OBJECTIVE

To evaluate the effect of ethyl pyruvate (EP) in improving the survival and ameliorating distant organ damage and to investigate the role of high-mobility group box (HMGB) 1 in rats with established severe acute pancreatitis (SAP).

METHODS

Severe acute pancreatitis was induced by retrograde infusion of sodium taurodeoxycholate (5%, 1 mL/kg) into the biliopancreatic ducts in male Wistar rats. The rats were infused intravenously with EP of 40 mg/kg, 4 mg/kg, and 0.4 mg/kg initiating 12 hours, and EP of 40 mg/kg was administered beginning 2 hours before surgery (-2 hours) and 12, 24, and 36 hours after induction of SAP; then, the mortality was recorded. Serum tumor necrosis factor alpha, interleukin (IL) 6, and IL-1beta were measured using enzyme-linked immunosorbent assay. High-mobility group box 1 levels were measured using Western immunoblotting analysis.

RESULTS

Serum HMGB1 levels were increased dramatically after 12 hours, remained at high levels for 72 hours, and were significantly higher in rats with SAP than in those with mild and moderate pancreatitis (P < 0.01). Treatment with EP (40 mg/kg) conferred protection from lethality of SAP (EP survival [63%] vs vehicle survival [6.3%]; P < 0.001). No survival advantage occurred when treatment was initiated 36 hours after surgery, but administration beginning 2 hours before operation (-2 hours) and 12 and 24 hours after induction of SAP significantly increased survival. Ethyl pyruvate treatment significantly decreased serum HMGB1, tumor necrosis factor alpha, IL-1beta, and IL-6 levels and ameliorated extrapancreatic organ dysfunction in rats with SAP.

CONCLUSIONS

Ethyl pyruvate improves survival and ameliorates distant organ injury of SAP. These beneficial effects of EP are because of the modulation of HMGB1 and other inflammatory cytokine responses.

HMGB1, an evolutionarily conserved chromosomal protein, was recently re-discovered to act as a "danger signal" (alarmin) to alert the innate immune system for the initiation of host defense or tissue repair. Extracellular HMGB1 can be either passively released from damaged/necrotic cells or secreted by activated immune cells. Upon stimulation, dendritic cells (DCs), macrophages and natural killer (NK) cells secrete high levels of HMGB1 into the intercellular milieu. HMGB1 is potent to target DCs, macrophages, neutrophils and CD4(+) T cells. It also upregulates the expression of BCL-XL by which it may prevent the elimination of activated immune cells. As a result, HMGB1 has been suggested to be implicated in the pathogenesis of autoimmune disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and experimental allergic encephalomyelitis (EAE). Given the similarities of autoimmune response against beta cell self-antigens in type 1 diabetes (T1D), in this view we will discuss the possible implications of HMGB1 in T1D pathogenesis. Specifically, we will summarize and update the advancement of HMGB1 in the pathogenesis of autoimmune initiation and progression during T1D development, as well as islet allograft rejection of diabetic patients after islet transplantation. Elucidation of the role for HMGB1 in T1D pathogenesis would not only enhance the understanding of disease etiology, but also have the potential to shed new insight into the development of therapeutic strategies for prevention or intervention of this disorder.

OBJECTIVE

To investigate the effects of ulinastatin, a urinary trypsin inhibitor (UTI), on jugular venous superoxide radical (O₂⁻·) generation, oxidative stress, early inflammation, and endothelial activation in forebrain ischemia/reperfusion (FBI/R) rats.

METHODS

Fourteen Wistar rats were allocated to a control group (n = 7) and a UTI group (n = 7). Throughout the experiments, O₂⁻· in the jugular vein was measured by the produced current using a novel electrochemical O₂⁻· sensor. Forebrain ischemia was induced by occlusion of the bilateral common caroti darteries with hemorrhagic hypotension for 20 min, followed by reperfusion. In the UTI group, UTI (5 U/g) was administered intravenously immediately after reperfusion. At 60 min after reperfusion, plasma and brain were harvested, and malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule-1 (ICAM-1) were measured.

