In a proteomic search for heparan sulfate-binding proteins on monocytes, we identified HMGB1 (high mobility group protein B1). The extracellular role of HMGB1 as a cytokine has been studied intensively and shown to be important as a danger-associated molecular pattern protein. Here, we report that the activity of HMGB1 depends on heparan sulfate. Binding and competition studies demonstrate that HMGB1 interacts with CHO and endothelial cell heparan sulfate. By site-directed mutagenesis, we identified a loop region that connects the A-box and B-box domains of HMGB1 as responsible for heparan sulfate binding. HMGB1-induced Erk1/2 and p38 phosphorylation is abolished when endothelial heparan sulfate is removed or blocked pharmacologically, resulting in decreased HMGB1-induced endothelial sprouting. However, mutated HMGB1 that lacks the heparan sulfate-binding site retained its signaling activity. We show the major receptor for HMGB1, receptor for advanced glycation end products (RAGE), also binds to heparan sulfate and that RAGE and heparan sulfate forms a complex. Our data establishes that the functional receptor for HMGB1 consists of a complex of RAGE and cell surface heparan sulfate.

BACKGROUND

High mobility group protein box 1 (HMGB1) is a DNA binding protein located in nucleus. It is released into extracellular fluid where it acts as a novel proinflammatory cytokine which interacts with Toll like receptor 4 (TLR4) to activate nuclear factor-κB (NF-κB). This sequence of events is involved in tumor growth and progression. However, the effects of HMGB1, TLR4 and NF-κB on epidermal tumors remain unclear.

METHODS

Human epidermal tumor specimens were obtained from 96 patients. Immunohistochemistry was used to detect expression of HMGB1, TLR4 and NF-κB p65 in human epidermal tumor and normal skin specimens. Western blot analysis was used to detect the expression of NF-κB p65 in epithelial cell nuclei in human epidermal tumor and normal tissues.

RESULTS

Immunohistochemistry and western blot analysis indicated a progressive but statistically significant increase in p65 expression in epithelial nuclei in benign seborrheic keratosis (SK), precancerous lesions (PCL), low malignancy basal cell carcinoma (BCC) and high malignancy squamous cell carcinoma (SCC) (P <0.01). The level of extracellular HMGB1 in SK was significantly higher than in normal skin (NS) (P <0.01), and was higher than in SCC but without statistical significance. The level of TLR4 on epithelial membranes of SCC cells was significantly higher than in SK, PCL, BCC and NS (P <0.01). There was a significant positive correlation between p65 expression in the epithelial nuclei and TLR4 expression on the epithelial cell membranes (r = 0.3212, P <0.01).

CONCLUSIONS

These findings indicate that inflammation is intensified in parallel with increasing malignancy. They also indicate that the TLR4 signaling pathway, rather than HMGB1, may be the principal mediator of inflammation in high-grade malignant epidermal tumors. Combined detection of p65 in the epithelial nuclei and TLR4 on the epithelial membranes may assist the accurate diagnosis of malignant epidermal tumors.

Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them ("editing process") at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DC(HIV) become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DC(HIV). The escape of DC(HIV) from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DC(HIV) cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DC(HIV). Finally, we demonstrate that restoration of DC(HIV) susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific siRNA, or by inhibiting HMGB1 with blocking antibodies or glycyrrhizin, arguing for a key role of HMGB1 in TRAIL resistance and DC(HIV) survival. These findings provide evidence for a new strategy developed by HIV to escape immune attack, they challenge the question of the involvement of HMGB1 in the establishment of viral reservoirs in DCs, and they identify potential therapeutic targets to eliminate infected DCs.

The receptor for advanced glycation endproducts (RAGE), a multiligand member of the immunoglobulin superfamily, interacts with proinflammatory AGEs, the products of nonenzymatic glycation and oxidation of proteins; high-mobility group box 1 (HMGB1), also known as amphoterin and S100/calgranulins to amplify inflammation and tissue injury. Previous studies showed that blockade of RAGE suppressed recruitment of proinflammatory mechanisms in murine models. We tested the hypothesis that RAGE contributes to alloimmune responses and report that in vivo, acute rejection of fully allogeneic cardiac allografts in a murine model of heterotopic cardiac transplantation is significantly delayed by pharmacological antagonism of RAGE. In parallel, allogeneic T-cell proliferation in the mixed lymphocyte reaction is, at least in part, RAGE-dependent. These data provide the first insights into key roles for RAGE in allorecognition responses and suggest that antagonism of this receptor may exert beneficial effects in allogeneic organ transplantation.

