Van Den Berghe Research Articles

Abstract SY27-02: In vitro and in vivo regulation of regulated necrosis induced by TNF

Abstract Cells can die by multiple ways. Two prototypes of regulated cell death have been put forward viz. apoptosis and programmed necrosis. Nowadays, programmed necrosis, also called regulated necrosis or necroptosis (1), is recognized as a highly controlled form of necrotic cell death (2). Using the L929 fibrosarcoma cellular system of TNF-induced necrotic cell death, we identified crucial subcellular events during necroptosis such as mitochondrial ROS production and lysosomal membrane permeabilization (LMP) (3). The initial events that lead to TNF-induced necrosis involve the activation of the serine threonine kinases RIPK1 and RIPK3, which initiate the formation of the socalled necrosome complex due to homotypic interaction between the RHIM domains. Also other stimuli such as TLR3 and TLR4 ligands have been shown to require RIPK3 for regulated necrosis in macrophages, while in this cellular context RIPK1 fullfils a survival function (4). Although a direct link between RIPK1/RIPK3 kinase activity and the downstream subcellular events at the mitochondrial and lysosomal level remain to be established, at the moment more is known on the regulation of the formation of necrosome complex containing FADD, caspase-8, RIPK1 and RIPK3. This TNF-induced complex formation is highly regulated by ubiquitylation/deubiquitylation balances involving ubiquitylating (a.o. IAP, A20) and deubiquitylating enzymes (CYLD), kinases (a.o. RIP1, RIP3, TAK1) and caspase activating platforms (FADD, FLIPL, caspase-8) (5,6). Recently, other stimuli like etoposide and Smac mimetics where shown to induce spontaneously a cytosolic complex called complex called ripoptosome independent of endogenous TNF production and containing FADD, caspase-8, RIPK1 and RIPK3 (7,8). These regulatory mechanisms involving ubiquitylation, phosphorylation and caspase-8-mediated proteolysis desensitize/sensitize and retard/accellerate necroptosis, like brakes and gears on the necrotic program. They determine the cellulular outcome being survival, apoptosis or necroptosis, and apparently also operate in vivo (9,10,11) and are involved in hyperinflammation in gut (12,13) and skin (14). We studied whether the regulatory mechanisms of TNF-induced necroptosis identified in vitro were also applicable for in vivo. Therefore we injected high dose of TNF causing systemic inflammatory response syndrome (SIRS), resulting in septic shock. We demonstrated that deletion of apoptotic executioner or inflammatory caspases had no impact on lethal SIRS despite lowered levels of apoptosis or circulating IL-1β. In contrast, deletion of the RIPK3 gene conferred complete survival and protection against necrotic cell death, reflected by reduced levels in the serum of mitochondrial DNA, lysosomal enzymes and other markers of organ damage. Interesting, also circulating inflammatory cytokines such as IL-6 and IL-1 were dramatically lowered in RIPK3 knockout mice, demonstrating that induction of necroptosis is preceding excessive inflammatory cytokine production. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, had a similar protective effect on mortality and resulted in reduced levels of markers of organ damage and circulating inflammatory cytokines. These results demonstrate that RIPK1/RIPK3-mediated necroptosis plays an indispensable role in TNF-induced SIRS as the determinant between life and death. The crucial role of necroptosis in SIRS in infectious sepsis was further underscored by the protective effect of RIPK3 deficiency in caecal ligation puncture (CLP) model. Altogether, our findings demonstrate that regulated necrosis or necroptosis in vivo is a crucial process and that components of the necroptotic cell death pathway are potential therapeutic targets for treatment of SIRS and sepsis (15).