RESULTS

O₂⁻· current increased gradually during forebrain ischemia in both groups. The current increased markedly in the control group immediately after reperfusion but was significantly attenuated in the UTI group after reperfusion. Brain and plasma malondialdehyde, HMGB1, and ICAM-1 were significantly attenuated in the UTI group compared with those in the control group, except for brain HMGB1, which was associated with the amount of O₂⁻· generated during FBI/R.

CONCLUSIONS

UTI suppressed jugular venous O₂⁻· generation, oxidative stress, early inflammation, and endothelial activation in FBI/R rats. Therefore, UTI might be a useful agent for the therapy of the cerebral ischemia/reperfusion pathophysiology.

The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by [Pt(1R,2R-diaminocyclohexane)](2+), cis-[Pt(NH(3))(cyclohexylamine)](2+), [Pt(ethylenediamine)](2+), cis-[Pt(NH(3))(cyclobutylamine)](2+), and cis-[Pt(NH(3))(2-picoline)](2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-[Pt(NH(3))- (cyclohexylamine)](2+), but not [Pt((1R,2R)-diaminocyclohexane)](2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.

The amount of steroid hormone receptor proteins does not always correlate with the response of breast cancers to endocrine therapy. This may partly be due to the fact that binding of the estrogen receptor (ER) to estrogen responsive elements (ERE) of its target genes is mediated by additional cellular proteins. One of these is the high mobility group protein HMGB1, known to interact with ER thus dramatically increasing its binding to ERE. This is the first report analysing the expression patterns of HMGB1 in breast cancer cells. Northern blot analyses of the 1.4 kb and the 2.4 kb transcripts of HMGB1 in 13 breast cancer samples revealed a strong intertumoural variation by a factor of 8.5 and 14.5, respectively. This variation may contribute to the different response, of estrogen receptor-positive breast tumours to endocrine therapy, making HMGB1 a marker of considerable clinical interest.

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

High-mobility group box 1 protein (HMGB1), a DNA-binding nuclear and cytosolic protein, is a pro-inflammatory cytokine released by monocytes and macrophages. HMGB1 as well as its B box domain induce maturation of human dendritic cells (DCs). This report demonstrates that the B box domain induces phenotypic maturation of murine bone marrow-derived dendritic cells (BM-DCs) as evidenced by increased CD86, CD40 and MHC-II expression. The B box domain enhanced secretion of pro-inflammatory cytokines and chemokines: IL-1beta, IL-2, IL-5, IL-8, IL-12 and tumor necrosis factor (TNF)-alpha, but not IL-6 and IL-10. Furthermore, four peptides whose sequences correspond to different regions of HMGB1 induced production of IL-1beta, IL-2 and IL-12 (p70), but not IL-10 and IL-6 in mouse BM-DCs. Interestingly, these peptides differed in their capacity to induce TNF-alpha, IL-5, IL-18 and IL-8. B box domain as well as peptide-activated DCs acted as potent stimulators of allogeneic T cells in a mixed leukocyte reaction. DCs exposed to HMGB1 peptides induced proliferation of ovalbumin-specific syngeneic T cells. These DC-activating peptides could serve as an adjuvant in immunotherapeutic or vaccine context and the selective activity of these different peptides suggests a means to customize the functional properties of DCs.

