OBJECTIVE

High-mobility group protein B1 (Hmgb1) is released from necrotic cells and induces an inflammatory response. Although Hmgb1 has been implicated in ischemia/reperfusion (IR) injury of the brain, its role in IR injury of the retina remains unclear. Here, the authors provide evidence that Hmgb1 contributes to retinal damage after IR.

METHODS

Retinal IR injury was induced by unilateral elevation of intraocular pressure and the level of Hmgb1 in vitreous humor was analyzed 24 hours after reperfusion. To test the functional significance of Hmgb1 release, ischemic or normal retinas were treated with the neutralizing anti-Hmgb1 antibody or recombinant Hmgb1 protein respectively. To elucidate in which cell type Hmgb1 exerts its effect, primary retinal ganglion cell (RGC) cultures and glia RGC cocultures were treated with Hmgb1. To clarify the downstream signaling pathways involved in Hmgb1-induced effects in the ischemic retina, receptor for advanced glycation end products (Rage)-deficient mice (RageKO) were used.

RESULTS

Hmgb1 is accumulated in the vitreous humor 24 hours after IR. Inhibition of Hmgb1 activity with neutralizing antibody significantly decreased retinal damage after IR, whereas treatment of retinas or retinal cells with Hmgb1 induced a loss of RGCs. The analysis of RageKO versus wild-type mice showed significantly reduced expression of proinflammatory genes 24 hours after reperfusion and significantly increased survival of ganglion cell layer neurons 7 days after IR injury.

CONCLUSIONS

These results suggest that an increased level of Hmgb1 and signaling via the Rage contribute to neurotoxicity after retinal IR injury.

The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulum stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01-1 μg/ml) for 24h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.

PAMAM-Arg is a cationic arginine-grafted polyamidoamine (PAMAM) dendrimer. In the previous study, we reported that PAMAM-Arg facilitates transfection in a range of mammalian cell types. In the present study, we investigated the transfection efficiency of PAMAM-Arg in primary cortical cultures, which are known to be extremely vulnerable to exogenous gene transfection. PAMAM-Arg/DNA complexes showed particularly high transfection efficiencies and low cytotoxicity in primary cortical cells, as compared to other gene carriers such as, native PAMAM, polyethylenimine (BPEI), and Lipofectamine. Efficient transfection was not limited to neurons but extended to all three glial cells, astrocytes, microglia, and oligodendrocytes, present in these primary cortical cultures. The potential use of PAMAM-Arg was demonstrated by efficient gene knock-down by transfecting HMGB1 shRNA-expressing plasmid. The numbers of green fluorescent protein (GFP)-positive and HMGB1-negative cells indicated that PAMAM-Arg/shRNA-expressing plasmid complex suppressed target gene expression in over 40% of cells, which is the highest level achieved to date in primary cortical culture by any gene carrier. Here, we present evidence of the successful delivery and expression of both a reporter gene and of a shRNA-expressing plasmid in primary cortical cells, which demonstrates the potential of PAMAM-Arg for mediating gene delivery to primary neuronal cells.

OBJECTIVE

To investigate the effect of delayed ethyl pyruvate (EP) delivery on distant organ injury, survival time and serum high mobility group box 1 (HMGB1) levels in rats with experimental severe acute pancreatitis (SAP).

METHODS

A SAP model was induced by retrograde injection of artificial bile into the pancreatic ducts of rats. Animals were divided randomly into three groups (n = 32 in each group): sham group, SAP group and delayed EP treatment group. The rats in the delayed EP treatment group received EP (30 mg/kg) at 12 h, 18 h and 30 h after induction of SAP. Animals were sacrificed, and samples were obtained at 24 h and 48 h after induction of SAP. Serum HMGB1, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (Cr) levels were measured. Lung wet-to-dry-weight (W/D) ratios and histological scores were calculated to evaluate lung injury. Additional experiments were performed between SAP and delayed EP treatment groups to study the influence of EP on survival times of SAP rats.

RESULTS

Delayed EP treatment significantly reduced serum HMGB1 levels, and protected against liver, renal and lung injury with reduced lung W/D ratios (8.22 +/- 0.42 vs 9.76 +/- 0.45, P < 0.01), pulmonary histological scores (7.1 +/- 0.7 vs 8.4 +/- 1.1, P < 0.01), serum AST (667 +/- 103 vs 1 368 +/- 271, P < 0.01), ALT (446 +/- 91 vs 653 +/- 98, P < 0.01) and Cr (1.2 +/- 0.3 vs 1.8 +/- 0.3, P < 0.01) levels. SAP rats had a median survival time of 44 h. Delayed EP treatment significantly prolonged median survival time to 72 h (P < 0.01).

