We show for the first time that necroptosis is strongly associated with intestinal inflammation in children with IBD and contributes to strengthen the inflammatory process. We believe that RIP3 and MLKL could represent attractive targets for the management of human IBD.

The amyloid-␤ (A␤) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-␤ precursor protein (APP) through consecutive proteolytic cleavages by ␤-site APP-cleaving enzyme and ␥-secretase. Unexpectedly ␥-secretase inhibitors can increase the secretion of A␤ peptides under some circumstances. This "A␤ rise" phenomenon, the same inhibitor causing an increase in A␤ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the A␤ rise depends on the ␤-secretase-derived C-terminal fragment of APP (␤CTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of A␤, known as "p3," formed by ␣-secretase cleavage, did not exhibit a rise. In addition to the A␤ rise, low ␤CTF or C99 expression decreased ␥-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, ␥-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of A␤ under conditions of low substrate expression. The A␤ rise was also observed in rat brain after dosing with the ␥-secretase inhibitor BMS-299897. The A␤ rise and potency shift are therefore relevant factors in the development of ␥-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the A␤ rise, including the "incomplete processing" and endocytic models, are discussed.Evidence suggests that the amyloid-␤ (A␤) 9 peptide plays a key role in Alzheimer disease. A␤ is generated by proteolytic processing of the amyloid-␤ precursor protein (APP) through consecutive cleavages by the ␤-site APP-cleaving enzyme (BACE) and ␥-secretase. APP is cleaved by BACE to form a ␤-secretase-derived C-terminal fragment of APP (␤CTF), which undergoes further cleavage by ␥-secretase to form A␤. In addition, APP is cleaved by ␣-secretase to form ␣CTF, which undergoes ␥-secretase cleavage to produce an N-terminally truncated form of A␤ known as "p3." Using the conventional amino acid numbering of A␤, BACE cleavage leads to A␤ peptides with N-terminal ends at positions 1 and 11, whereas ␣-secretase leads to p3 peptides with an N-terminal end at position 17. Cleavage of ␤CTF and ␣CTF by ␥-secretase produces a mixture of different C termini in the resulting A␤ and p3 peptides. For example, the predominant ␥-secretase cleavage of ␤CTFs at position 40 produces A␤-(1-40) and A␤-(11-40), whereas other ␥-secretase cleavage sites produce a range of less abundant A␤ peptides, such as the disease-associated A␤-(1-42) (1, 2).

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

The nucleotide-binding oligomerization domain (NOD) protein, NOD2, belonging to the intracellular NOD-like receptor family, detects conserved motifs in bacterial peptidoglycan and promotes their clearance through activation of a proinflammatory transcriptional program and other innate immune pathways, including autophagy and endoplasmic reticulum stress. An inactive form due to mutations or a constitutive high expression of NOD2 is associated with several inflammatory diseases, suggesting that balanced NOD2 signaling is critical for the maintenance of immune homeostasis. In this review, we discuss recent developments about the pathway and mechanisms of regulation of NOD2 and illustrate the principal functions of the gene, with particular emphasis on its central role in maintaining the equilibrium between intestinal microbiota and host immune responses to control inflammation. Furthermore, we survey recent studies illustrating the role of NOD2 in several inflammatory diseases, in particular, inflammatory bowel disease, of which it is the main susceptibility gene.

4-(1,3-Dimethoxyprop-2-ylamine)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine (DMP696) is a highly selective and potent, nonpeptide corticotropin-releasing factor 1 (CRF 1 ) antagonist. In this study, we measured in vivo CRF 1 receptor occupancy of DMP696 by using ex vivo ligand binding and quantitative autoradiography and explored the relationship of receptor occupancy with plasma and brain exposure and behavioral efficacy. In vitro affinity (IC 50 ) of DMP696 to brain CRF 1 receptors measured using the brain section binding autoradiography in this study is similar to that assessed using homogenized cell membrane assays previously. The ex vivo binding assay was validated by demonstrating that potential underestimation of receptor occupancy with this procedure could be minimized by identifying an appropriate in vitro incubation time (40 min) based upon the dissociation kinetics of DMP696. Orally administrated DMP696 dose dependently occupied CRF 1 receptors in the brain, with ϳ60% occupancy at 3 mg/kg. In the defensive withdrawal test of anxiety, this dose of DMP696 produced approximately 50% reduction in the exit latency. The time course of plasma and brain drug levels paralleled that of receptor occupancy, with peak exposure at 90 min after dosing. The plasma-free concentration of DMP696 corresponding to 50% CRF 1 receptor occupancy (in vivo IC 50 , 1.22 nM) was similar to the in vitro IC 50 (ϳ1.0 nM). Brain concentrations of DMP696 were over 150-fold higher than the plasma-free levels. In conclusion, doses of DMP696 occupying over 50% brain CRF 1 receptors are consistent with doses producing anxiolytic efficacy in the defense withdrawal test of anxiety, and the IC 50 value estimated in vivo based on plasmafree drug concentrations is consistent with the in vitro IC 50 value.Corticotropin releasing factor (CRF), a 41-amino acid peptide, plays a pivotal role in the behavioral, endocrine, immune, and autonomic responses of the body to stress (Owens and Nemeroff, 1991). In addition to the hypothalamic paraventricular nucleus where it was originally identified, CRF is also widely distributed across brain regions (Chalmers et al., 1996;Heinrichs and De Souza, 1999;Gilligan et al., 2000a). The physiological functions of CRF are mediated by at least two G-protein coupled receptors, CRF 1 and CRF 2 (including splice variants CRF 2␣ , CRF 2␤ , CRF 2␥ ), both of which are linked to adenylyl cyclase activation but have distinct brain distributions. CRF 1 receptors are widespread in the cortex, limbic system, cerebellum, and pituitary, whereas CRF 2 receptors are dominant in subcortical areas including the lateral septum (CRF 2␣ ), ventromedial hypothalamus (CRF 2␣ ), and choroid plexus (CRF 2␤ ) (De Souza, 1987;Chalmers et al., 1995;Primus et al., 1997;Rominger et al., 1998).Increasing evidence suggests that the CRF system is involved in pathophysiology of anxiety disorders (Heinrichs and De Souza, 1999;Gilligan et al., 2000a). Intracerebroventricular administration of CRF induces stress beha...

It was shown for the first time in our study that HMGB1 is secreted by human inflamed intestinal tissues and abundantly found in the stools of IBD patients. Hence, it can be considered as a novel marker for intestinal inflammation. We can also suggest that the presence of HMGB1 in large amounts in the fecal stream of IBD patients is mainly due to active secretion of the protein stored in the nucleus rather than a "de novo" synthesis.

This is the first study showing the presence of adhesive-invasive bacteria strains in the inflamed tissues of children with IBD. Collective features of these strains indicate that they belong to the AIEC spectrum, suggesting their possible role in disease pathogenesis.

Pediatric UC is extensive and severe at the diagnosis, with an overall high rate of disease extension at the follow-up. Endoscopic atypical features are common in young children. The colectomy rate is related to the location and severity of the disease at the diagnosis.