Recently, several reports have suggested that HMGB1 (the high-mobility group box-1) plays a key role in tumor angiogenesis through multiple mechanisms, including up-regulation of proangiogenic factors. This study was conducted to investigate the prognostic role and the effects of chemotherapy on serum (ELISA) angiogenic factors: HMGB1, survivin and VEGF (Vascular Endothelial Growth Factor) in patients with advanced stage non-small cell lung cancer (NSCLC). The study entered 40 patients (31 man) and 15 healthy volunteers (control group). Peripheral blood samples were taken before and after four cycles of chemotherapy. The mean serum HMGB1 and VEGF levels were significantly higher in patients with advanced NSCLC than in controls (p=0.024, p=0.028, respectively). The levels of survivin in NSCLC patients were comparable to controls. No correlation was found between HMGB1, survivin and VEGF concentrations and the histological type and staging of lung cancer. Similarly, no correlation was revealed between the concentrations of HMGB1, survivin and VEGF and the effect of chemotherapy. However, in patients with NSCLC, HMGB1 positevely correlated with survivin (R=0.814, p=0.007) before chemotherapy, and negatively with VEGF (R=-0.841, p=0.035) after chemotherapy. When the cut-off values of serum HMGB1, survivin and VEGF (2.38 ng/ml, 81.92 pg/ml, 443.26 pg/ml, respectively) were used, the prognoses of high and low groups were not different. Concluding, patients with NSCLC have a higher serum concentration of HMGB1 and VEGF, while survivin levels are comparable to healthy individuals. In our opinion, determination of HMGB1, survivin and VEGF concentrations has no clinical significance in the prognosis of the survival time in lung cancer.

BACKGROUND

High-mobility group box 1 (HMGB1) is an important mediator of the inflammatory response. Its expression is increased in diabetic cardiomyopathy (DCM), but its role is unclear. We investigated the potential role and mechanism of HMGB1 in diabetes-induced myocardial fibrosis and dysfunction in mice.

METHODS

In vivo, type 1 diabetes was induced by streptozotocin (STZ) in mice. HMGB1 expression was knocked down by lentivirus-mediated short-hairpin RNA (shRNA). Cardiac function was assessed by echocardiography. Total collagen deposition was assessed by Masson's trichrome and Picrosirius red staining. HMGB1, collagen I and III, and transforming growth factor β1 (TGF-β1) expression was quantified by immunostaining and western bolt analysis. In vitro, isolated neonatal cardiac fibroblasts were treated with high glucose (HG) or recombinant HMGB1 (rHMGB1). Pharmacologic (neutralizing anti-HMGB1 antibody) or genetic (shRNA-HMGB1) inhibition of HMGB1 was used to investigate the role of HMGB1 in HG-induced functional changes of cardiac fibroblasts.

RESULTS

In vivo, HMGB1 was diffusely expressed in the myocardium of diabetic mice. HMGB1 silencing ameliorated left ventricular dysfunction and remodeling and decreased collagen deposition in diabetic mice. In vitro, HG induced HMGB1 translocation and secretion in both viable cardiomyocytes and fibroblasts. Administration of rHMGB1 dose-dependently increased the expression of collagens I and III and TGF-β1 in cardiac fibroblasts. HMGB1 inhibition reduced HG-induced collagen production, matrix metalloproteinase (MMP) activities, proliferation, and activated mitogen-activated protein kinase signaling in cardiac fibroblasts.

CONCLUSIONS

HMGB1 inhibition could alleviate cardiac fibrosis and remodeling in diabetic cardiomyopathy. Inhibition of HMGB1 might have therapeutic potential in the treatment of the disease.

The architectural chromosomal protein high-mobility group box 1 protein (HMGB1) acts as an alarmin when released from cells. It is involved in the pathogenesis of inflammatory and autoimmune diseases. HMGB1 can undergo post-translational modifications including oxidation. However, the mechanisms and functional relevance of HMGB1 oxidation are not yet understood. Increased concentrations of reactive oxygen species (ROS) have been reported during apoptosis and necrosis. Hence, we investigated the oxidative status of HMGB1 in dead cells. Immunoblot analyses under reducing and non-reducing conditions revealed that HMGB1 is oxidized in dead cells. Moreover, tagging of oxidized cysteine residues by a maleimide moiety linked to polyethylene glycol showed that HMGB1 passively released from primary and secondary necrotic cells was predominantly oxidized. Also HMGB1 in plasma of patients with systemic lupus was reversibly oxidized. In conclusion, HMGB1 undergoes reversible oxidative modifications at cysteine residues during cell death, which may modulate its biological properties.

