Transcriptional repression of NFKBIA triggers constitutive IKK- and proteasome-independent p65/RelA activation in senescence.

The IκB kinase (IKK)-NF-κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double-strand breaks elicit two subsequent phases of NF-κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first-phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re-expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF-κB family member p65/RelA, in part mediated by GSK3ß, results in transcriptional silencing of NFKBIA and IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.

The IκB kinase complex is a regulator of mRNA stability.

The IκB kinase (IKK) is considered to control gene expression primarily through activation of the transcription factor NF-κB. However, we show here that IKK additionally regulates gene expression on post-transcriptional level. IKK interacted with several mRNA-binding proteins, including a Processing (P) body scaffold protein, termed enhancer of decapping 4 (EDC4). IKK bound to and phosphorylated EDC4 in a stimulus-sensitive manner, leading to co-recruitment of P body components, mRNA decapping proteins 1a and 2 (DCP1a and DCP2) and to an increase in P body numbers. Using RNA sequencing, we identified scores of transcripts whose stability was regulated via the IKK-EDC4 axis. Strikingly, in the absence of stimulus, IKK-EDC4 promoted destabilization of pro-inflammatory cytokines and regulators of apoptosis. Our findings expand the reach of IKK beyond its canonical role as a regulator of transcription.

A cytoplasmic ATM-TRAF6-cIAP1 module links nuclear DNA damage signaling to ubiquitin-mediated NF-κB activation.

As part of the genotoxic stress response, cells activate the transcription factor NF-κB. The DNA strand break sensor poly(ADP-ribose)-polymerase-1 (PARP-1) and the kinase ataxia telangiectasia mutated (ATM) act as proximal signal mediators. PARP-1 assembles a nucleoplasmic signalosome, which triggers PIASy-mediated IKKγ SUMOylation. ATM-dependent IKKγ phosphorylation and subsequent ubiquitination were implicated to activate the cytoplasmic IκB kinase (IKK) complex by unknown mechanisms. We show that activated ATM translocates in a calcium-dependent manner to cytosol and membrane fractions. Through a TRAF-binding motif, ATM activates TRAF6, resulting in Ubc13-mediated K63-linked polyubiquitin synthesis and cIAP1 recruitment. The ATM-TRAF6-cIAP1 module stimulates TAB2-dependent TAK1 phosphorylation. Both nuclear PARP-1- and cytoplasmic ATM-driven signaling branches converge at the IKK complex to catalyze monoubiquitination of IKKγ at K285. Our data indicate that exported SUMOylated IKKγ acts as a substrate. IKKγ monoubiquitination is a prerequisite for genotoxic IKK and NF-κB activation, but also promotes cytokine signaling.

A nuclear poly(ADP-ribose)-dependent signalosome confers DNA damage-induced IkappaB kinase activation.

Upon genotoxic stresses, cells activate IkappaB kinases (IKKs) and the transcription factor NF-kappaB to modulate apoptotic responses. The SUMO-1 ligase PIASy and the kinase ataxia talengiectasia mutated (ATM) have been implicated to SUMOylate and phosphorylate nuclear IKKgamma (NEMO) in a consecutive mode of action, which in turn results in activation of cytoplasmic IKK holocomplexes. However, the nuclear signals and scaffold structures that initiate IKKgamma recruitment and activation are unknown. Here, we show that poly(ADP-ribose)-polymerase-1 (PARP-1) is the DNA proximal regulator, which senses DNA strand breaks and, through poly(ADP-ribose) (PAR) synthesis, assembles IKKgamma, PIASy, and ATM in a dynamic manner. Signalosome formation involves direct protein-protein interactions and binding to ADP-ribose polymers through PAR binding motifs (PARBM). Activated PARP-1 and a PARBM in PIASy are required to trigger IKKgamma SUMOylation, which in turn permits IKK and NF-kappaB activation, as well as NF-kappaB-regulated resistance to apoptosis.

The IκB kinase complex in NF-κB regulation and beyond.