BACKGROUND

Coronary ischemia-reperfusion (I/R) injury causes cardiomyocyte necrosis in a multi-step process that includes an inflammatory reaction. A recent study has suggested that high-mobility group box 1 (HMGB1) is a late mediator of lethal sepsis and an early mediator of inflammation and necrosis following I/R injury. In the present study a neutralizing monoclonal antibody (mAb) for HMGB1 was used to clarify the role of HMGB1 in cardiac I/R injury.

RESULTS

Rats underwent 30 min of left coronary artery occlusion followed by 60 min reperfusion. An intravenous injection of anti-HMGB1 mAb or control IgG was administered just before reperfusion. The infarct size was enlarged in the anti-HMGB1 mAb group in comparison with the control group (p<0.05). The treatment of anti-HMGB1 mAb significantly increased the plasma troponin-T and norepinephrine (NE) content in the heart in comparison with the control (p<0.05). Moreover, the production of dihydroxyphenylglycol was reduced in the anti-HMGB1-treated group (p<0.05).

CONCLUSIONS

This study shows for the first time the effects of treatment with neutralizing anti-HMGB1 mAb on I/R injury in the rat heart. The findings support the novel view that I/R-induced HMGB1 may be an important factor in the modulation of interstitial NE.

High-mobility group box 1 (HMGB1) acts as an early mediator of inflammation and organ damage in hepatic ischemia-reperfusion (I/R) injury. Glycyrrhizin is a natural anti-inflammatory and antiviral triterpene in clinical use. The purpose of this study was to investigate the effect of glycyrrhizin on liver injury caused by I/R and production of HMGB1 by Kupffer cells in rats. In the first test period, rats were given saline or glycyrrhizin 20 min before segmental hepatic warm I/R. Serum alanine aminotransferase and HMGB1 levels and hepatic histopathological findings were evaluated after I/R. Furthermore, expression of HMGB1 in the liver was assessed by immunohistochemical staining after I/R. Kupffer cells were isolated by collagenase digestion and differential centrifugation, and production of HMGB1 was assessed. In another set of experiments, the effect of inhibition of Kupffer cells by injection of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP) on liver injury and expression of HMGB1 were investigated after I/R. Liver injury was prevented in the glycyrrhizin group compared with the control group. Furthermore, serum HMGB1 levels were also significantly blunted in the glycyrrhizin group compared with the control group. Cells expressing HMGB1 were detected in the hepatic sinusoid by immunohistochemistry and recognized morphologically as Kupffer cells. Furthermore, the expression of HMGB1 was reduced in the glycyrrhizin group compared with the control group. Production of HMGB1 was reduced in Kupffer cells isolated from the glycyrrhizin group compared with the control group. It is noteworthy that treatment with lipo-MDP significantly blunted serum HMGB1 levels and prevented liver injury after I/R. These results suggest that glycyrrhizin has the therapeutic potential to prevent warm I/R-induced injury during hepato-biliary surgery.

We investigated whether quercetin protects from steatosis and limits the expression of proinflammatory and fibrogenic genes in C57BL/6J mice with nonalcoholic steatohepatitis (NASH) induced by feeding a methionine-choline-deficient (MCD) diet. Quercetin (50 mg/kg) was given by oral route daily. Mice were randomly divided into 4 groups that received for 2 or 4 wk: the control diet plus vehicle, control diet plus quercetin, MCD diet plus vehicle, and MCD diet plus quercetin. At both 2 and 4 wk, feeding the MCD diet resulted in liver steatosis, inflammatory cell accumulation, oxidative stress evaluated by the concentration of TBARS, and fibrosis evidenced by the staining of α-smooth muscle actin-positive cells in the liver. At both 2 and 4 wk, the MCD diet induced an increase in the mRNA levels of Il6, Tnf, Ptgs2, and Hmgb1 and increased the protein concentrations of Toll-like receptor-4, c-Jun terminal kinase, and p65 NFκB subunit compared with control rats. Feeding the mice the MCD diet also triggered an increase of Col1a1, Col3a1, Plod3, Tgfb1, Smad3, Smad7, Pdgfb, Ctgf, Areg, Mmp9, and Timp1 mRNA levels. These effects were totally or partially prevented by treatment with quercetin. The data obtained suggest that attenuation of multiple profibrotic and proinflammatory gene pathways contributes to the beneficial effects of quercetin in mice with MCD diet-induced steatohepatitis.

