Fecal calprotectin and lactoferrin determination may be useful in predicting impending clinical relapse-especially during the following 3 months-in both CD and UC patients.

In IBD patients, AZA or MP treatment induces abnormality of LTs in a relatively high proportion of the cases, but the development of true hepatotoxicity/liver injury is exceptional. Moreover, most of the cases of thiopurine-induced hepatotoxicity in IBD patients are mild, and the abnormalities in LTs spontaneously return to normal values despite AZA/MP being maintained, therapy withdrawal being necessary in only approximately 4% of the patients.

T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubuleorganizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1β phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1β phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1β re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1β regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization.

Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor B (NF-B) in several tissues including liver and macrophages. Maximal activation was observed 1 h after treatment but declined at 3 and 6 h. The levels of IB␣ and IB␤ were analyzed during this period in an attempt to correlate NF-B activity with IB resynthesis. Degradation of IB␣ was very rapid and was followed by recovery 1 h after LPS administration. IB␤ degradation, which has been associated with persistent NF-B activation, was complete at 1 h. However, a rapid recovery of IB␤ in these tissues was observed at 3 h in parallel with the abrogation of NF-B activity. Immunolocalization of newly synthesized IB␤ by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of IB␤ by Western blot using high resolution polyacrylamide gel electrophoresis showed the presence of two bands in cytosolic extracts of LPS-treated macrophages at 3 h, but only one band with the same mobility as the control was detected at 6 h. Moreover, treatment of extracts of resynthesized IB␤ with alkaline phosphatase resulted in the accumulation of the protein of slightly higher electrophoretic mobility, indicating the prevalence of a rapid phosphorylation of the newly synthesized IB␤. At the mRNA level, up-regulation of IB␤ was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor ␣, but not interleukin-1 or interferon-␥, promoted an important degradation of IB␤ followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS-and tumor necrosis factor ␣-specific pathways involved in a rapid IB␤ degradation and resynthesis and might explain the transient period of activation of NF-B in these tissues upon stimulation with these factors. This rapid control of NF-B function may contribute to the attenuation of the inflammatory response of these cells. Nuclear factor B (NF-B)1 participates in the regulation of the expression of multiple immediate early genes involved in the immune, acute phase, and inflammatory responses (1). NF-B is a heterodimer of proteins of the Rel family of transcription factors. In mammalian cells, they include p65 (Rel A), Rel B, the proto-oncogene c-Rel, p50/p105 (NF-B1), and p52/ p100 (NF-B2) (1, 2). NF-B proteins are constitutively present in the cell, but they are retained in the cytoplasm associated with inhibitory proteins known as IB (3, 4). Activated NF⅐B complexes, typically composed of p50 and p65, are translocated to the nucleus in response to mitogens, cytokines (IL-1␤, IL-2, and TNF-␣), and bacterial lipopolysaccharide and lipopeptides (1, 5-8). Activation of NF-B appears to require phosphorylation and degradation of the IB proteins, thereby allowing the rapid translocation of NF-B from the cytoplasm to the nucleus (7, 9 -11). Several IB proteins have been characterized including IB␣, IB␤, IB␥, and the candidate oncogene 4,12). All these proteins share a characteristic ankyrin repeat motif,...

Aliment Pharmacol Ther 2011; 34: 544–554 Summary Background  Low thiopurine‐methyl‐transferase (TPMT) activity and high 6‐thioguanine‐nucleotide (6TGN) concentrations have been linked to therapeutic success in inflammatory bowel disease patients treated with thiopurines; however, this has not been implemented in clinical practice. Aim  To identify a therapeutic threshold value for TPMT or 6TGN concentrations, and their capability to predict treatment safety and efficacy. Methods  Prospective multicentre study including steroid‐resistant/dependent patients starting thiopurines. The TPMT activity was determined at inclusion (>5 U/mL required). Azathioprine metabolites [6TGN, 6‐methyl‐mercaptopurine ribonucleotides (6MMP), and 6TGN/6MMP and 6TGN/TPMT ratios] were periodically monitored during steroid tapering and after withdrawal for 6 months or until a new flare occurred. Results  A total of 113 patients were analysed (62% clinical response). Areas under the receiver operating characteristic (ROC) curve (AUC) relating clinical response and metabolite levels at 2, 4 and 6 months after steroid withdrawal were less than 0.7. The AUCs relating final response and initial TPMT activity or metabolite concentrations at 2, 4, 8 and 16 weeks after starting thiopurines were less than 0.7. No cut‐off point with worthwhile sensitivity/specificity was found. Eight (7%) patients developed thiopurine‐related toxicity that could not be linked to TPMT activity or 6TGN levels. Conclusions  Our results do not support determination of TPMT activity or 6TGN concentrations to predict treatment outcome, and no useful serum metabolites threshold value to adjust the drug’s dose was identified.

A concerted activation of transcription factors involved in the transactivation of type II NO synthase (iNOS) gene occurred after partial hepatectomy (PH), resulting in the transient expression of iNOS. The corresponding mRNA and protein levels of iNOS reached a maximum at 4 h and 8 h post-PH respectively. This induction was preceded by an early and transient activation of nuclear factor kappaB (NF-kappaB). Analysis of the kappaB inhibitory (I) proteins showed an important role for IkappaBalpha in the process of NF-kappaB activation, whereas the contribution of IkappaBbeta was less evident. Interferon regulatory factor 1, which has been described as an important activator of iNOS expression, was up-regulated after PH but failed to bind to the corresponding DNA binding sequences of the iNOS promoter. The transcriptional control of iNOS after PH, was compared with the events associated with the hepatic expression of this enzyme in animals challenged with lipopolysaccharide, showing a differential pattern of transcription-factor activation and IkappaB degradation between both models. Transfection of hepatoma cell lines with iNOS promoter constructs, followed by stimulation with post-PH sera, revealed the requirement of NF-kappaB activation for iNOS expression. These data suggest that there is an important role for the restricted NF-kappaB activation in the temporal pattern of iNOS expression in regenerating liver.

Incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS), S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-

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