The IκB kinase (IKK) complex is the signal integration hub for NF-κB activation. Composed of two serine-threonine kinases (IKKα and IKKß) and the regulatory subunit NEMO (also known as IKKγ), the IKK complex integrates signals from all NF-κB activating stimuli to catalyze the phosphorylation of various IκB and NF-κB proteins, as well as of other substrates. Since the discovery of the IKK complex components about 15 years ago, tremendous progress has been made in the understanding of the IKK architecture and its integration into signaling networks. In addition to the control of NF-κB, IKK subunits mediate the crosstalk with other pathways, thereby extending the complexity of their biological function. This review summarizes recent advances in IKK biology and focuses on emerging aspects of IKK structure, regulation and function.

A New Application for Albumin Dialysis in Extracorporeal Organ Support: Characterization of a Putative Interaction Between Human Albumin and Proinflammatory Cytokines IL-6 and TNFα.

Albumin dialysis in extracorporeal organ support is often performed in the treatment of liver failure as it facilitates the removal of toxic components from the blood. Here, we describe a possible effect of albumin dialysis on proinflammatory cytokine levels in vitro. Initially, albumin samples were incubated with different amounts of cytokines and analyzed by enzyme-linked immunosorbent assay (ELISA). Analysis of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) levels indicated that increased concentrations of albumin reduce the measureable amount of the respective cytokines. This led to the hypothesis that the used proinflammatory cytokines may interact with albumin. Size exclusion chromatography of albumin spiked with cytokines was carried out using high-performance liquid chromatography analysis. The corresponding fractions were evaluated by immunoblotting. We detected albumin and cytokines in the same fractions indicating an interaction of the small-sized cytokines IL-6 and TNFα with the larger-sized albumin. Finally, a two-compartment albumin dialysis in vitro model was used to analyze the effect of albumin on proinflammatory cytokines in the recirculation circuit during 6-h treatment. These in vitro albumin dialysis experiments indicated a significant decrease of IL-6, but not of TNFα, when albumin was added to the dialysate solution. Taken together, we were able to show a putative in vitro interaction of human albumin with the proinflammatory cytokine IL-6, but with less evidence for TNFα, and demonstrated an additional application for albumin dialysis in liver support therapy where IL-6 removal might be indicated.

It takes two to tango: IκBs, the multifunctional partners of NF-κB.

The inhibitory IκB proteins have been discovered as fundamental regulators of the inducible transcription factor nuclear factor-κB (NF-κB). As a generally excepted model, stimulus-dependent destruction of inhibitory IκBs and processing of precursor molecules, both promoted by components of the signal integrating IκB kinase complex, are the key events for the release of various NF-κB/Rel dimers and subsequent transcriptional activation. Intense research of more than 20 years provides evidence that the extending family of IκBs act not simply as reversible inhibitors of NF-κB activation but rather as a complex regulatory module, which assures feedback regulation of the NF-κB system and either can inhibit or promote transcriptional activity in a stimulus-dependent manner. Thus, IκB and NF-κB/Rel family proteins establish a complex interrelationship that allows modulated NF-κB-dependent transcription, tailored to the physiological environment.

The Relationship between Supportive Care Needs and Health-Related Quality of Life in Cancer Patients.

The aim of this study was to analyze the relationship between quality of life (QoL) and supportive care needs (SCNs) in cancer patients. It is difficult to relate SCNs to detriments in QoL since SCNs and QoL assessment tools generally comprise different dimensions that cannot be directly related to each other. Therefore, we developed a short questionnaire with eight dimensions for uniformly measuring SCNs, QoL, and the subjective importance of these dimensions. A total of 1108 cancer patients with mixed diagnoses assessed eight dimensions of health-related QoL concerning SCNs, satisfaction, and importance. Among the eight dimensions of QoL, physical functioning received the highest SCN assessments (M = 3.4), while autonomy (M = 20.7) and social relationships (M = 1.88) were the dimensions with the lowest SCN mean scores on the 1-5 scale. For each of the eight dimensions, high levels of SCNs were reported by those patients who had low levels of satisfaction with that dimension (r between -0.32 and -0.66). The subjective importance of the dimensions was not consistently correlated with SCNs (r between -0.19 and 0.20). Females reported higher SCNs than males in six of the eight specific dimensions. Patients with prostate and male genital cancers reported the lowest SCNs. These results suggest gender-specific SCN patterns that warrant further exploration. This study highlights the value of a unified assessment instrument for SCNs and QoL, providing a robust basis for future cancer care strategies.