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.

Hemoglobin is released from lysed RBCs in numerous clinical settings. High mobility group box 1 (HMGB1) is a nuclear and cytosolic DNA-binding protein released from injured cells that has been shown to play an important role in inducing inflammation. Because both of these endogenous molecules are frequently present in sites of necrosis and inflammation, we studied their interaction on the activation of macrophages. We report in this article that hemoglobin and HMGB1 synergize to activate mouse macrophages to release significantly increased proinflammatory cytokines. Addition of microbial ligands that activate through TLR2 or TLR4 resulted in further significant increases, in a "three-way" synergy between endogenous and microbial ligands. The synergy was strongly suppressed by hemopexin (Hx), an endogenous heme-binding plasma protein. The findings suggest that hemoglobin may play an important role in sterile and infectious inflammation, and that endogenous Hx can modulate this response. Administration of Hx may be beneficial in clinical settings characterized by elevated extracellular hemoglobin and HMGB1.

High-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecules, favors tissue regeneration via recruitment and activation of leukocytes and stem cells. Here we demonstrate, in a model of acute sterile muscle injury, that regeneration is accompanied by active reactive oxygen species (ROS) production counterbalanced and overcome by the generation of antioxidant moieties. Mitochondria are initially responsible for ROS formation. However, they undergo rapid disruption with almost complete disappearance. Twenty-four hours after injury, we observed a strong induction of MURF1 and atrogin-1 ubiquitin ligases, key signals in activation of the proteasome system and induction of muscle atrophy. At later time points, ROS generation is maintained by nonmitochondrial sources. The antioxidant response occurs in both regenerating fibers and leukocytes that express high levels of free thiols and antioxidant enzymes, such as superoxide dismutase 1 (SOD1) and thioredoxin. HMGB1, a protein thiol, weakly expressed in healthy muscles, increases during regeneration in parallel with the antioxidant response in both fibers and leukocytes. A reduced environment may be important to maintain HMGB1 bioactivity. Indeed, oxidation abrogates both muscle stem cell migration in response to HMGB1 and their ability to differentiate into myofibers in vitro. We propose that the early antioxidant response in regenerating muscle limits HMGB1 oxidation, thus allowing successful muscle regeneration.

High mobility group B1 (HMGB1) and its counter-receptor, receptor for advanced glycation end products (RAGE), represent suitable targets for investigation, integrating many aspects of modern biology, particularly that associated with chronic diseases involving inflammation, dysregulated cell death and cancer. Also known as amphoterin, HMGB1 was initially identified over 25 years ago as a transcriptional regulatory molecule causing DNA bending, and facilitating binding of several transcriptional complexes, in particular members of the nuclear hormone receptor family. Although loosely bound to chromatin, it is released from necrotic cells but not apoptotic cells and is actively secreted by activated macrophages in a partially tumor necrosis factor-dependent manner. It serves as a late mediator of septic death present within the serum and inflammatory sites of patients with arthritis, correlating with the inflammatory response, to signal tissue injury, causes sickness behavior, and acts as an endogenous pyrogen. Although known to interact with RAGE on endothelial cells causing activation and leukocyte recruitment, RAGE itself has most recently been shown to serve as a counter-receptor for leukocyte integrins, suggesting that signaling through this molecule is potentially important for cell adhesion and clustering as well as recruitment of inflammatory cells. Targeting the HMGB1 ligand or its receptor represents an important potential application in cancer therapeutics, given its widespread overexpression, as well as that of its receptor in virtually every tumor type carefully examined. This, coupled with its ability to accelerate tumor growth in immunodeficient murine models, suggests that it is a possible therapeutic target in patients with cancer.

OBJECTIVE

High mobility group box 1 (HMGB1) has been implicated as a late mediator in sepsis. We here sought to determine the extent of HMGB1 release in patients with sepsis stratified to the three most common infectious sources and to determine HMGB1 concentrations at the site of infection during peritonitis or pneumonia.

METHODS

Observational studies in patients and healthy humans challenged with lipopolysaccharide.

METHODS

Three intensive care units and one clinical research unit.

METHODS

Three patient populations were studied: 1) 51 patients with sepsis due to pneumonia (n = 29), peritonitis (n = 12), or urinary tract infection (n = 10); 2) 17 patients with peritonitis; and 3) four patients with community-acquired pneumonia. In addition, eight healthy subjects were studied after intravenous injection of lipopolysaccharide (4 ng/kg).

METHODS

One population of healthy volunteers received lipopolysaccharide intravenously.