Acute lung injury (ALI) frequently occurs after liver transplantation and major liver surgery. Proinflammatory mediators released by damaged liver after liver ischemia/reperfusion (I/R) injury might contribute to this form of ALI, but the underlying mechanisms have not been well characterized. High-mobility group box protein 1 (HMGB1), a recently identified proinflammatory cytokine, was found to be significantly higher in the serum after liver I/R injury. This study investigated whether HMGB1 was involved as a stimulating factor, and whether its downstream Toll-like receptor 4 (TLR4), p38 mitogen-activated protein kinase (p38MAPK), and activator protein-1 (AP-1) signaling pathways act as mediators in the development of liver I/R injury-induced ALI. Extensive ALI and lung inflammation was induced in a rat model of liver I/R injury. Serum HMGB1 was significantly higher after liver I/R injury, and more importantly, expression of HMGB1 mRNA and protein in the lung tissue was also significantly increased. We further found that liver I/R injury enhanced the expression of TLR4 mRNA and protein, and the activity of p38MAPK and AP-1 in the lung tissue. Inhibition of TLR4 expression in the lung tissue by infection with pGCSIL-GFP-lentivirus-expressing small hairpin RNAs targeting the TLR4 gene (TLR4-shRNA lentivirus) significantly attenuated ALI, lung inflammation, and activity of p38MAPK and AP-1 in the lung tissue. These findings indicate that HMGB1 might contribute to the underlying mechanism for liver I/R injury-induced ALI and that its downstream TLR4, p38MAPK, and AP-1 signaling pathways are potentially important mediators in the development of ALI.

OBJECTIVE

Glycyrrhizin (GL) has been found to inhibit extracellular HMGB1 cytokine's activity, and protect spinal cord, liver and brain against I/R-induced injury in experimental animals. The purpose of this study was to investigate the protective effect of GL in rat myocardial I/R-induced injury and to elucidate the underlying mechanisms.

METHODS

Male adult Sprague-Dawley rats underwent a 30-min left coronary artery occlusion followed by a 24-h reperfusion. The rats were treated with glycyrrhizin or glycyrrhizin plus recombinant HMGB1 after 30 min of ischemia and before reperfusion. Serum HMGB1, TNF-α and IL-6 levels, and hemodynamic parameters were measured at the onset and different time points of reperfusion. At the end of the experiment, the heart was excised, and the infarct size and histological changes were examined. The levels of Bcl2, Bax and cytochrome c, as well as phospho-ERK1/2, phospho-JNK and phospho-P38 in the heart tissue were evaluated using Western blot analysis, and myocardial caspase-3 activity was measured colorimetrically using BD pharmingen caspase 3 assay kit.

RESULTS

Intravenous administration of GL (10 mg/kg) significantly reduced the infarct size, but did not change the hemodynamic parameters at different time points during reperfusion. GL significantly decreased the levels of serum HMGB1, TNF-α and IL-6. GL changed the distribution of Bax and cytochrome c expression between the mitochondrial and cytosolic fractions in the heart tissue, resulting in inhibition of myocardial apoptosis. Moreover, expression of phospho-JNK, but not ERK1/2 and P38 was decreased by GL in the heart tissue. All of the effects produced by GL treatment were reversed by co-administration with the recombinant HMGB1 (100 μg). Intravenous administration of SP600125, a selective phospho-JNK inhibitor (0.5 mg/kg), attenuated HMGB1-dependent Bax translocation and the subsequent apoptosis.

CONCLUSIONS

These results demonstrate that GL alleviates rat myocardial I/R-induced injury via directly inhibiting extracellular HMGB1 cytokine activity and blocking the phospho-JNK/Bax pathway.