Controlling nucleation and crystal growth of a distinct polyoxovanadate cluster: an in situ energy dispersive X-ray diffraction study under solvothermal conditions.

The formation of the antimonato polyoxovanadates [V(14)Sb(8)(C(6)H(15)N(3))(4)O(42)(H(2)O)]·4H(2)O (1), (C(6)H(17)N(3))(2)[V(15)Sb(6)(C(6)H(15)N(3))(2)O(42)(H(2)O)]·2.5H(2)O (2), {C(6)H(15)N(3)}(4)[V(16)Sb(4)O(42)]2H(2)O (3) (C(6)H(15)N(3)=1-(2-aminoethyl)piperazine, AEP) has been studied under solvothermal conditions by using in situ energy dispersive X-ray diffraction (EDRXD). The syntheses were performed with an identical ratio for Sb(2)O(3) and NH(4)VO(3). If the reactions slurries are not stirred during the solvothermal reaction and by applying 70-75% amine concentration, the products contain all three compounds, whereas 3 is observed at 80%. Under stirring conditions, variation of the concentration of AEP led to crystallization of the three different compounds at distinct concentrations, that is, 1 is formed at 75%, 1 and 2 between 75 and 80% and 3 at 80%. At an amine concentration of 77.5%, first reflections of 2 occurred and at later stages, compound 1 started to crystallize. The sample with the lowest number of V(IV) species was formed at the lowest amine concentration, whereas crystallization of 3 required the highest concentration. The formation of the compounds occurred without crystalline intermediates and/or precursors. With increasing reaction temperature, the incubation time was significantly reduced.

Immunoadsorption and plasmapheresis at the ICU - A description of the frequencies and indications in a single center experience and a case report.

Immunologically mediated diseases can lead to severe courses that have to be treated in an intensive care unit. The use of extracorporeal organ support systems (ventilation, ECMO) is common. A therapeutic principle for these diseases is the removal of disease-causing antibodies. This can be done nonspecifically by plasmapheresis or specifically by immune adsorption. While most intensive care units have the facilities for plasmapheresis (membrane plasma filtration), immunoadsorption is much less common. Over a period of 10 years, the numbers of immunoadsorption and plasmapheresis treatments performed in a single center intensive care unit are shown according to their indication (IA: 18 Pts, 58 treatments. PA: 54 Pts, 148 treatments). A case study of a patient with granulomatosis with polyangiitis shows the successful treatment with immunoadsorption. The advantages of immunoadsorption in patients with complex coagulation disorders and a critical clinical picture in terms of SIRS and ARDS are shown.

Comparison of changes in albumin binding capacity during hemodiafiltration or hemodialysis with middle cut off membranes.

BACKGROUND: Albumin is important for the transport of protein-bound substances (PBS). Albumin binding capacity (ABiC) is reduced in dialysis patients. This can contribute to worsening of uremic symptoms. It is presumed that open-porous middle cut off filters that is, HDx (Baxter-Theranova) remove high molecular substances more efficiently than conventional treatment. To evaluate HDx for the improvement of ABiC and removal of PBS, HDx was compared to hemodiafiltration (Fresenius-FX80, HDF). METHODS: We included 32 chronic patients on HDF. After inclusion patients were treated with HDx for 14 days. Blood samples were drawn before/after treatments at study entry, first HDx and sixth HDx, to determine ABiC and other study parameters. RESULTS: ABiC improved in HDx (68.4% vs 72.4%) and HDF (69.9% vs 72.4%) without differences between both therapies. No reduction of albumin concentration during HDx treatment was observed. CONCLUSION: HDx is accepted as a safe and equally efficient therapy for removing albumin bound uremic toxins compared to HDF with high flux dialyzers.

