Transcription factor CTF1 acts as a chromatin domain boundary that shields human telomeric genes from silencing.

Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.

Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.

By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.

p53-induced protein with a death domain (PIDD) isoforms differentially activate nuclear factor-kappaB and caspase-2 in response to genotoxic stress

Cells respond to DNA damage in a complex way and the fate of damaged cells depends on the balance between pro- and antiapoptotic signals. This is of crucial importance in cancer as genotoxic stress is implied both in oncogenesis and in classical tumor therapies. p53-induced protein with a death domain (PIDD), initially described as a p53-inducible gene, is one of the molecular switches able to activate a survival or apoptotic program. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and we describe here a third isoform. These three isoforms are differentially expressed in tissues and cell lines. Genotoxic stress only affects PIDD isoform 3 mRNA levels, whereas isoforms 1 and 2 mRNA levels remain unchanged. All isoforms are capable of activating nuclear factor-kappaB in response to genotoxic stress, but only isoform 1 interacts with RIP-associated ICH-1/CED-3 homologous protein with a death domain and activates caspase-2. Isoform 2 counteracts the pro-apoptotic function of isoform 1, whereas isoform 3 enhances it. Thus, the differential splicing of PIDD mRNA leads to the formation of at least three proteins with antagonizing/agonizing functions, thereby regulating cell fate in response to DNA damage

Brain-derived neurotrophic factor-like immunoreactivity is localized mainly in small sensory neurons of rat dorsal root ganglia.

Brain-derived neurotrophic factor (BDNF) is a protein capable of supporting the survival and fiber outgrowth of peripheral sensory neurons. It has been argued that histological detection of BDNF has proven difficult because of its low molecular weight and relatively low expression. In the present study we report that rapid removal of dorsal root ganglia (DRG) from the rat, followed by rapid freezing and appropriate fixation with cold acetone, preserves BDNF in situ without altering protein antigenicity. Under these conditions, specific BDNF-like immunoreactivity was detected in DRG both in vivo and in vitro. During DRG development in vivo, BDNF-like immunoreactivity (BDNF-LI) was observed only in a subset of sensory neurons. BDNF-LI was confined to small neurons, after neurons became morphologically distinct on the basis of size. BDNF-L immunoprecipitate was detected only in neuronal cells, and not in satellite or Schwann cells. While in vivo BDNF localization was restricted to small neurons, practically all neurons in DRG cell culture displayed BDNF-LI. Small or large primary afferent neurons exhibited a faint but clear BDNF-LI during the whole life span of cultures. Again, non-neuronal cells were devoid of BDNF-LI. In conclusion, in DRG in vivo, specific BDNF-LI was confined to small B sensory neurons. In contrast, all DRG sensory neurons displayed BDNF-LI in vitro. The finding that BDNF expressed in all DRG neurons in vitro but not in vivo suggests that BDNF expression may be modulated by environmental factors.

Pretherapeutic gamma-glutamyltransferase is an independent prognostic factor for patients with renal cell carcinoma.

BACKGROUND: Gamma-glutamyltransferase (GGT) regulates apoptotic balance and promotes cancer progression and invasion. Higher pretherapeutic GGT serum levels have been associated with worse outcomes in various malignancies, but there are no data for renal cell carcinoma (RCC). METHODS: Pretherapeutic GGT serum levels and clinicopathological parameters were retrospectively evaluated in 921 consecutive RCC patients treated with nephrectomy at a single institution between 1998 and 2013. Gamma-glutamyltransferase was analysed as continuous and categorical variable. Associations with RCC-specific survival were assessed with Cox proportional hazards models. Discrimination was measured with the C-index. Decision-curve analysis was used to evaluate the clinical net benefit. The median postoperative follow-up was 45 months. RESULTS: Median pretherapeutic serum GGT level was 25 U l(-1). Gamma-glutamyltransferase levels increased with advancing T (P<0.001), N (P=0.006) and M stages (P<0.001), higher grades (P<0.001), and presence of tumour necrosis (P<0.001). An increase of GGT by 10 U l(-1) was associated with an increase in the risk of death from RCC by 4% (HR 1.04, P<0.001). Based on recursive partitioning-based survival tree analysis, we defined four prognostic categories of GGT: normal low (<17.5 U l(-1)), normal high (17.5 to <34.5 U l(-1)), elevated (34.5 to <181.5 U l(-1)), and highly elevated (⩾181.5 U l(-1)). In multivariable analyses that adjusted for the effect of standard features, both continuously and categorically coded GGT were independent prognostic factors. Adding GGT to a model that included standard features increased the discrimination by 0.9% to 1.8% and improved the clinical net benefit. CONCLUSIONS: Pretherapeutic serum GGT is a novel and independent prognostic factor for patients with RCC. Stratifying patients into prognostic subgroups according to GGT may be used for patient counselling, tailoring surveillance, individualised treatment planning, and clinical trial design.

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.