Understanding the causes of hyperglycemia in burn patients

DOI of original article: 10.1016/j.jss.2012.0 * Corresponding author. Department of Surge Center, 10 Union Square East, Suite 2M, New E-mail address: mleitman@chpnet.org (I. 0022-4804/$ e see front matter a 2013 Elsev doi:10.1016/j.jss.2012.03.042 Hyperglycemia is commonly encountered in the critically ill patient. Although this is believed to be an adaptive stress response, hyperglycemia is in fact correlated with adverse outcomes [1]. Accordingly, critically ill patients are believed to benefit from enhanced glucose control. Several randomized controlled trials (RCTs) of strict glucose control with insulin therapy have been performed, most notably the 2001 landmark study by Van den Berghe et al., which found that intensive insulin therapy to maintain glucose at 80e110 mg/dL significantly reduced morbidity and mortality in intensive care unit (ICU) patients [2]. However, subsequent RCTs have reported conflicting results [1]. A metaanalysis of RCTs concluded that only surgical ICU patients, not medical ICU patients, enjoyed the benefit of strict glycemic control [3]. The large multicenter NICESUGAR trial demonstrated that an intermediate glucose target (140e180 mg/dL) was ideal, and resulted in lower mortality than stricter control (80e110 mg/dL) [4]. At the present time, due to conflicting data, the optimal target glucose level remains unclear. These data have been extrapolated to burn patients, despite the fact that burn patients were underrepresented in the RCTs of glycemic control in the ICU. Hyperglycemia was

International Don Quixote

International Don Quixote

Glycaemic control in ICUs in large English hospitals: a follow-up telephone survey

Following van den Berghe's landmark paper in 2001 (Leuven study) [1], the critical care community became very interested in 'tight' glycaemic control [2]; however, recent negative studies have dampened this interest [3]. In view of more recent analyses, which offer possible explanations for equivocal results [4], it is possible there will be renewed interest in glycaemic control. The purpose of this survey is to assess the utilisation of tight glycaemic control protocols in a sample of ICUs in England, as a reflection of current UK intensive care practice.

Preliminary ICU experience of a novel intravascular blood glucose sensor

A need for continuous blood glucose monitoring has always been expressed by critical care practitioners. The results from several iterations of a novel optical fluorescence-based intravascular blood glucose sensor were examined for correlation with an accepted laboratory assay. Ever since Van Den Berghe's group demonstrated reductions in hospital mortality and morbidity from the application of tight glycaemic control [1], many groups have attempted to replicate those results with limited success. Practitioners have speculated upon the reasons behind this observation, and have cited manpower implications and incidence of hypoglycaemic episodes as contributing factors [2]. Investigators have speculated that a continuous blood glucose sensor might contribute towards safe effective glycaemic control [3].

Liberalism: A Counter-History by Domenico Losurdo

Liberalism: A Counter-History by Domenico Losurdo

Contemporary Gastronomic Identities: Some Concluding Remarks

In this article we extend the papers by Steven Van den Berghe, Priscilla Parkhurst Ferguson and Virginie Amilien to highlight the most salient changes and evolution of the restaurant industry, emphasizing the first decade of the twenty-first century. Reflecting some of the themes raised by the preceding articles we ask: what are the identities of the contemporary restaurant and how are they expressed? Focusing on the American context we trace four major developments: the new charismatic celebrity chef, food enthusiasts and the expansion of a unique food culture, “moral” eating and related food politics and the changing structures of kitchen life.

D469del-COMP Retention in Chondrocytes Stimulates Caspase-Independent Necroptosis

Mutations in the cartilage oligomeric matrix protein gene (COMP) cause pseudoachondroplasia (PSACH). This dysplasia results from the intracellular retention of mutant COMP protein and premature death of growth-plate chondrocytes. Toward better understanding of these underlying mechanisms, we examined D469del-COMP activation of the unfolded protein response and cell death pathways in rat chondrosarcoma cells. Using an inducible expression system, we examined the effects of D469del-COMP retention after 4 days of mRNA expression and then 5 days without inducing agent. Retention of D469del-COMP stimulated Chop (Ddit3) and Gadd34 (Ppp1r15a) and triggered reactivation of protein translation that exacerbated intracellular retention. High levels of Nox4 and endoplasmic reticulum receptor stress-inducible Ero1β generated reactive oxygen species, causing oxidative stress. Increased expression of Gadd genes and presence of γH2AX indicated that DNA damage was occurring. The presence of cleaved apoptosis inducing factor (tAIF) and the absence of activated caspases indicated that retention of D469del-COMP triggers cell death in chondrocytes by necroptosis, a caspase-independent programmed necrosis. Loss of growth-plate chondrocytes by necroptosis was also found in our pseudoachondroplasia mouse model. These results suggest a model in which D469del-COMP expression induces persistent endoplasmic reticulum stress, oxidative stress, and DNA damage, thus priming chondrocytes for necroptosis. We define for the first time the precise mechanisms underlying D469del-COMP pathology in pseudoachondroplasia and suggest that oxidative stress and AIF may be promising therapeutic targets.

