Resveratrol, the most important ingredient extracted from Polygonum cuspidatum, exerts cytoprotective effects via anti-inflammatory actions in vitro. In this study, we investigated this effect of resveratrol on the lipopolysaccharide (LPS)-induced inflammatory response and its underlying molecular mechanism of action in RAW264.7 murine macrophages. Results showed that resveratrol down-regulated the expression of inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6), therefore, suppressed the production of nitric oxide and the secretion of IL-6 in LPS-stimulated RAW264.7 cells in a dose-dependent manner. Resveratrol also inhibited the translocation of high-mobility group box 1 (HMGB1) from the nucleus to the cytoplasm and of nuclear transcription factor kappa-B (NF-κB) p65 from the cytoplasm to the nucleus; it suppressed the phosphorylation of IκBα. Furthermore, these actions were mediated by suppressing the phosphorylation of signal transducer and activator of transcription (STAT)-1 and -3. In conclusion, these data indicate that resveratrol exerts anti-inflammatory effects, at least in part by reducing the release of HMGB1 and modulating the NF-κB and Janus kinase/STAT signaling pathways. Resveratrol could potentially be developed as a useful agent for the chemoprevention of inflammatory diseases.

In a wide variety of diseases, cell death represents both an outcome and an important step in pathogenesis. This duality occurs because cell death leads to the extracellular release of molecules and structures that can potently induce the innate immune system. These mediators include the alarmins which are endogenous cellular constituents that exit activated or dying cells to stimulate toll-like receptors (TLRs) as well as non-TLR receptors. Of alarmins, the nonhistone protein HMGB1 is the prototype. Like DNA and RNA, HMGB1 can translocate from cells as they die. The activity of HMGB1 may reflect its interaction with other molecules such as LPS, DNA, and cytokines. In addition to alarmins, dead and dying cells can release subcellular organelles called microparticles that contain cytoplasmic and nuclear constituents, including DNA and RNA. These particles can impact on many cell types to induce inflammation. The release of HMGB1 and microparticles shows important similarities, occurring with cell death as well as stimulation of certain but not all TLRs. Furthermore, nitric oxide can induce the release of both. These observations suggest that the products of dead cells can serve as important mediators to drive immune responses and promote inflammation and autoreactivity.

High-mobility group protein box-1 (HMGB1) is a proinflammatory involved in many inflammatory diseases. However, its roles in intracerebral hemorrhage (ICH) remain unknown. The purpose of this study was to examine the correlation between changes in serum levels of HMGB1 following acute ICH and the severity of stroke as well as the underlying mechanism. Changes in serum levels of HMGB1 in 60 consecutive patients with primary hemispheric ICH within 12 hours of onset of symptoms were determined. The correlation of HMGB1 with disease severity, IL-6, and TNF-α was analyzed. Changes in HMGB1 levels were detected with ELISA and Western blot. Compared with normal controls, patients with ICH had markedly elevated levels of HMGB1, which was significantly correlated with the levels of IL-6 and TNF-α, NIHSS score at the 10th day, and mRS score at 3 months. In comparison with the control group, the levels of HMGB1 in the perihematomal tissue in mice with ICH increased dramatically, peaked at 72 hours, and decreased at 5 days. Meanwhile, heme could stimulate cultured microglia to release large amounts of HMGB1 whereas Fe(2+/3+) ions failed to stimulate HMGB1 production from microglia. Our findings suggest that HMGB1 may play an essential role in the ICH-caused inflammatory injury.

Manipulation of protein stability with small molecules has a great potential for both basic research and clinical therapy. Recently, we have developed a series of hybrid small molecules named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser) that induces degradation of target proteins via ubiquitin-proteasome system. Here we report the activities of SNIPER(ER) that targets estrogen receptor alpha (ERα) for degradation. SNIPER(ER) induced degradation of ERα and inhibited estrogen-dependent expression of pS2 gene in an estrogen-dependent breast cancer cell line MCF-7. A proteasome inhibitor MG132 and siRNA-mediated downregulation of cIAP1 abrogated the SNIPER(ER)-induced ERα degradation, suggesting that the ERα is degraded by proteasome subsequent to cIAP1-mediated ubiquitylation. Intriguingly, after the ERα degradation, the SNIPER(ER)-treated MCF-7 cells undergo rapid cell death. Detailed analysis indicated that SNIPER(ER) caused necrotic cell death accompanied by a release of HMGB1, a marker of necrosis, from the cells. Following the ERα degradation, reactive oxygen species (ROS) was produced in the SNIPER(ER)-treated MCF-7 cells, and an anti-oxidant N-acetylcysteine inhibited the necrotic cell death. These results indicate that SNIPER(ER) induces ERα degradation, ROS production and necrotic cell death, implying a therapeutic potential of SNIPER(ER) as a lead for the treatment of ERα-positive breast cancers.