CONCLUSIONS

Delayed EP therapy protects against distant organ injury and prolongs survival time via reduced serum HMGB1levels in rats with experimental SAP. EP may potentially serve as an effective new therapeutic option against the inflammatory response and multiple organ dysfunction syndrome (MODS) in SAP patients.

Necrosis is a characteristic feature of advanced solid tumors. Released necrotic factors, also referred to as damage associated molecular patterns (DAMPs), are known to critically impact the tumor microenvironment by enhancing angiogenesis or influencing the immune response. We have recently shown that DAMPs can act as chemoattractants and activators of granulocytes. We demonstrate that necrotic material from both normal and tumor cells promotes proliferation and trafficking of human mesenchymal stem cells (MSCs). We characterize the protein high mobility group box 1 (HMGB1) as a crucial member of DAMPs within necrotic material. In addition, we show that DAMPs interfere with expression of indoleamine 2, 3-dioxygenase (IDO) in MSCs. The biological activity of necrotic material toward MSCs is abolished once these DAMPs are oxidized. MSCs found within tumor tissue can act as immunoregulatory cells and are able to promote tumor metastasis, thus playing a crucial role within the tumor microenvironment. Here, we reveal DAMPs to be crucial factors in the setting of MSC biology within the tumor microenvironment. The tumor microenvironment is characterized by reducing and hypoxic conditions that protect DAMPs from oxidation. Based on our results, oxidizing conditions should be considered for therapeutic approaches that target the tumor microenvironment.

Lipid mediators such as prostaglandin E2 (PGE2) play a central role during atherogenesis as a consequence of inflammation. PGE2 is produced from phospholipids by a cascade of enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E synthase (PGES). It is released by several cell types, including vascular smooth muscle cells (VSMCs). Recent work has shown that the secretory PLA2-IIA (sPLA2-IIA), the most abundant isoform of secreted PLA2 in VSMCs, acts as a potent cytokine and activates VSMCs through a positive feedback loop. High mobility group protein 1 (HMGB1), also known as amphoterin, is a ubiquitous protein that plays various roles in the nucleus. HMGB1 is released by necrotic cells and by immune cells in response to various inflammatory mediators and acts as a potent proinflammatory cytokine. The present study investigates the role of HMGB1 in the activation of sPLA2-IIA expression and PGE2 production in VSMCs. Recombinant HMGB1 slightly activated the sPLA2-IIA, COX-2, and mPGES-1 genes but dramatically stimulated these genes in VSMCs that had been incubated with the proinflammatory cytokine IL-1beta for 24 h. This effect was accompanied by significantly increased PGE2 release. Induction of the three known receptors of HMGB1, namely RAGE, TLR-2, and TLR-4, by IL-1beta suggests that proinflammatory cytokines sensitize VSMCs to HMGB1. This provides new insights into the role of HMGB1 in VSMCs, suggesting it may be essential for the progression of atherosclerosis.

Our seminal discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal systemic inflammation has prompted a new field of investigation for the development of experimental therapeutics. We previously reported that a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS), selectively inhibited endotoxin-induced HMGB1 release and conferred protection against lethal endotoxemia and sepsis. To investigate the underlying mechanisms by which TSN-SS effectively inhibits HMGB1 release, we examined whether TSN-SS stimulates HMGB1 uptake by macrophages and whether genetic depletion of HMGB1 receptors [e.g., toll-like receptors (TLR)2, TLR4, or the receptor for advanced glycation end product (RAGE)] or pharmacological inhibition of endocytosis impairs TSN-SS-facilitated HMGB1 cellular uptake. TSN-SS stimulated internalization of exogenous HMGB1 protein into macrophage cytoplasmic vesicles that subsequently co-localized with microtubule-associated protein light chain 3 (LC3)-positive punctate structures (likely amphisomes). Meanwhile, it time-dependently elevated cellular levels of internalized HMGB1, leading to elevated LC3-II production and aggregation. Although genetic depletion of TLR2, TLR4, and/or RAGE did not impair TSN-SS-mediated HMGB1 uptake, specific inhibitors of the clathrin- and caveolin-dependent endocytosis significantly impaired TSN-SS-mediated HMGB1 uptake. Co-treatment with a lysosomal inhibitor, bafilomycin A1, led to enhanced accumulation of endogenous LC3-II and internalized exogenous HMGB1 in TSN-SS/rHMGB1-treated macrophages. Taken together, these findings suggest that TSN-SS may facilitate HMGB1 endocytic uptake, and subsequently delivered it to LC3-positive vacuoles (possibly amphisomes) for degradation via a lysosome-dependent pathway.