A new dichromatic species of Oaxacanthaxia Bellamy 1991 <br />(Coleoptera: Buprestidae) from Quintana Roo, México

x Twenty years ago, a new buprestid genus and species, Oaxacanthaxia viridis Bellamy 1991 was described for specimens collected by Jim Cope during the summer of 1989 in the Isthmus of Tehuantepec, Oaxaca, México (Bellamy 1991). The summer following that publication (1992), a group of 10 buprestiphiles from Australia, Czech Republic, Germany, the United States and Russia combined forces to collect in southern México (Guerrerro and Oaxaca), hoping to find, among many other taxa, O. viridis. That quest was partly successful (only one female specimen collected by Dave Verity), but completely unexpectedly Ted MacRae, Gayle Nelson and Dave Verity collected a second species of Oaxacanthaxia, which was described as O. nigroaenea by Nelson & MacRae (1994). Twelve years later, Niehuis & Gottwald (2006) described a new species from Nicaragua: O. vandenberghei, named for Manfred Niehuis’ nephew Eric van den Berghe, who had lived and collected for several years in Nicaragua. Most recently Hornburg & Gottwald (2008) described O. aenea from Venezuela, greatly expanding the range of the genus.

Dual Face Apoptotic Machinery: From Initiator of Apoptosis to Guardian of Necroptosis

In this issue of Immunity, Bonnet etal. (2011) show that skin-specific ablation of the adaptor protein FADD sensitizes keratinocytes to RIPK3-dependent necrotic cell death, which leads to severe skin inflammation.

Selective Glucocorticoid Receptor Agonists for Treatment of Inflammatory Bowel Disease – in vitro studies

Selective Glucocorticoid Receptor Agonists for Treatment of Inflammatory Bowel Disease – in vitro studies

The Ripoptosome: Death Decision in the Cytosol

In this issue of Molecular Cell, Tenev et al. and Feoktistova et al. describe the Ripoptosome, a cytosolic death-inducing RIP1-, FADD-, and caspase-8-containing complex that spontaneously assembles upon cIAP depletion, challenging the view that such complexes exclusively originate from receptor activation.

Tumor Necrosis Factor α-Mediated Induction of Interleukin 17C in Human Keratinocytes Is Controlled by Nuclear Factor κB

IL-17C is a member of the IL-17 family of cytokines. The expression of IL-17C has been demonstrated to be strongly induced by TNFα in human keratinocytes, and recently the level of IL-17C was found to be increased in the inflammatory skin disease psoriasis. However, little is known about the molecular mechanisms involved in the regulation of IL-17C. Here, we show that pretreatment of cultured human keratinocytes with the inhibitor of κB kinase 2 inhibitor, SC-514, resulted in a significant reduction in both IL-17C mRNA and protein expression, indicating the significance of this pathway in the regulation of IL-17C. NF-κB binding sites were identified upstream from the IL-17C gene, and by electrophoretic mobility shift assay NF-κB was shown to bind to all three identified binding sites. Moreover, NF-κB binding to these sites was inducible by TNFα. Supershift analysis revealed binding of the NF-κB subunits p65 and p50 to all three NF-κB binding sites. To determine the contribution of NF-κB in IL-17C expression, we conducted luciferase gene reporter experiments and demonstrated that a 3204-bp promoter fragment of IL-17C containing three putative NF-κB binding sites was strongly activated by TNFα. Interestingly, mutations of the three NF-κB binding sites revealed that one specific NF-κB binding site was crucial for the TNFα-mediated IL-17C induction because mutation of this specific site completely abolished TNFα-induced IL-17C promoter activation. We conclude that the activation of NF-κB (p65/p50) is crucial for the TNFα-induced stimulation of IL-17C expression in human keratinocytes.