RESULTS

Patients with severe sepsis due to pneumonia displayed elevated circulating HMGB1 concentrations at both days 0 and 3 after inclusion. Patients with sepsis due to peritonitis had elevated HMGB1 levels at day 0 but not at day 3, whereas urinary tract infection was associated with a delayed HMGB1 response, with elevated levels only at day 3. HMGB1 concentrations did not differ between survivors and nonsurvivors and were not correlated to either disease severity or concurrently measured cytokine levels. In line with these observations, although intravenous lipopolysaccharide injection clearly elevated plasma cytokine levels, HMGB1 remained undetectable. In patients with peritonitis, HMGB1 concentrations in abdominal fluid were more than ten-fold higher than in concurrently obtained plasma. In pneumonia patients, HMGB1 levels were higher in bronchoalveolar lavage fluid obtained from the site of infection than in lavage fluid from healthy controls.

CONCLUSIONS

In severe sepsis, the kinetics of HMGB1 release may differ depending on the primary source of infection. In patients with severe infection, HMGB1 release may predominantly occur at the site of infection.

HMGB1/Amphoterin is a ubiquitous, highly conserved DNA-binding protein that can be also released to the extracellular space by various cell types. Extracellular HMGB1 regulates migratory responses of several cell types through binding to RAGE that communicates with the cytoskeleton to regulate cell motility. HMGB1-induced cell signalling has been associated with mechanisms of several diseases, including cancer, sepsis, rheumatoid arthritis, stroke and atherosclerosis. This article reviews the evidence linking the functional roles of HMGB1 to RAGE signalling. Furthermore, we discuss the molecular and cellular mechanisms that may explain the roles of HMGB1/RAGE in diverse disease processes.

Chronic obstructive pulmonary disease (COPD), a progressive lung disease characterized by sustained neutrophilic airway inflammation, is caused by chronic exposure to noxious stimuli, e.g., cigarette smoke. This chronic exposure can induce immunogenic cell death of structural airway cells, inducing the release of damage-associated molecular patterns (DAMPs). Levels of several DAMPs, including S100 proteins, defensins, and high-mobility group box-1 (HMGB1), are increased in extracellular lung fluids of COPD patients. As DAMPs can attract and activate immune cells upon binding to pattern recognition receptors, we propose that their release may contribute to neutrophilic airway inflammation. In this review, we discuss the novel role of DAMPs in COPD pathogenesis. Relevant DAMPs are categorized based on their subcellular origin, i.e. cytoplasm, endoplasmic reticulum, nucleus, and mitochondria. Furthermore, their potential role in the pathophysiology of COPD will be discussed.

OBJECTIVE

An abnormal generation of reactive oxygen species (ROS) is thought to contribute to systemic sclerosis (SSc), fostering autoimmunity, fibrosis, and vascular inflammation. The function of the prototypic damage-associated molecular pattern, high mobility group box 1 (HMGB1), depends on its redox status. Here we investigate whether oxidative stress regulates the cross-talk between leukocytes and platelets via HMGB1, thus contributing to vessel inflammation in SSc.

RESULTS

The oxidation of HMGB1 amplified its ability to activate neutrophils, as detected assessing the redistribution of primary granule molecules and the transactivation of the β2 integrin chain CD18. Activated platelets are a source of bioactive HMGB1 and via P-selectin stimulated neutrophils to generate ROS. Oxidized extracellular HMGB1, soluble or associated to platelet membrane or to platelet-derived microparticles (PDμPs), further increased leukocyte activation. Leukocyte activation abated in the presence of inhibitors of HMGB1 or of catalase, which catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. The redistribution of the content of primary granules and the transactivation of β2 integrins characterized blood leukocytes of SSc patients and membrane HMGB1 was significantly higher in patients with pulmonary hypertension or with diffuse SSc. HMGB1(+) microparticles (μPs) purified from SSc patients, but not HMGB1(-) μPs purified from control subjects, activated in vitro healthy neutrophils, and HMGB1 inhibitors reversed the effects of μPs.

CONCLUSIONS

ROS dramatically increase the ability of extracellular HMGB1 to activate blood leukocytes. This event might contribute to maintain the microvascular injury of patients with SSc.