BACKGROUND

High-mobility group box 1 protein (HMGB1) belonging to endogenous danger signals prolongs eosinophil survival and acts as a chemoattractant.

OBJECTIVE

The authors evaluated the role of HMGB1 in the pathogenesis of asthma characterized by eosinophilic airway inflammation.

METHODS

Firstly, HMGB1 expressions in induced sputum obtained from human asthmatics were determined. This was followed by an evaluation of the role of HMGB1 in a murine model of asthma using anti-HMGB1 antibodies. Then the effect of HMGB1 on the receptor of advanced glycation end products (RAGE) expressions on CD11b-CD11c(+) cells isolated from a murine model of asthma were measured to elucidate the mechanisms involved.

RESULTS

Sputum HMGB1 expressions were markedly higher in asthmatics than in normal controls, and were positively correlated with sputum eosinophilia and sputum TNF-α, IL-5 and IL-13 expressions. In a murine model of asthma, HMGB1 expressions in lung tissue and HMGB1 levels in bronchoalveolar lavage fluid were significantly elevated and eosinophilic airway inflammation, non-specific airway hyperresponsiveness, and pathological changes were attenuated by blocking HMGB1 activity. Furthermore, we found that enhanced RAGE expressions on CD11b-CD11c(+) also significantly decreased when HMGB1 activity was blocked.

CONCLUSIONS

Our findings suggest that HMGB1 plays a key role in the pathogenesis of clinical and experimental asthma characterized by eosinophilic airway inflammation.

Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so, and likely modulates their repair by the NER machinery. The abundance of HMGB1 suggests that it may play an important role in determining the sensitivity of cells to DNA damage under physiological, experimental, and therapeutic conditions.

Neurologic disease promoted by microbial pathogens, sterile injury, or neurodegeneration rapidly induces innate immunity in adjacent healthy tissue, which in turn contributes extensively to neurologic injury. With more recent focus on innate immune processes, it appears that necrotic, but not apoptotic, death mechanisms provoke inflammatory responses likely due to the release or production of endogenous ligands that activate resident immune cells of the central nervous system. These ligands comprise a diverse set of proteins, nucleic acids, and glycosaminoglycans, including heat shock proteins, HMGB1, RNA, DNA, hyaluronan, and heparin sulfate, that stimulate innate immune mechanisms largely through Toll-like receptors (TLRs). The blockade of interactions between endogenous ligands and TLRs may enable neuroprotective therapeutic strategies for a variety of neurologic diseases.

Acquisition of drug-resistant phenotypes is often associated with chemotherapy in osteosarcoma. Studies show that high-mobility group box 1 (HMGB1) plays an important role in facilitating autophagy and promotes drug resistance in osteosarcoma cells. In this study, we determined the targeting role of miR-22 to HMGB1 and the regulation of miR-22 on the HMGB1-mediated cell autophagy and on the cell proliferation, migration, and invasion of osteosarcoma cells. Results demonstrated that miR-22 well paired with the 3'-UTR of HMGB1 downregulated the HMGB1 expression and blocked the HMGB1-mediated autophagy during chemotherapy in osteosarcoma cells in vitro. Further study showed that the blockage of autophagy by miR-22 inhibited the osteosarcoma cell proliferation, migration, and invasion. In summary, this study implied the negative regulation of miR-22 on the HMGB1-mediated autophagy in osteosarcoma cells.

BACKGROUND

High mobility group box 1(HMGB1) overexpression has been reported in a variety of human cancers. However, the role of HMGB1 in hepatocellular carcinoma (HCC) remains unclear. The aim of present study was to analyze HMGB1 protein expression in tumor, para-tumor and normal tissue and to assess its prognostic significance for HCC after curative hepatectomy.