Exponentially fitted methods applied to fourth-order boundary value problems

Fourth-order boundary value problems are solved by means of exponentially fitted methods of different orders. These methods, which depend on a parameter, can be constructed following a six-step flow chart of Ixaru and Vanden Berghe. Special attention is paid to the expression for the error term and to the choice of the parameter in order to make the error as small as possible. Some numerical examples are given to illustrate the practical implementation issues of these methods.

1 A mouse model highlights a role for p53 in 5q − syndrome

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

The need for increased vigilance in managing hyperglycaemia during acute coronary syndrome in the emergency department: An introduction to the evidence

Summary Until recently, increased blood glucose levels in those with and without diabetes with critical illnesses were perceived to be adaptive and benign responses to physiological stress in the critical care environment. In a landmark study published in 2001, Van den Berghe and colleagues demonstrated stress hyperglycaemia was not beneficial for patients who were experiencing critical illness. Instead, they found it resulted in significant increases in mortality. Several clinical trials and meta-analyses have since reported early management of hyperglycaemia in acute coronary syndrome (ACS), and many other critical illnesses to be advantageous. Not all evidence supports tight glycaemic control however. In this paper, we examine possible mechanisms by which increased blood glucose may harm patients with ACS and explain how insulin may be protective. We introduce evidence for and against increased glucose control in the emergency department. Given the increased mortality and morbidity associated with high blood glucose in ACS, we recommend increased diligence in the management of hyperglycaemic patients with ACS upon presentation to emergency departments.

The Histone Modifications Governing TFF1 Transcription Mediated by Estrogen Receptor

Transcription regulation by histone modifications is a major contributing factor to the structural and functional diversity in biology. These modifications are encrypted as histone codes or histone languages and function to establish and maintain heritable epigenetic codes that define the identity and the fate of the cell. Despite recent advances revealing numerous histone modifications associated with transcription regulation, how such modifications dictate the process of transcription is not fully understood. Here we describe spatial and temporal analyses of the histone modifications that are introduced during estrogen receptor α (ERα)-activated transcription. We demonstrated that aborting RNA polymerase II caused a disruption of the histone modifications that are associated with transcription elongation but had a minimal effect on modifications deposited during transcription initiation. We also found that the histone H3S10 phosphorylation mark is catalyzed by mitogen- and stress-activated protein kinase 1 (MSK1) and is recognized by a 14-3-3ζ/14-3-3ε heterodimer through its interaction with H3K4 trimethyltransferase SMYD3 and the p52 subunit of TFIIH. We showed that H3S10 phosphorylation is a prerequisite for H3K4 trimethylation. In addition, we demonstrated that SET8/PR-Set7/KMT5A is required for ERα-regulated transcription and its catalyzed H4K20 monomethylation is implicated in both transcription initiation and elongation. Our experiments provide a relatively comprehensive analysis of histone modifications associated with ERα-regulated transcription and define the biological meaning of several key components of the histone code that governs ERα-regulated transcription.

Glucose for the brain: fuel or poison?

Glycaemic control has been an area of active research over the past decade. To a large extent, this was kick-started by the seemingly impossible beneficial effects of tight glycaemic control demonstrated by van den Berghe et al in 2001. Subsequent to this, numerous trials were conducted which have not shown the same degree of benefit and, in addition, numerous concerns have been raised with regard to the potential harmful effects related to hypoglycaemic events.

Heteromeric Interactions Required for Abundance and Subcellular Localization of Human CDC50 Proteins and Class 1 P4-ATPases

Members of the P(4) family of P-type ATPases (P(4)-ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P(4)-ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P(4)-ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P(4)-ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P(4)-ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P(4)-ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.