The Transcription Factor NFATc2 Controls Apoptosis and Activation of T Cells by IL-6 in Colitis

The nuclear factor of activated T cells (NFAT) family of transcription factors controls calcium signaling in T lymphocytes. In this study, we have identified a crucial regulatory role of the transcription factor NFATc2 in T cell-dependent experimental colitis. Similar to ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-induced chronic intestinal inflammation. Furthermore, NFATc2 deficiency suppressed colitis induced by oxazolone administration. This finding was associated with enhanced T cell apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by mucosal T lymphocytes. Further studies using knockout mice showed that IL-6, rather than IL-23 and -17, are essential for oxazolone colitis induction. Administration of hyper-IL-6 blocked the protective effects of NFATc2 deficiency in experimental colitis, suggesting that IL-6 signal transduction plays a major pathogenic role in vivo. Finally, adoptive transfer of IL-6 and wild-type T cells demonstrated that oxazolone colitis is critically dependent on IL-6 production by T cells. Collectively, these results define a unique regulatory role for NFATc2 in colitis by controlling mucosal T cell activation in an IL-6-dependent manner. NFATc2 in T cells thus emerges as a potentially new therapeutic target for inflammatory bowel diseases.

Macrophage migration inhibitory factor deficiency leads to age-dependent impairment of glucose homeostasis in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many cells and tissues including pancreatic beta-cells, liver, skeletal muscle, and adipocytes. This study investigates the potential role of MIF in carbohydrate homeostasis in a physiological setting outside of severe inflammation, utilizing Mif knockout (MIF-/-) mice. Compared with wild-type (WT) mice, MIF-/- mice had a lower body weight, from birth until 4 months of age, but subsequently gained weight faster, resulting in a higher body weight at 12 months of age. The lower weight in young mice was related to a higher energy expenditure, and the higher weight in older mice was related to an increased food intake and a higher fat mass. Fasting blood insulin level was higher in MIF-/- mice compared with WT mice at any age. After i.p. glucose injection, the elevation of blood insulin level was higher in MIF-/- mice compared with WT mice, at 2 months of age, but was lower in 12-month-old MIF-/- mice. As a result, the glucose clearance during intraperitoneal glucose tolerance tests was higher in MIF-/- mice compared with WT mice until 4 months of age, and was lower in 12-month-old MIF-/- mice. Insulin resistance was estimated (euglycemic-hyperinsulinemic clamp tests), and the phosphorylation activity of AKT was similar in MIF-/- mice and WT mice. In conclusion, this mouse model provides evidence for the role of MIF in the control of glucose homeostasis.

Recommendations for the treatment of Crohn's disease with tumor necrosis factor antagonists: an expert consensus report.

Background: Symptom relief is the traditional treatment goal in Crohn's disease (CD). New goals including mucosal healing and bowel preservation are now achievable with tumor necrosis factor (TNF) antagonists. Infliximab and adalimumab are approved as second-line treatments for severe, active CD. Certolizumab pegol is approved only in the U.S. and Switzerland as second-line treatment for moderate-to-severe, active CD. Data from trials of infliximab suggest that high-risk patients and patients with active inflammation (CRP elevation and/or ileocolonic ulcers) may benefit from earlier use of this drug.

Studies of the renal effects of angiotensin II receptor blockade: the confounding factor of acute water loading on the action of vasoactive systems.

The acute renal tubular effects of two pharmacologically distinct angiotensin II receptor antagonists have been evaluated in normotensive volunteers on various salt diets. In the first study, the renal response to a single oral dose of losartan (100 mg) was assessed in subjects on a low (50 mmol Na/d) and on a high (200 mmol Na/d) salt intake. In a second protocol, the renal effects of 50 mg irbesartan were investigated in subjects receiving a 100 mmol Na/d diet. Both angiotensin II antagonists induced a significant increase in urinary sodium excretion. With losartan, a modest, transient increase in urinary potassium and a significant increase in uric acid excretion were found. In contrast, no change in potassium and uric acid excretions were observed with irbesartan, suggesting that the effects of losartan on potassium and uric acid are due to the intrinsic pharmacologic properties of losartan rather than to the specific blockade of renal angiotensin II receptors. Assessment of segmental sodium reabsorption using lithium as a marker of proximal tubular reabsorption demonstrated a decreased distal reabsorption of sodium with both antagonists. A direct proximal tubular natriuretic effect of the angiotensin II antagonist could be demonstrated only with irbesartan. This apparent discrepancy allowed us to reveal the importance of acute water loading as a possible confounding factor in renal studies. The results of the present analysis show that acute water loading per se may enhance renal sodium excretion and hence modify the level of activity of the renin-angiotensin system expected from a given sodium diet. Since acute water loading is a common practice in clinical renal studies, this confounding factor should be taken into account when investigating the renal effects of vasoactive systems.

An Adeno-Associated Virus-Based Intracellular Sensor of Pathological Nuclear Factor-κB Activation for Disease-Inducible Gene Transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Gene-frequencies of coagulation-factor XIIIb in Switzerland

Coagulation factor XIIIB polymorphism was studied by agarose isoelectric focusing and immunofixation in 592 unrelated individuals from Switzerland. The gene frequencies observed were: FXIIIB*1 = 0.769, FXIIIB*3 = 0.139, FXIIIB*2 = 0.085, FXIIIB*4 = 0.007.

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

OBJECTIVE: Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS: First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS: These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.