Cancer stem cells/cancer-initiating cells (CICs) and their microenvironmental niche play a vital role in malignant tumour recurrence and metastasis. Cancer-associated fibroblasts (CAFs) are major components of the niche of breast cancer-initiating cells (BCICs), and their interactions may profoundly affect breast cancer progression. Autophagy has been considered to be a critical process for CIC maintenance, but whether it is involved in the cross-talk between CAFs and CICs to affect tumourigenesis and pathological significance has not been determined. In this study, we found that the presence of CAFs containing high levels of microtubule-associated protein 1 light chain 3 (LC3II), a marker of autophagosomes, was associated with more aggressive luminal human breast cancer. CAFs in human luminal breast cancer tissues with high autophagy activity enriched BCICs with increased tumourigenicity. Mechanistically, autophagic CAFs released high-mobility group box 1 (HMGB1), which activated its receptor, Toll-like receptor (TLR) 4, expressed by luminal breast cancer cells, to enhance their stemness and tumourigenicity. Furthermore, immunohistochemistry of 180 luminal breast cancers revealed that high LC3II/TLR4 levels predicted an increased relapse rate and a poorer prognosis. Our findings demonstrate that autophagic CAFs play a critical role in promoting the progression of luminal breast cancer through an HMGB1-TLR4 axis, and that both autophagy in CAFs and TLR4 on breast cancer cells constitute potential therapeutic targets. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chemotherapy, radiation therapy, as well as targeted anticancer agents can induce clinically relevant tumor-targeting immune responses, which critically rely on the antigenicity of malignant cells and their capacity to generate adjuvant signals. In particular, immunogenic cell death (ICD) is accompanied by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which altogether confer a robust adjuvanticity to dying cancer cells, as they favor the recruitment and activation of antigen-presenting cells. ICD-associated DAMPs include surface-exposed calreticulin (CALR) as well as secreted ATP, annexin A1 (ANXA1), type I interferon, and high-mobility group box 1 (HMGB1). Additional hallmarks of ICD encompass the phosphorylation of eukaryotic translation initiation factor 2 subunit-α (EIF2S1, better known as eIF2α), the activation of autophagy, and a global arrest in transcription and translation. Here, we outline methodological approaches for measuring ICD markers in vitro and ex vivo for the discovery of next-generation antineoplastic agents, the development of personalized anticancer regimens, and the identification of optimal therapeutic combinations for the clinical management of cancer.

BACKGROUND

High-sensitivity sandwich ELISA methods have been developed using chemiluminescent substrates. HMGB1 (high mobility group box 1) protein has been shown to play a critical role in several inflammatory diseases and it may be involved in the development of atherosclerosis.

METHODS

Anti-human HMGB1 monoclonal antibodies and anti-peptide polyclonal antibodies against the peptide sequence (KPDAAKKGVVKAEK) with high antigenicity and different from the sequence of HMGB2 were developed, and the antibodies were used to construct sandwich ELISA methods with a chromogenic substrate (TMBZ) and a chemiluminescent substrate (PS-atto). Highly purified human HMGB1 was used as a standard material and high-sensitivity CRP was measured to compare with HMGB1.

RESULTS

The analytical characteristics of the ELISA method we developed were validated inter-assay and intra-assay CVs were <10%, and the detection limit was 0.3 microg/l by the chemiluminescent method and 1 microg/l with the chromogenic substrates. HMGB1 was detected in the serum of patients with acute coronary syndrome (ACS). When a cut-off of 0.6 microg/l HMGB1 upon admission to the intensive care unit (ICU) was used, the risk of developing an acute cardiac event within 1 month after discharge of ACS patients with an abnormal HMGB1 was significantly higher than for the patients with normal values (P<0.0001). The usefulness of HMGB1 as an acute prognostic marker was suggested.

CONCLUSIONS

The assay is easy to perform and suitable for use in the hospital laboratory and for screening large populations. HMGB1 is detectable in the serum of ACS patients and that the serum concentration of HMGB1 may be a prognostic indicator in ACS patients.