METHODS

The levels of HMGB1 mRNA and protein in tumor, para-tumor and normal tissue were evaluated in 11 HCC cases by Reverse Transcription-polymerase chain reaction (RT-PCR) and Western blot. Additionally, HMGB1 protein expression in 161 HCC was analyzed by immunohistochemistry and correlated with clinicopathological characteristics and survivals. Student's t-test, spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data.

RESULTS

By RT-PCR and Western blot, the levels of HMGB1 mRNA and protein were significantly higher in HCC, compared to that in para-tumor (p < 0.001) and normal tissue (p < 0.001). Immunohistochemical staining revealed that high expression of HMGB1 was detected in 42.9% (69/161) HCC cases. High expression of HMGB1 was significantly associated with incomplete encapsulation (p = 0.035) and advanced TNM stage (p = 0.036). Multivariate analysis showed that high expression of HMGB1 was an independent prognostic factor for both overall (p = 0.009, HR = 1.834, 95%CI: 1.167-2.881) and disease-free survival (p = 0.018, HR = 1.622, 95%CI: 1.088-2.419), along with tumor size. Subgroup analysis revealed that high expression of HMGB1 predicted poorer overall survival only for tumor >5 cm (p = 0.031), but not for tumor ≤5 cm (p = 0.101).

CONCLUSIONS

HMGB1 protein might contribute to the malignant progression of HCC, high expression of HMGB1 predicts poor prognosis for patients with HCC after curative hepatectomy, especially for patients with tumor >5 cm.

Sepsis, a leading cause of death in hospitalized patients, is characterized by lethal systemic inflammatory responses. JAK2 is an essential tyrosine kinase modulating immune responses. However, the implications of JAK2 in infectious disorders remain undetermined. Here, we report that JAK2 inhibitors rescue animals from polymicrobial sepsis in a clinically relevant time frame. JAK2 inhibition with AG490 prevents NF-kappaB activation, modulates macrophage activation, and restrains the production of inflammatory cytokines. The inhibition of JAK2 blunted TNF production in both macrophages and splenocytes in a concentration-dependent manner. JAK2 inhibition specifically prevents LPS-induced STAT3 tyrosine phosphorylation without affecting serine phosphorylation in macrophages. JAK2 inhibitor prevents the activation of the canonical p65RelA/p50NF-kappaB1 pathway but not the other NF-kappaB proteins. In vivo, JAK2 inhibition restrains serum TNF levels by modulating TNF production in the lung and the spleen and protects mice from lethal endotoxemia in a concentration-dependent manner. AG490 also inhibits extracellular release of HMGB1 from macrophages and prevents an increase in serum HMGB1 levels during sepsis. JAK2 inhibition started at 24 h after the onset of sepsis rescued the mice from polymicrobial sepsis. Our study is the first experimental evidence that JAK2 inhibitors may provide a pharmacological advantage for the treatment of sepsis in a clinically relevant time frame.

OBJECTIVE

High mobility group box-1(HMGB1) was identified as a DNA-binding protein that functions as a cofactor for proper transcriptional regulation in somatic cells. Extracellular HMGB1 acts as a potent proinflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Ethyl pyruvate (EP), a stable aliphatic ester derived from pyruvic acid, was first described as a pharmacological inhibitor of HMGB1 secretion. We designed this study to identify changes in HMGB1 expression in rat kidney tissues after ischemia reperfusion injury and effects of EP on the expression of HMGB1.

METHODS

Sprague-Dawley rats (200-300 g) were subjected to 40 minutes of renal warm ischemia. The animals were divided into 3 groups: sham group without warm ischemia, EP group (EP given before ischemia), and ischemic control group. Kidneys were harvested and serum creatinine and TNF-alpha measured at 6 hours, 1 day, 3 days, and 5 days after reperfusion. We performed immunohistochemical staining of HMGB1.

RESULTS

Serum creatinine and TNF-alpha level were elevated in the ischemic control group and the EP injection group. In the EP injection group, serum creatinine and TNF-alpha levels were lower than the ischemic control group. In the 40-minute ischemia-reperfusion model, HMGB1 expression increased at 6 hours after reperfusion and decreased gradually at 1, 3, and 5 days after reperfusion. HMGB1 expression was more distinct at the outer medullary area. intraperitoneal EP injection had no effect on HMGB1 expression.

CONCLUSIONS

From these results, we deduced that the preventive effect of EP on rat kidney ischemia-reperfusion injury was not due to the decreased expression of HMGB1 but the prevention of HMGB1 release.