High mobility group box 1 (HMGB1)-Toll-like receptor 4 (TLR4) signaling has been recently found to induce interleukin (IL)-17A secretion in drug-induced hepatitis and myocardial I/R injury. The purpose of this study is to evaluate whether HMGB1-TLR4 signaling could induce IL-17A secretion and lead to brain I/R injury. We also sought to investigate whether glycyrrhizin elucidated its neuroprotective effects through HMGB1-TLR4-IL-17A signaling pathway. Various biochemical estimations, neurological status, and assessment of cerebral infarct size were carried out 72h after middle cerebral artery occlusion (MCAO) stroke. Apoptotic cells were assessed using a terminal deoxynucleotidyl transferase, dUTP nick and labeling (TUNEL) kit. The expression of HMGB1, IL-17A, bcl-2, bax and cleaved caspase-3, were determined by Western blot assay. In the present study we found that glycyrrhizin significantly decreased HMGB1 protein expression. Glycyrrhizin markedly reduced whereas recombinant HMGB1 (rHMGB1) increased IL-17A expression. HMGB1 induced increase of IL-17A was significantly diminished in TLR4-mutant C3H/HeJ mice. Brain injury and neurological deficits were largely abrogated by glycyrrhizin or IL-17A knockout. In contrast, rHMGB1 or recombinant mouse IL-17A markedly increased I/R injury. However, rHMGB1 had no effects on infarct size and neurological deficits in Il17a(-/-) mice following brain I/R injury. In addition, IL-17A knockout mice significantly increased bcl-2 protein expression and had fewer apoptotic cells, whereas recombinant IL-17A-treated mice significantly increased bax and cleaved caspase-3 protein expression and had more apoptotic cells. Together these results indicate that glycyrrhizin has neuroprotective efficacy in the postischemic brain through HMGB1-TLR4-IL-17A signaling pathway.

Hypoxia is a hallmark of cancer that is strongly associated with invasion, metastasis, resistance to therapy and poor clinical outcome. Tumour hypoxia affects immune responses and promotes the accumulation of macrophages in the tumour microenvironment. However, the signals linking tumour hypoxia to tumour-associated macrophage recruitment and tumour promotion are incompletely understood. Here we show that the damage-associated molecular pattern High-Mobility Group Box 1 protein (HMGB1) is released by melanoma tumour cells as a consequence of hypoxia and promotes M2-like tumour-associated macrophage accumulation and an IL-10 rich milieu within the tumour. Furthermore, we demonstrate that HMGB1 drives IL-10 production in M2-like macrophages by selectively signalling through the Receptor for Advanced Glycation End products (RAGE). Finally, we show that HMGB1 has an important role in murine B16 melanoma growth and metastasis, whereas in humans its serum concentration is significantly increased in metastatic melanoma. Collectively, our findings identify a mechanism by which hypoxia affects tumour growth and metastasis in melanoma and depict HMGB1 as a potential therapeutic target.

This study investigated the immunomodulating effect of melatonin on toll-like receptor (TLR)-stimulated signal transduction. Rats were subjected to 60 min of ischemia followed by 1 or 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min prior to ischemia and immediately before reperfusion. Melatonin treatment significantly reduced the level of serum alanine aminotransferase activity. Increased levels of TLR3 and TLR4 protein expression induced by ischemia/reperfusion (I/R) were attenuated by melatonin. Serum level of high-mobility group box 1 (HMGB1), a potent alarmin of the TLR system, increased significantly in the I/R group, and melatonin inhibited this release. Melatonin suppressed the increase in myeloid differentiation factor 88 (MyD88) protein expression, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation and nuclear translocation of nuclear factor κB (NF-κB) and phosphorylated c-Jun, a component of activator protein 1. The increased level of toll-receptor-associated activator of interferon (TRIF) expression, phosphorylation of interferon (IFN) regulatory factor 3 (IRF3) and serum IFN-β was attenuated by melatonin. Melatonin attenuated the levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and inducible nitric oxide synthase (iNOS) protein and mRNA expression, while the level of heme oxygenase-1 (HO-1) was augmented. Our results suggest that melatonin ameliorates I/R-induced liver damage by modulation of TLR-mediated inflammatory responses.

