Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

Prognostic value of postoperative carcinoembryonic antigen concentration and extent of invasion of resection margins after hepatic resection for colorectal metastases.

OBJECTIVE: To evaluate the prognostic value of postoperative concentration of carcinoembryonic antigen (CEA) and extent of surgical margins after resection of liver metastases from colorectal cancer. DESIGN: Retrospective study. SETTING: Teaching hospital, Switzerland. SUBJECTS: 49 patients with hepatic metastases after primary colorectal cancer. INTERVENTIONS: Resection of hepatic metastases MAIN OUTCOME MEASURES: Assessment of prognostic value of variables by univariate and multivariate analysis. RESULTS: Median survival was 24 months (range 5-86 months). Resection margins were clear (> 1-cm) in 10, close (< 1-cm) in 25 and invaded in 9 patients. On univariate analysis, a postoperative concentration of CEA of <4ng/ml was correlated with prolonged survival (p < 0.001), but the width of the resection margin was not of prognostic importance. There was no correlation between width of resection margins and postoperative concentration of CEA (p = 0.5). On multivariate analysis, postoperative concentrations of CEA of 4 ng/ml or more were associated with increased risk of death (relative risk 7.3; 95% confidence interval (CI) 2.8-18.7, p < 0.001). CONCLUSION: Postoperative CEA offers better prognostic discrimination than the width of resection margins after resection of liver metastases from colorectal tumours. Some patients with invaded resection margins did survive for 3 years, but no patient did whose CEA concentration was 4 ng/ml or more. The definition of a potentially curative hepatic resection should include a postoperative CEA concentration of <4 ng/ml (within the reference range).

Can the trabecular bone score be considered as a major clinical risk factor of osteoporotic fractures? A meta-like analysis.

To have an added value over BMD, a CRF of osteoporotic fracture must be predictable of the fracture, independent of BMD, reversible and quantifiable. Many major recognized CRF exist. Out of these factors many of them are indirect factor of bone quality. TBS predicts fracture independently of BMD as demonstrated from previous studies. The aim of the study is to verify if TBS can be considered as a major CRF of osteoporotic fracture. Existing validated datasets of Caucasian women were analyzed. These datasets stem from different studies performed by the authors of this report or provided to our group. However, the level of evidence of these studies will vary. Thus, the different datasets were weighted differently according to their design. This meta-like analysis involves more than 32000 women (≥50years) with 2000 osteoporotic fractures from two prospective studies (OFELY&MANITOBA) and 7 cross-sectional studies. Weighted relative risk (RR) for TBS was expressed for each decrease of one standard deviation as well as per tertile difference (TBS=1.300 and 1.200) and compared with those obtained for the major CRF included in FRAX®. Overall TBS RR obtained (adjusted for age) was 1.79 [95%CI-1.37-2.37]. For all women combined, RR for fracture for the lowest compared with the middle TBS tertile was 1.55[1.46-1.68] and for the lowest compared with the highest TBS tertile was 2.8[2.70-3.00]. TBS is comparable to most of the major CRF and thus could be used as one of them. Further studies have to be conducted to confirm these first findings.

The distribution of congenital anomalies within the VACTERL association among tumor necrosis factor antagonist-exposed pregnancies is similar to the general population.

OBJECTIVE: To compare the distribution of congenital anomalies within the VACTERL association (vertebral defects, anal atresia, cardiac, tracheoesophageal, renal, and limb abnormalities) between patients exposed to tumor necrosis factor-α (TNF-α) antagonist and the general population. METHODS: Analysis for comparison of proportional differences to a previous publication between anomaly subgroups, according to subgroup definitions of the European Surveillance of Congenital Anomalies (EUROCAT), a population-based database. RESULTS: Most EUROCAT subgroups belonging to the VACTERL association contained only one or 2 records of TNF-α antagonist exposure, so comparison of proportions was imprecise. Only the category "limb abnormalities" showed a significantly higher proportion in the general population. CONCLUSION: The high number of congenital anomalies belonging to the VACTERL association from a report of pregnancies exposed to TNF-α antagonists could not be confirmed using a population-based congenital anomaly database.

Maintenance of hepatic nuclear factor 6 in postnatal islets impairs terminal differentiation and function of beta-cells.