High mobility group box (HMGB)-1 plays an important role in sepsis-associated death in experimental studies. Heme oxygenase-1 (HO-1) inducers were reported to reduce HMGB1 release in experimental sepsis. Previously, we reported on the importance of the β₁-adrenergic receptor and protein kinase A pathway in the regulation of HO-1 expression by isoproterenol (ISO) in RAW 264.7 cells. We investigated whether ISO reduces HMGB1 release in LPS-activated RAW 264.7 cells and improves survival rate in septic mice due to HO-1 induction. ISO concentration-dependently increased HO-1 via Nrf-2 translocation and inhibited release of HMGB1 through the β₁-adrenergic receptor (β₁-AR) in LPS-activated RAW 264.7 cells. This conclusion was supported by the finding that dobutamine but not salbutamol increased HO-1 expression in both RAW 264.7 cells. ISO failed to inhibit HMGB1 release when HO-1 expression was suppressed by ZnPPIX, an HO-1 inhibitor in RAW 264.7 cells. ISO significantly inhibited phosphorylation of IκB-α and NF-κB-driven luciferase activity in LPS-activated RAW 264.7 cells. In addition, LY294002, a PI3K inhibitor, and SB203580, a p38 MAPK inhibitor, significantly inhibited not only HO-1 induction but also HMGB1 release by ISO. Importantly, ISO increased HO-1 protein expression in heart and lung tissues, reduced HMGB1 in plasma and increased survival rate in CLP-treated septic mice, which was significantly reversed by co-treatment with ZnPPIX. Taken together, we conclude that inhibition of HMGB1 release during sepsis via β₁-AR-mediated HO-1 induction is a novel mechanism for the beneficial effects of ISO in the treatment of sepsis.

High mobility group box 1 (HMGB1) is a critical mediator of lethal sepsis. Previously, we showed that apoptotic cells can activate macrophages to release HMGB1. During sepsis, apoptosis occurs primarily in lymphoid organs, including the spleen and thymus. Currently, it is unclear whether this accelerated lymphoid organ apoptosis contributes to systemic release of HMGB1 in sepsis. In this study, we report that splenectomy significantly reduces systemic HMGB1 release and improves survival in mice with polymicrobial sepsis. Treatment with a broad-spectrum caspase inhibitor reduces systemic lymphocyte apoptosis, suppresses circulating HMGB1 concentrations, and improves survival during polymicrobial sepsis, but fails to protect septic mice following splenectomy. These findings indicate that apoptosis in the spleen is essential to the pathogenesis of HMGB1-mediated sepsis lethality.

High mobility group box protein 1 (HMGB1), transcriptional activity regulatory protein is associated with most cancers including prostate cancer. To investigate the effects of down-regulation of HMGB1 expression, we have transfected LNCaP cells with four short hairpin RNA (shRNA) targeting HMGB1 plasmid vectors. Transfection with the four shRNAs efficiently and specifically reduced the HMGB1 expression in LNCaP cells. The gene silencing effects on HMGB1 expression were subsequently confirmed by RT-PCR and immunoblotting analyses. Down-regulation of HMGB1 expression resulted in the inhibition of cell growth in LNCaP prostate cancer cells and the decreased cell number was due to transfected cells undergoing apoptosis via caspase-3-dependent pathways. These findings suggest that HMGB1 is critical for the survival of prostate cancer cells and targeted knockdown of HMGB1 mRNA can be used as a strategy to kill prostate cancer cells. Our findings may have some potential therapeutic relevance for treating prostate cancer.

BACKGROUND

High-mobility group box-1 protein (HMGB1) has been known as a chromosomal protein for many years. HMGB1 has recently been shown to be a proinflammatory cytokine with a role in the immunopathogenesis of sepsis. Lipopolysaccharide-binding protein (LBP) has a central role in the innate immune response when the host is challenged by bacterial pathogens. Procalcitonin (PCT) has been suggested as a marker of severe bacterial infections and sepsis. The aim of the present study was to investigate levels of HMGB1, LBP and PCT in a well-characterised sepsis cohort. The study plan included analysis of the levels of the inflammatory markers in relation to the severity of infection, to the prognosis and to the ability to identify patients with bacteraemia.