Nuclear factor kappaB-dependent gene expression profiling of Hodgkin's disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity.

Constitutive nuclear nuclear factor (NF)-kappaB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-kappaB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkin's disease (HD) cell lines to identify NF-kappaB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-kappaB. The NF-kappaB-dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-kappaB target genes. Using chromatin immunoprecipitation we demonstrate that NF-kappaB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-kappaB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-kappaB in the pathogenesis of HD and potentially of other tumors with constitutive NF-kappaB activation.

Low-field magnetic resonance imaging: increased safety for pacemaker patients?

AIMS: The number of low-field (<0.5 T) magnetic resonance (MR) scanners installed worldwide is increasing due to a favourable cost and safety profile and improved patient comfort using an open-scanner design. Therefore, the aim of our study was to evaluate a strategy for the safe performance of magnetic resonance imaging (MRI) at a field strength of 0.2 T, in pacemaker (PM) patients without limitations on scan region, PM dependency, or the presence of abandoned leads. METHODS AND RESULTS: One hundred and fourteen PM patients, including PM-dependent patients and patients with abandoned leads, examined at a 0.2 T MR scanner due to an urgent clinical need for an MRI examination, were evaluated. All PMs were reprogrammed before MRI: if heart rate was <60 bpm, the asynchronous mode (with a rate of 80 bpm) was programmed to avoid MR-induced inhibition; if heart rate was >60 bpm, sense-only mode (ODO/OVO/OAO) was used to avoid MR-induced competitive pacing and potential proarrhythmia. Patients were monitored with electrocardiogram (ECG) and pulse oximetry. All PMs were interrogated before and after MRI, including measurement of lead impedance, pacing capture threshold (PCT), and battery voltage. All MRI scans were completed safely. No induction of arrhythmias or inhibition of PM output occurred. There were no statistically significant changes in lead impedance, PCT, or battery voltage (P>0.05). CONCLUSION: Low-field MRI of PM patients, including high-risk PM patients and MRI scan regions, can be performed with an acceptable risk-benefit ratio under controlled conditions.

Treatment of thyrotoxic crisis with plasmapheresis and single pass albumin dialysis: a case report.

Thyrotoxic crisis (thyroid storm) is a life-threatening condition. Standard therapy is based on thiamazole, prednisolone, and nonselective beta-blockers. Extracorporeal plasmapheresis is an additional tool for removing circulating thyroxine in patients who do not respond quickly to conventional standard therapy. As thyroxine can be bound by albumin, the aims of the present therapy report were to investigate the potential of extracorporeal single-pass albumin dialysis (SPAD) to remove thyroid hormones and to compare it with plasmapheresis. A 68-year-old female with thyrotoxic crisis refractory to conventional therapy underwent two sessions of plasmapheresis without clinical response. For the treatment dose to be increased, the patient was then treated with a modified continuous veno-venous hemodialysis with a dialysate containing 4% of human serum albumin (SPAD) intended to bind and remove thyroxines continuously. In total, the patient received three sessions of plasmapheresis and four SPAD treatments. Thyroxine levels were detected in the patient and in exchanged plasma or albumin dialysate, respectively, to calculate the amount removed. The main finding was that SPAD treatments were tolerated well by the patient. Due to continuous approach, SPAD sessions removed more thyroid hormone than plasmapheresis did, resulting in the improvement of the clinical status of the patient (reduction of heart rate and catecholamine dosage), which enabled bridging the patient to thyroidectomy as the ultimate surgical treatment. This is the first clinical report of the use of albumin dialysis in thyroid storm. SPAD represents a safe and efficient alternative to plasmapheresis as it can be performed continuously in this critical condition.

Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors.

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer's disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD.