METHODS

We measured the expression and responses of Toll-Like Receptor 4 (TLR4) activation in the intervertebral disc (IVD) in vitro and in vivo. We hypothesize that stimulation of the IVD with the TLR4 ligand lipopolysaccharide (LPS) results in upregulation of a coordinated set of proinflammatory mediators and inhibition of matrix expression, both consistent with a molecular profile of degeneration.

OBJECTIVE

To characterize early inflammatory and morphological changes induced by TLR4 activation in the IVD.

BACKGROUND

TLR4 is a pattern recognition receptor activated in innate immunity that has been implicated in disease mechanisms of inflammatory cartilaginous degeneration. However, no study to date has examined the expression and responses of TLR4 in the IVD.

METHODS

IVD cells were stimulated with LPS in a dose-dependent manner, and inflammatory cytokine levels were measured by quantitative reverse transcription-polymerase chain reaction. Histological and inflammatory changes due to in vivo injection of LPS into the rat caudal IVD were measured by enzyme-linked immunosorbent assay and immunoblotting.

RESULTS

Baseline TLR4 expression in IVD tissue varied according to cell type. LPS stimulation resulted in significant increases in tumor necrosis factor α (TNF)-α, interleukin (IL)-1β, IL-6, and nitric oxide levels and significant inhibition in aggrecan and collagen-2. Intradiscal injection of LPS was found to cause moderate degenerative changes in the IVD, with increases in tissue levels of IL-1β, TNF-α, high mobility group box 1 protein (HMGB1), and macrophage migration inhibitory factor (MIF).

CONCLUSIONS

This study provides the first evidence that IVD cells express TLR4 and are responsive to TLR4 activation by upregulating a coordinated set of inflammatory cytokines. This study suggests that intradiscal injection of LPS offers a model for triggering inflammation of the IVD, demonstrating that inflammatory insults alone may potentially trigger degenerative changes of the IVD.

The blood-brain barrier (BBB) dysfunction represents an early feature of Alzheimer's disease (AD) that precedes the hallmarks of amyloid beta (amyloid β) plaque deposition and neuronal neurofibrillary tangle (NFT) formation. A damaged BBB correlates directly with neuroinflammation involving microglial activation and reactive astrogliosis, which is associated with increased expression and/or release of high-mobility group box protein 1 (HMGB1) and thrombin. However, the link between the presence of these molecules, BBB damage, and progression to neurodegeneration in AD is still elusive. Therefore, we aimed to profile and validate non-invasive clinical biomarkers of BBB dysfunction and neuroinflammation to assess the progression to neurodegeneration in mild cognitive impairment (MCI) and AD patients.

We determined the serum levels of various proinflammatory damage-associated molecules in aged control subjects and patients with MCI or AD using validated ELISA kits. We then assessed the specific and direct effects of such molecules on BBB integrity in vitro using human primary brain microvascular endothelial cells or a cell line.

We observed a significant increase in serum HMGB1 and soluble receptor for advanced glycation end products (sRAGE) that correlated well with amyloid beta levels in AD patients (vs. control subjects). Interestingly, serum HMGB1 levels were significantly elevated in MCI patients compared to controls or AD patients. In addition, as a marker of BBB damage, soluble thrombomodulin (sTM) antigen, and activity were significantly (and distinctly) increased in MCI and AD patients. Direct in vitro BBB integrity assessment further revealed a significant and concentration-dependent increase in paracellular permeability to dextrans by HMGB1 or α-thrombin, possibly through disruption of zona occludins-1 bands. Pre-treatment with anti-HMGB1 monoclonal antibody blocked HMGB1 effects and leaving BBB integrity intact.

