the need for implementing the role of the continuum in theoretical descriptions or to a wrong determination of the atomic mass of F26

Quasi-free (p,pN) scattering of light neutron-rich nuclei around N = 14

Background: For many years, quasifree scattering reactions in direct kinematics have been extensively used to study the structure of stable nuclei, demonstrating the potential of this approach. The RB3 collaboration has performed a pilot experiment to study quasifree scattering reactions in inverse kinematics for a stable C12 beam. The results from that experiment constitute the first quasifree scattering results in inverse and complete kinematics. This technique has lately been extended to exotic beams to investigate the evolution of shell structure, which has attracted much interest due to changes in shell structure if the number of protons or neutrons is varied. Purpose: In this work we investigate for the first time the quasifree scattering reactions (p,pn) and (p,2p) simultaneously for the same projectile in inverse and complete kinematics for radioactive beams with the aim to study the evolution of single-particle properties from N=14 to N=15. Method: The structure of the projectiles O23, O22, and N21 has been studied simultaneously via (p,pn) and (p,2p) quasifree knockout reactions in complete inverse kinematics, allowing the investigation of proton and neutron structure at the same time. The experimental data were collected at the R3B-LAND setup at GSI at beam energies of around 400 MeV/u. Two key observables have been studied to shed light on the structure of those nuclei: the inclusive cross sections and the corresponding momentum distributions. Conclusions: The knockout reactions (p,pn) and (p,2p) with radioactive beams in inverse kinematics have provided important and complementary information for the study of shell evolution and structure. For the (p,pn) channels, indications of a change in the structure of these nuclei moving from N=14 to N=15 have been observed, i.e., from the 0d5/2 shell to the 1s1/2. This supports previous observations of a subshell closure at N=14 for neutron-rich oxygen isotopes and its weakening for the nitrogen isotopes

Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model

Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs

Signaling mediated by the NF-κB sub-units NF-κB1, NF-κB2 and c-Rel differentially regulate Helicobacter felis-induced gastric carcinogenesis in C57BL/6 mice

The classical nuclear factor-kappaB (NF-κB) signaling pathway has been shown to be important in a number of models of inflammation-associated cancer. In a mouse model of Helicobacter-induced gastric cancer, impairment of classical NF-κB signaling in the gastric epithelium led to the development of increased preneoplastic pathology, however the role of specific NF-κB proteins in Helicobacter-associated gastric cancer development remains poorly understood. To investigate this C57BL/6, Nfkb1−/−, Nfkb2−/− and c-Rel−/− mice were infected with Helicobacter felis for 6 weeks or 12 months. Bacterial colonization, gastric atrophy and preneoplastic changes were assessed histologically and cytokine expression was assessed by qPCR. Nfkb1−/− mice developed spontaneous gastric atrophy when maintained for 12 months in conventional animal house conditions. They also developed more pronounced gastric atrophy after short-term H. felis colonization with a similar extent of preneoplasia to wild-type (WT) mice after 12 months. c-Rel−/− mice developed a similar degree of gastric atrophy to WT mice; 3 of 6 of these animals also developed lymphoproliferative lesions after 12 months of infection. Nfkb2−/− mice developed minimal gastric epithelial pathology even 12 months after H. felis infection. These findings demonstrate that NF-κB1- and NF-κB2-mediated signaling pathways differentially regulate the epithelial consequences of H. felis infection in the stomach, while c-Rel-mediated signaling also appears to modulate the risk of lymphomagenesis in gastric mucosa-associated lymphoid tissue

Structure of 13Be studied in proton knockout from 14B

International audienceThe neutron-unbound isotope Be13 has been studied in several experiments using different reactions, different projectile energies, and different experimental setups. There is, however, no real consensus in the interpretation of the data, in particular concerning the structure of the low-lying excited states. Gathering new experimental information, which may reveal the Be13 structure, is a challenge, particularly in light of its bridging role between Be12, where the N=8 neutron shell breaks down, and the Borromean halo nucleus Be14. The purpose of the present study is to investigate the role of bound excited states in the reaction product Be12 after proton knockout from B14, by measuring coincidences between Be12, neutrons, and γ rays originating from de-excitation of states fed by neutron decay of Be13. The Be13 isotopes were produced in proton knockout from a 400 MeV/nucleon B14 beam impinging on a CH2 target. The Be12-n relative-energy spectrum dσ/dEfn was obtained from coincidences between Be12(g.s.) and a neutron, and also as threefold coincidences by adding γ rays, from the de-excitation of excited states in Be12. Neutron decay from the first 5/2+ state in Be13 to the 2+ state in Be12  at 2.11 MeV is confirmed. An energy independence of the proton-knockout mechanism is found from a comparison with data taken with a 35 MeV/nucleon B14 beam. A low-lying p-wave resonance in Be13(1/2−) is confirmed by comparing proton- and neutron-knockout data from B14 and Be14