Cytokine adsorption is a promising tool in the therapy of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis is a life-threatening clinical syndrome caused by severe hypercytokinemia brought on by a highly stimulated but ineffective immune response. Animal studies and case series have demonstrated that a reduction in blood cytokine levels achieved with an extracorporeal adsorption cartridge that contains blood-compatible porous polymer beads (CytoSorb®) can effectively attenuate the inflammatory response during sepsis and possibly improve outcomes. We report a case series of two patients in which three episodes of severe hemophagocytic lymphohistiocytosis triggered by infections with herpesviridae were treated successfully with cytokine adsorption. A marked decrease in interleukin-6 plasma levels and a stable or decreasing need of vasopressor therapy were the most significant results of this treatment. Importantly, treatment was safe and well-tolerated, without any adverse events.

Striking back at the activator: how IkappaB kinase terminates antigen receptor responses.

Antigen recognition by the T cell receptor (TCR) elicits several intracellular signaling cascades, one of which activates the transcription factor NF-kappaB through IkappaB kinases (IKK). NF-kappaB regulates lymphocyte differentiation, proliferation, and apoptosis; thus, tight temporal control of its activation is required to prevent harmful immune cell dysregulation. Although considerable insight into the IKK and NF-kappaB activation process has emerged, less is known about the temporal regulation and termination of immunoreceptor signaling. Two recent studies have revealed that the scaffold protein Bcl10--which, together with CARMA1 and Malt1, forms the TCR-induced IKK-activating CBM complex--is a negative feedback substrate for IKK. IKKbeta initially contributes to CBM formation--which is required for full IKK activation--and then, through carboxyl-terminal Bcl10 phosphorylation, disrupts this structure to terminate signaling. IKK triggers Bcl10 degradation by the ubiquitin-proteasome system through phosphorylation of Bcl10 at other sites. Thus, inactivation through negative feedback mechanisms is an intrinsic property of the TCR-induced NF-kappaB pathway.

Inducible shRNA expression for application in a prostate cancer mouse model.

RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110alpha and p110beta, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.

A simple approach to industrial laser safety.

Industrial applications of lasers include marking, welding, cutting, and other material processing. Lasers used in these ways have significant power output but are generally designed to limit operator exposure to direct or scattered laser radiation to harmless levels in order to meet the Federal Laser Product Performance Standard (21CFR1040) for Class 1 laser products. Interesting challenges occur when companies integrate high power lasers into manufacturing or process control equipment. A significant part of the integration process is developing engineering and administrative controls to produce an acceptable level of laser safety while balancing production, maintenance, and service requirements. 3M Company uses a large number of high power lasers in numerous manufacturing processes. Whether the laser is purchased as a Class 1 laser product or whether it is purchased as a Class 4 laser and then integrated into a manufacturing application, 3M Company has developed an industrial laser safety program that maintains a high degree of laser safety while facilitating the rapid and economical integration of laser technology into the manufacturing workplace. This laser safety program is based on the requirements and recommendations contained in the American National Standard for Safe Use of Lasers, ANSI Z136.1. The fundamental components of the 3M program include hazard evaluation, engineering, administrative, and procedural controls, protective equipment, signs and labels, training, and re-evaluation upon change. This program is implemented in manufacturing facilities and has resulted in an excellent history of laser safety and an effective and efficient use of laser safety resources.

Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma.

Multiple myeloma (MM) is an aggressive incurable plasma cell malignancy with a median life expectancy of less than seven years. Antibody-based therapies have demonstrated substantial clinical benefit for patients with hematological malignancies, particular in B cell Non-Hodgkin's lymphoma. The lack of immunotherapies specifically targeting MM cells led us to develop a human-mouse chimeric antibody directed against the B cell maturation antigen (BCMA), which is almost exclusively expressed on plasma cells and multiple myeloma cells. The high affinity antibody blocks the binding of the native ligands APRIL and BAFF to BCMA. This finding is rationalized by the high resolution crystal structure of the Fab fragment in complex with the extracellular domain of BCMA. Most importantly, the antibody effectively depletes MM cells in vitro and in vivo and substantially prolongs tumor-free survival under therapeutic conditions in a xenograft mouse model. A BCMA-antibody-based therapy is therefore a promising option for the effective treatment of multiple myeloma and autoimmune diseases.