We previously described a caspase-independent death induced in macrophages by a high intracellular burden of Mycobacterium tuberculosis (Mtb). This death, with features of apoptosis and necrosis, releases viable bacilli for spreading infection. Interferon (IFN)-γ promotes survival of macrophages with a low intracellular Mtb load by inhibiting bacterial replication. Macrophages in naïve hosts are unable to restrict Mtb replication following aerosol transmission, but IFN-γ is increasingly present when adaptive immunity is expressed in the lungs ~2 weeks post-infection. We therefore investigated the effects of IFN-γ on macrophages challenged with Mtb at high multiplicity of infection (MOI). In contrast to the response at low MOI, IFN-γ accelerated the death of heavily infected macrophages and altered the characteristics of the dying cells. IFN-γ increased caspase-dependent DNA cleavage and apoptotic vesicle formation, but it also increased mitochondrial injury and release of LDH and HMGB1 in a caspase-independent manner. Adaptive immunity in tuberculosis (TB), mediated primarily by IFN-γ, has differential effects on Mtb-induced macrophage cell death depending on the intracellular bacillary load. While IFN-γ generally promotes host defense, our data suggest that its effects on heavily infected macrophages could also accelerate necrosis and spreading infection in TB disease.

Thiopurine treatment of human leukemia cells deficient in components of the mismatch repair system (Nalm6) initiated apoptosis after incorporation into DNA, as revealed by caspase activation and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. To elucidate the cellular sensor(s) responsible for recognition of DNA damage in cells with an inactive mismatch repair system, we isolated a multiprotein nuclear complex that preferentially binds DNA with thioguanine incorporated. The components of this nuclear multiprotein complex, as identified by protein mass spectroscopy, included high mobility group proteins 1 and 2 (HMGB1, HMGB2), heat shock protein HSC70, protein disulfide isomerase ERp60, and glyceraldehyde 3-phosphate dehydrogenase. The same complex was also shown to bind synthetic oligodeoxyribonucleotide duplexes containing the nonnatural nucleosides 1-beta-D-arabinofuranosylcytosine or 5-fluoro-2'-deoxyuridine. Fibroblast cell line derived from Hmgb1(-/-) murine embryos had decreased sensitivity to thiopurines, with an IC(50) 10-fold greater than Hmgb1-proficient cells (P < 0.0001) and exhibited comparable sensitivity to vincristine, a cytotoxic drug that is not incorporated into DNA. These findings indicate that the HMGB1-HMGB2-HSC70-ERp60-glyceraldehyde 3-phosphate dehydrogenase complex detects changes in DNA structure caused by incorporation of nonnatural nucleosides and is a determinant of cell sensitivity to such DNA modifying chemotherapy.

High-mobility group box 1 (HMGB1) was originally identified as a highly conserved nuclear DNA-binding protein that participates in DNA replication, repair and transcriptional regulation of gene expression. Although the nuclear role of HMGB1 is not quite understood, recent studies characterized the emerging role of extracellular HMGB1 as a prototypical danger signal that regulates inflammatory and repair responses. Under conditions of infection, injury and sterile inflammation, HMGB1 can be passively released from damaged cells or actively secreted from activated immune cells. Inflammasomes, large caspase-1-activating protein complexes, were recently shown to play a critical role in mediating the extracellular release of HMGB1 from activated and infected immune cells.

HMGB1 has been formerly known for its intracellular function - as the intranuclear non-histone DNA binding protein, which contributes to stabilization of nucleosomes, mediation of DNA bending and is regarded to have an essential position in DNA repair. Lately, its participation in innate and specific immune responses has been revealed. Passively released from necrotic cells or actively produced by various cell types it acts as an alarmin and is responsible for production of pro-inflammaory cytokines. HMGB1 is able to interact with RAGE and TLRs, receptors that belong into family of pattern recognition receptors and are involved in activation of pathways leading to production of pro-inflammatory cytokines. Its key role has been revealed in mediation of sepsis and as it is released later than other pro-inflammatory cytokines it became known as a "late mediator of sepsis". HMGB1 also contributes to the development of atherosclerosis and autoimmune diseases, e.g. its association with immunopathogenesis of SLE and RA has been suggested. Beside its negative function, HMGB1 protein seems to be able to attract stem cells to the area of inflammation and thus promotes regeneration processes. This paradoxical function of HMGB1 protein has also been revealed in growth and spread of many types of tumours. HMGB1 represents a potential target in therapy of various disorders related to inflammation (Fig. 2, Ref. 137).