METHODS

Patients suspected of having severe infections and admitted to a department of internal medicine were included in a prospective manner. Demographic data, comorbidity, routine biochemistry, microbiological data, infection focus, severity score and mortality on day 28 were recorded. Plasma and serum were sampled within 24 hours after admission. Levels of all studied markers (HMGB1, LBP, PCT, IL-6, C-reactive protein, white blood cell count and neutrophils) were measured with commercially available laboratory techniques.

RESULTS

A total of 185 adult patients were included in the study; 154 patients fulfilled our definition of infection. Levels of HMGB1, LBP and PCT were higher in infected patients compared with a healthy control group (P < 0.0001). Levels of HMGB1, LBP and PCT were higher in the severe sepsis group compared with the sepsis group (P < 0.01). No differences were observed in levels of the inflammatory markers in fatal cases compared with survivors. Levels of all studied markers were higher in bacteraemic patients compared with nonbacteraemic patients (P < 0.05). PCT performed best in a receiver-operator curve analysis discriminating between bacteraemic and nonbacteraemic patients (P < 0.05). HMGB1 correlated to LBP, IL-6, C-reactive protein, white blood cell count and neutrophils (P < 0.001). LBP correlated to PCT, IL-6 and C-reactive protein (P < 0.001).

CONCLUSIONS

Levels of HMGB1, PCT and LBP were higher in infected patients compared with those in healthy controls, and levels were higher in severe sepsis patients compared with those in sepsis patients. Levels of all studied inflammatory markers (HMGB1, LBP, PCT, IL-6) and infection markers (C-reactive protein, white blood cell count, neutrophils) were elevated among bacteraemic patients. PCT performed best as a diagnostic test marker for bacteraemia.

Success in treating severe sepsis will require relevant tools to monitor the patient immunoinflammatory status. This study aimed to investigate the feasibility of measuring a panel of immunological mediator mRNAs in whole blood and to study their prognostic values in septic shock patients. At the onset of shock, compared to healthy volunteers, mRNA levels in septic shock patients were increased for IL-10, IL-1beta, and high mobility group B1 (HMGB1) and decreased for transforming growth factor beta 1, the Th1, and Th2 transcription factors, T-bet and GATA-3, respectively. Single parameter analysis highlighted an increased expression of IL-10 and HMGB1 mRNA in nonsurvivors and a significant rise over time of GATA3 in survivors. Combining the expression levels of four genes, hierarchical cluster analysis showed that up to 95% of the patients with a similar outcome displayed transcriptional similarities. These results illustrate both the potential of whole blood mRNA quantification assays and the interest of a multiparametric strategy to better stratify septic patients.

BACKGROUND This study aimed to determine the role of miR-129-5p in irradiation-induced autophagy in breast cancer cells and to investigate its downstream regulation in autophagy-related radiosensitivity. MATERIAL AND METHODS Relative miR-129-5p expression in breast cancer cell lines MCF-7, MDA-MB-231, BT474, and BT549, and in 1 non-tumorigenic breast epithelial cell line, MCF-10A, was compared. The effect of miR-129-5p on irradiation-induced autophagy and radiosensitivity of the cancer cells was explored. The regulative effect of miR-129-5p on HMGB1 and the functional role of this axis in autophagy and radiosensitivity were also studied. RESULTS Ectopic expression of miR-129-5p sensitized MDA-MD-231 cells to irradiation, while knockdown of miR-129-5p reduced radiosensitivity of MCF-7 cells. MiR-129-5p overexpression inhibited irradiation-induced autophagy. HMGB1 is a direct functional target of miR-129-5p in breast cancer cells. MiR-129-5p may suppress autophagy and decrease radioresistance of breast cancer cells by targeting HMGB1. CONCLUSIONS The miR-129-5p/HMGB1 axis can regulate irradiation-induced autophagy in breast cancer and might be an important pathway in regulating radiosensitivity of breast cancer cells.

Chemotherapy has historically been thought to induce cancer cell death in an immunogenically silent manner. However, recent studies have demonstrated that therapeutic outcomes with specific chemotherapeutic agents (e.g. anthracyclines) correlate strongly with their ability to induce a process of immunogenic cell death (ICD) in cancer cells. This process generates a series of signals that stimulate the immune system to recognize and clear tumor cells. Extensive studies have revealed that chemotherapy-induced ICD occurs via the exposure/release of calreticulin (CALR), ATP, chemokine (C-X-C motif) ligand 10 (CXCL10) and high mobility group box 1 (HMGB1). This review provides an in-depth look into the concepts and mechanisms underlying CALR exposure, activation of the Toll-like receptor 3/IFN/CXCL10 axis, and the release of ATP and HMGB1 from dying cancer cells. Factors that influence the impact of ICD in clinical studies and the design of therapies combining chemotherapy with immunotherapy are also discussed.