Our current studies indicate that thrombin and HMGB1 are causal proximate proinflammatory mediators of BBB dysfunction, while sTM levels may indicate BBB endothelial damage; HMGB1 and sRAGE might serve as clinical biomarkers for progression and/or therapeutic efficacy along the AD spectrum.

OBJECTIVE

To assess the prevalence and predictive value of natural autoantibodies to high-mobility group box 1 (HMGB1) during sepsis.

METHODS

Anti-HMGB1 and anti-human serum albumin (HSA) autoantibodies were detected by ELISA in 178 plasma samples longitudinally collected from 40 critically ill patients with septic shock. One hundred thirty-two plasma samples from healthy donors were used as control.

RESULTS

IgGs to HMGB1 were detected in 15/40 patients (37.5%). The prevalence of anti-HMGB1 antibodies was significantly higher in the patients who survived (55%) compared to the patients who did not (20%) (p<0.0001). The detection of anti-HMGB1 antibodies during the course of the disease was significantly associated with patient survival (p=0.038). Moreover, there is a progressive and significant emergence of anti-HMGB1 antibodies during the course of the disease, mostly in patients who survived.

CONCLUSIONS

This study shows that autoantibodies to HMGB1 are produced during sepsis and are associated with a favorable outcome in patients undergoing septic shock.

BACKGROUND

Red blood cell (RBC) transfusions are associated with increased risk of acute respiratory distress syndrome (ARDS) in the critically ill, yet the mechanisms for enhanced susceptibility to ARDS conferred by RBC transfusions remain unknown.

OBJECTIVE

To determine the mechanisms of lung endothelial cell (EC) High Mobility Group Box 1 (HMGB1) release following exposure to RBCs and to determine whether RBC transfusion increases susceptibility to lung inflammation in vivo through release of the danger signal HMGB1.

METHODS

In vitro studies examining human lung EC viability and HMGB1 release following exposure to allogenic RBCs were conducted under static conditions and using a microengineered model of RBC perfusion. The plasma from transfused and nontransfused patients with severe sepsis was examined for markers of cellular injury. A murine model of RBC transfusion followed by LPS administration was used to determine the effects of RBC transfusion and HMGB1 release on LPS-induced lung inflammation.

RESULTS

After incubation with RBCs, lung ECs underwent regulated necrotic cell death (necroptosis) and released the essential mediator of necroptosis, receptor-interacting serine/threonine-protein kinase 3 (RIP3), and HMGB1. RIP3 was detectable in the plasma of patients with severe sepsis, and was increased with blood transfusion and among nonsurvivors of sepsis. RBC transfusion sensitized mice to LPS-induced lung inflammation through release of the danger signal HMGB1.

CONCLUSIONS

RBC transfusion enhances susceptibility to lung inflammation through release of HMGB1 and induces necroptosis of lung EC. Necroptosis and subsequent danger signal release is a novel mechanism of injury following transfusion that may account for the increased risk of ARDS in critically ill transfused patients.

High-mobility group box 1 (HMGB1) was initially described as a damage-associated-molecular-pattern (DAMP) mediator that worsens acute brain injury after stroke. But, recent findings suggest that HMGB1 can play a surprisingly beneficial role during stroke recovery by promoting endothelial progenitor cell (EPC) function and vascular remodeling in cortical gray matter. Here, we ask whether HMGB1 may also influence EPC responses in white matter injury. The standard lysophosphatidylcholine (LPC) injection model was used to induce focal demyelination in the corpus callosum of mice. Immunostaining showed that within the focal white matter lesions, HMGB1 was up-regulated in GFAP-positive reactive astrocytes, along with the accumulation of Flk1/CD34-double-positive EPCs that expressed pro-recovery mediators such as brain-derived neurotrophic factor and basic fibroblast growth factor. Astrocyte-EPC signaling required the HMGB1 receptor RAGE as treatment with RAGE-neutralizing antibody significantly decreased EPC accumulation. Moreover, suppression of HMGB1 with siRNA in vivo significantly decreased EPC numbers in damaged white matter as well as proliferated endothelial cell numbers. Finally, in vitro cell culture systems confirmed that HMGB1 directly affected EPC function such as migration and tube formation. Taken together, our findings suggest that HMGB1 from reactive astrocytes may attract EPCs to promote recovery after white matter injury.