Three major morphological types of cell death can be distinguished: type I (apoptotic cell death), type II (autophagic cell death), and type III (necrotic cell death). Details of the pathways of apoptotic and autophagic cell death have been described, and distinct biochemical markers have been identified. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic parameters (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). Moreover, a confounding factor is that apoptotic cells in the absence of phagocytosis proceed to secondary necrosis, which has many morphological features of primary necrotic cells. Secondary necrotic cells have already gone through an apoptotic stage, and so it is generally advisable in cell death research to perform time kinetics of cell death parameters. This chapter concentrates on methods that can distinguish apoptosis from necrosis on three different levels (morphological, biochemical, and analysis of cell-cell interactions) and emphasizes that only a combination of several techniques can correctly characterize cell death type. First, we describe analysis of apoptotic versus necrotic morphology by time-lapse microscopy, flow fluorocytometry, and transmission electron microscopy. We also discuss various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow fluorocytometry), cellular markers such as DNA fragmentation (flow fluorocytometry), caspase activation, Bid cleavage, and cytochrome c release (Western blotting). Next, we describe how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. Finally, we discuss cell-cell interactions during cell death and describe a quantitative method for examining dead cell clearance by flow fluorocytometry. A selection of techniques that can be used to study internalization mechanisms used by phagocytes to engulf dying cells is also presented, such as scanning and transmission electron microscopy and fluorescence microscopy.

Innate immunity is an arsenal of molecules and receptors expressed by professional phagocytes, glial cells and neurons and involved in host defence and clearance of toxic and dangerous cell debris. However, any uncontrolled innate immune responses within the central nervous system (CNS) are widely recognized as playing a major role in the development of autoimmune disorders and neurodegeneration, with multiple sclerosis (MS) and Alzheimer's diseases (AD) being primary examples. Critically, neuroimmune regulatory proteins (NIReg) may control the adverse immune responses in health and diseases. NIRegs are found mainly on neurons, glia, endothelia and ependymal cells and include GPI-anchored molecules (CD24, CD90, complement regulators CD55 and CD59), molecules of the immunoglobulin superfamily (siglec CD22, Siglec 10, CD200, ICAM-5) and others (CD47, fractalkine, TAM receptor tyrosine kinase and complement C3a and factor H). These regulators modulate the innate immune response in the CNS and for instance critically control the level of phagocytosis and inflammation engaged by resident microglia and infiltrating immune cells. Others will sequester and neutralize proinflammatory molecules such as HMGB1 and DNA. Moreover, some NIRegs can instigate the recruitment of stem cells to mediate tissue repair. In the absence of these regulators, when neurons die by apoptosis, become infected or damaged, microglia and infiltrating immune cells are free to cause injury and an adverse inflammatory response in acute and chronic settings. The therapeutic applications of NIRegs should be exploited given their natural and selective healing properties.

OBJECTIVE

Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel.

METHODS

Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers.

RESULTS

A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified.

CONCLUSIONS

Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFβ (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1-/- chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.

Novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires induction of immunogenic cell death (ICD), coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study shows that in human pancreatic cancer cells (pancreatic ductal adenocarcinoma [PDAC] cells n=4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0±0.5 times (58%±9% of total content; up to 100 ng/ml) in all infected cultures, whether nondying, necrotic, or apoptotic. An alternative secretory route allowed H-1PV to overcome the failure of gemcitabine to trigger HMGB1 release, without impeding cytotoxicity or other ICD activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided with but was uncoupled from an autocrine interleukin-1β (IL-1β) loop. That and the pattern of viral determinants maintained in gemcitabine-treated cells suggested the activation of an inflammasome/caspase 1 (CASP1) platform alongside DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signaled through secretion of the alarmin HMGB1 and, besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD process. A transient arrest of cells in the cyclin A1-rich S phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV, which is not pathogenic in humans, into multimodal anticancer treatments.

OBJECTIVE

The current therapeutic concepts targeting aggressive malignancies require an induction of immunogenic cell death characterized by exposure of calreticulin (CRT) as well as release of ATP and HMGB1 from dying cells. In pancreatic tumor cells (PDAC cells) infected with the oncolytic parvovirus H-1PV, only HMGB1 was released by all infected cells, whether nondying, necrotic, or succumbing to one of the programmed death pathways, including contraproductive apoptosis. Our data suggest that active secretion of HMGB1 from PDAC cells is a sentinel reaction emerging during early stages of the viral life cycle, irrespective of cell death, that is compatible with and complements cytotoxic regimens. Consistent induction of HMGB1 secretion raised the possibility that this reaction might be a general "alarming" phenomenon characteristic of H-1PV's interaction with the host cell; release of IL-1β points to the possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.