OBJECTIVE

High mobility group box chromosomal protein 1 (HMGB1) has been implicated as a mediator of inflammation in rheumatoid arthritis (RA), while its role in juvenile idiopathic arthritis (JIA) has not been described. To evaluate the role of HMGB1 in the inflammatory process in JIA and its potential as a therapeutic target, we investigated whether extracellular HMGB1 is detectable in JIA and if so, to correlate the levels with established inflammatory markers and clinical measures.

METHODS

Matching samples of blood and synovial fluid (SF) were collected from 23 patients with JIA. Levels of HMGB1, soluble receptor for advanced glycation endproducts, S100A12, myeloid-related protein 8/14, and other inflammatory mediators were analyzed.

RESULTS

Significantly increased HMGB1 levels were recorded in SF compared to blood samples from patients with JIA. The amount of HMGB1 was highest in patients with early disease onset irrespective of disease duration. In contrast, the proinflammatory S100 protein and interleukin 8 were highest in patients in early phases of disease. Matrix metalloproteinase-3, a marker of cartilage destruction, was higher in patients with late disease onset, indicating similarities with RA in that patient subgroup.

CONCLUSIONS

Levels of extracellular HMGB1 are increased in the inflamed joints of patients with JIA. This warrants further studies of HMGB1 as a mediator of JIA pathogenesis as well as a biomarker for inflammatory activity and as a target for therapy. The variation in levels of HMGB1 and S100 proteins in relation to disease onset indicates a difference in inflammatory phenotype during disease progression.

It is generally believed that some inflammatory antigens can recognize Toll-like receptors on synovial fibroblasts (SFs) and then activate downstream signals, leading to the formation of RASFs and inducing rheumatoid arthritis (RA). The objective of the current work was to study on the hypothesis that outer PAMP (LPS) binds to the inner DAMP (HMGB1) and becomes a complex that recognizes TLRs/RAGE on SFs, thus initiating a signaling cascade that leads to the secretion of inflammatory cytokines and chemokines, production of tissue-destructive enzymes, and formation of RASFs, finally resulting in RA. Osteoarthritis synovial fibroblasts (OASFs) were co-cultured with HMGB1-LPS complex in vitro for five generations to induce the transformation of human SFs to RA-like SFs (tOASFs). Then, changes of tOASFs in cell cycle and apoptosis-autophagy balance were investigated in vitro, and the pathogenicity of tOASFs was evaluated in a SCID mouse model in vivo. In vitro cell cycle analysis showed more tOASFs passing through the G1/S checkpoint and moving to S or G2 phase. Flow cytometry and confocal microscopy showed that apoptosis was reduced and autophagy was enhanced significantly in tOASFs as compared with those in OASFs. The expression of certain receptors and adhesion molecules in tOASFs was upregulated. In vivo experiments showed that tOASFs attached to, invaded, and degraded the co-implanted cartilage. In addition, histochemistry showed excessive proliferation of tOASFs and the expression of matrix metalloproteinases (MMPs). Based on the above findings, we conclude that HMGB1-LPS complex could promote the formation of RASFs.

RIG-I-like receptors (RLRs) sense virus-derived RNA or polyinosinic-polycytidylic acid (poly IC) to exert antiviral immune responses. Here, we examine the mechanisms underlying the adjuvant effects of poly IC. Poly IC was taken up by dendritic cells (DCs), and it induced lysosomal destabilization, which, in turn, activated an RLR-dependent signaling pathway. Upon poly IC stimulation, cathepsin D was released into the cytoplasm from the lysosome to interact with IPS-1, an adaptor molecule for RLRs. This interaction facilitated cathepsin D cleavage of caspase 8 and the activation of the transcription factor NF-κB, resulting in enhanced cytokine production. Further recruitment of the kinase RIP-1 to this complex initiated the necroptosis of a small number of DCs. HMGB1 released by dying cells enhanced IFN-β production in concert with poly IC. Collectively, these findings suggest that cathepsin D-triggered, IPS-1-dependent necroptosis is a mechanism that propagates the adjuvant efficacy of poly IC.