Cells can die through two mechanisms: necrosis and apoptosis. Necrosis occurs in response to stimuli that cause cellular damage and result in the release of intracellular proteins, which stimulate the inflammatory response. Necrosis is associated with tissue damage and organ deterioration. Apoptosis is a programmed cell death during which the cellular membrane remains intact, but activated enzymes destroy the cell from within. Bustin discusses how the mobility of the nuclear protein high mobility group B1 (HMGB1) may be a key signal for determining whether certain cells undergo necrotic or apoptotic death. Necrosis may result when HMGB1 is released from the cells and stimulates the inflammatory response. HMGB1 appears to have extracellular signaling functions, such as stimulation of cell motility and tumor metastasis and activation of the cytokine signaling pathways. Through its action on the cytokine signaling targets, HMGB1 may be an important therapeutic target for diseases of cytokine excess, including septic shock.

Sivelestat sodium hydrate is a selective inhibitor of neutrophil elastase (NE), and is effective in acute lung injury associated with systemic inflammatory response syndrome (SIRS). The effect of Sivelestat for postoperative clinical courses after transthoracic esophagectomy was investigated. Consecutive patients with carcinoma of the thoracic esophagus who underwent transthoracic esophagectomy between 2003 and 2004 were assigned to the Sivelestat-treated group (n = 18), and those between 1998 and 2003 were assigned to the control group (n = 25). The morbidity rate, duration of postoperative SIRS, mechanical ventilation, and intensive care unit (ICU) stay, and the sum of the sequential organ failure assessment scores at all time points after the operation were compared. Serum NE activities and serum concentrations of TNF-alpha, IL-1beta, IL-6, and high mobility group box chromosomal protein 1 (HMGB1) were measured. Postoperative complications developed in three patients in the control group, and one in the Sivelestat-treated group. The durations of SIRS, mechanical ventilation, and ICU stay were significantly shorter in the Sivelestat-treated group. Even in patients without complications, the durations of mechanical ventilation, and ICU stay were also significantly shorter, and the arterial oxygen pressure/fraction of inspired oxygen ratio at postoperative day 1 was significantly higher in the Sivelestat-treated group. Serum NE activities and serum concentrations of IL-1beta, IL-6, and HMGB1 were significantly suppressed in the Sivelestat-treated group. Postoperative Sivelestat treatment after transthoracic esophagectomy improves the condition of SIRS and postoperative clinical courses, even in patients without complications.

Osteosarcoma is the most common malignant bone tumor for children and adolescents, and the frequent acquisition of drug-resistant phenotypes and the occurrence of "secondary malignancies" are often associated with chemotherapy and are significant obstacles to achieving favorable outcomes. Thus, it is urgent to identify the molecular mechanisms underlying the chemoresistance of osteosarcoma. In this study, we showed that miR-22 and high-mobility group box 1 (HMGB1) were deregulated in osteosarcoma cells, post-chemotherapy; the upregulated HMGB1 mediated autophagy and contributed to chemotherapy resistance in osteosarcoma in vitro. However, possibly as a compensatory effect, miR-22 was also upregulated during the chemotherapy, and the overexpressed miR-22 targeted the 3' UTR of HMGB1 and inhibits the HMGB1-promoted autophagy. Our study suggests a complexity in the regulation of autophagy by miR-22 and HMGB1 during chemotherapy resistance in osteosarcoma. These results reveal novel potential role of miR-22 against chemotherapy resistance during the treatment of osteosarcoma.