The Onecut homeodomain transcription factor hepatic nuclear factor 6 (Hnf6) is necessary for proper development of islet beta-cells. Hnf6 is initially expressed throughout the pancreatic epithelium but is downregulated in endocrine cells at late gestation and is not expressed in postnatal islets. Transgenic mice in which Hnf6 expression is maintained in postnatal islets (pdx1(PB)Hnf6) show overt diabetes and impaired glucose-stimulated insulin secretion (GSIS) at weaning. We now define the mechanism whereby maintenance of Hnf6 expression postnatally leads to beta-cell dysfunction. We provide evidence that continued expression of Hnf6 impairs GSIS by altering insulin granule biosynthesis, resulting in a reduced response to secretagogues. Sustained expression of Hnf6 also results in downregulation of the beta-cell-specific transcription factor MafA and a decrease in total pancreatic insulin. These results suggest that downregulation of Hnf6 expression in beta-cells during development is essential to achieve a mature, glucose-responsive beta-cell.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Prognostic accuracy of cerebral blood flow measurement by perfusion computed tomography, at the time of emergency room admission, in acute stroke patients.

The purpose of this study was to determine the prognostic accuracy of perfusion computed tomography (CT), performed at the time of emergency room admission, in acute stroke patients. Accuracy was determined by comparison of perfusion CT with delayed magnetic resonance (MR) and by monitoring the evolution of each patient's clinical condition. Twenty-two acute stroke patients underwent perfusion CT covering four contiguous 10mm slices on admission, as well as delayed MR, performed after a median interval of 3 days after emergency room admission. Eight were treated with thrombolytic agents. Infarct size on the admission perfusion CT was compared with that on the delayed diffusion-weighted (DWI)-MR, chosen as the gold standard. Delayed magnetic resonance angiography and perfusion-weighted MR were used to detect recanalization. A potential recuperation ratio, defined as PRR = penumbra size/(penumbra size + infarct size) on the admission perfusion CT, was compared with the evolution in each patient's clinical condition, defined by the National Institutes of Health Stroke Scale (NIHSS). In the 8 cases with arterial recanalization, the size of the cerebral infarct on the delayed DWI-MR was larger than or equal to that of the infarct on the admission perfusion CT, but smaller than or equal to that of the ischemic lesion on the admission perfusion CT; and the observed improvement in the NIHSS correlated with the PRR (correlation coefficient = 0.833). In the 14 cases with persistent arterial occlusion, infarct size on the delayed DWI-MR correlated with ischemic lesion size on the admission perfusion CT (r = 0.958). In all 22 patients, the admission NIHSS correlated with the size of the ischemic area on the admission perfusion CT (r = 0.627). Based on these findings, we conclude that perfusion CT allows the accurate prediction of the final infarct size and the evaluation of clinical prognosis for acute stroke patients at the time of emergency evaluation. It may also provide information about the extent of the penumbra. Perfusion CT could therefore be a valuable tool in the early management of acute stroke patients.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis.

Macrophages are essential effector cells of innate immunity that play a pivotal role in the recognition and elimination of invasive microorganisms. Mediators released by activated macrophages orchestrate innate and adaptive immune host responses. The cytokine macrophage migration inhibitory factor (MIF) is an integral mediator of the innate immune system. Monocytes and macrophages constitutively express large amounts of MIF, which is rapidly released after exposure to bacterial toxins and cytokines. MIF exerts potent proinflammatory activities and is an important cytokine of septic shock. Recent investigations of the mechanisms by which MIF regulates innate immune responses to endotoxin and gram-negative bacteria indicate that MIF acts by modulating the expression of Toll-like receptor 4, the signal-transducing molecule of the lipopolysaccharide receptor complex. Given its role in innate immune responses to bacterial infections, MIF is a novel target for therapeutic intervention in patients with septic shock.

VeriStrat(®) has a prognostic value for patients with advanced non-small cell lung cancer treated with erlotinib and bevacizumab in the first line: Pooled analysis of SAKK19/05 and NTR528.

BACKGROUND: VeriStrat(®) is a serum proteomic test used to determine whether patients with advanced non-small cell lung cancer (NSCLC) who have already received chemotherapy are likely to have good or poor outcomes from treatment with gefitinib or erlotinib. The main objective of our retrospective study was to evaluate the role of VS as a marker of overall survival (OS) in patients treated with erlotinib and bevacizumab in the first line. PATIENTS AND METHODS: Patients were pooled from two phase II trials (SAKK19/05 and NTR528). For survival analyses, a log-rank test was used to determine if there was a statistically significant difference between groups. The hazard ratio (HR) of any separation was assessed using Cox proportional hazards models. RESULTS: 117 patients were analyzed. VeriStrat classified patients into two groups which had a statistically significant difference in duration of OS (p=0.0027, HR=0.480, 95% confidence interval: 0.294-0.784). CONCLUSION: VeriStrat has a prognostic role in patients with advanced, nonsquamous NSCLC treated with erlotinib and bevacizumab in the first line. Further work is needed to study the predictive role of VeriStrat for erlotinib and bevacizumab in chemotherapy-untreated patients.