Stress conditions affecting the functions of the endoplasmic reticulum (ER) cause the accumulation of unfolded proteins. ER stress is counteracted by the unfolded-protein response (UPR). However, under prolonged stress the UPR initiates a proapoptotic response. Mounting evidence indicate that the ER chaperone calnexin is involved in apoptosis caused by ER stress. Here, we report that overexpression of calnexin in Schizosaccharomyces pombe induces cell death with apoptosis markers. Cell death was partially dependent on the Ire1p ER-stress transducer. Apoptotic death caused by calnexin overexpression required its transmembrane domain (TM), and involved sequences on either side of the ER membrane. Apoptotic death caused by tunicamycin was dramatically reduced in a strain expressing endogenous levels of calnexin lacking its TM and cytosolic tail. This demonstrates the involvement of calnexin in apoptosis triggered by ER stress. A genetic screen identified the S. pombe homologue of the human antiapoptotic protein HMGB1 as a suppressor of apoptotic death due to calnexin overexpression. Remarkably, overexpression of human calnexin in S. pombe also provoked apoptotic death. Our results argue for the conservation of the role of calnexin in apoptosis triggered by ER stress, and validate S. pombe as a model to elucidate the mechanisms of calnexin-mediated cell death.

BACKGROUND

High-mobility group box 1 (HMGB1) is a late mediator of lethal systemic inflammation. Acute liver failure (ALF) has been shown to trigger systemic inflammation in clinical and animal studies. To evaluate the possibility of HMGB1 cytoplasmic translocation in ALF, we determined whether HMGB1 is released in hepatocytes and end organ in patients with liver failure/injury.

METHODS

HepG2 cell were stimulated with LPS or TNF-α, the increase of HMGB1 extracellularly in the culture medium and intracellularly in various cellular fractions were determined by western blot or immunocytochemistry. To observe sub-cellular location of HMGB1 in hepatocytes, liver specimens were obtained from 6 patients with ALF caused by HBV infection, 10 patients with chronic viral hepatitis B, 6 healthy controls, as well as animals model of ALF by intraperitoneal administration of D-GalN (600 mg/kg) and LPS (0.5 mg/kg).

RESULTS

In HepG2 cell culture, LPS or TNF actively induced HMGB1 cytoplasmic translocation and release in a time- and dose-dependent fashion. In animal model of ALF, cytoplasmic HMGB1 translocation was observed in hepatocyts as early as 3 hours post onset of ALF. In patients with ALF caused by HBV infection, cytoplasmic HMGB1 translocation was similarly observed in some hepatocytes of the liver specimen.

CONCLUSIONS

Cytoplasmic HMGB1 translocation may occur during ALF, which may potentially contribute to the pathogenesis of liver inflammatory diseases.

The High Mobility Group B1 (HMGB1) protein plays important roles in both intracellular (reductive) and extracellular (oxidative) environments. We have carried out quantitative investigations of the redox chemistry involving Cys22 and Cys44 of the HMGB1 A-domain, which form an intramolecular disulfide bond. Using NMR spectroscopy, we analyzed the real-time kinetics of the redox reactions for the A-domain in glutathione and thioredoxin systems, and also determined the standard redox potential. Thermodynamic experiments showed that the Cys22-Cys44 disulfide bond stabilizes the folded state of the protein. These data suggest that the oxidized HMGB1 may accumulate even in cells under oxidative stress.

Neutrophil elastase (NE) plays an important role in the progression of acute lung injury (ALI). Sivelestat sodium hydrate (Sivelestat) is a highly specific synthetic inhibitor of NE. High mobility group box 1 (HMGB1) is one of the key mediators in the development of sepsis. The aim of this study was to evaluate the effect of sivelestat and to determine whether it can reduce lipopolysaccharide (LPS)-induced acute lung injury in rats. Rats were randomly divided into a negative control group, an LPS-induced sepsis group, and a group treated with sivelestat prior to LPS administration. Animals in the sivelestat group received a bolus of 10 mg/kg of sivelestat injected into the intraperitoneal cavity before the LPS treatment. Furthermore, rats were administered sivelestat at 0, 1, 3, and 6 h following LPS treatment. We measured cytokine and HMGB1 levels in the serum after the induction of sepsis. In addition, we observed histopathology, wet/dry weight ratio, inducible nitric oxide synthase and HMGB1 expression in the lung tissue. Lung histopathology was significantly improved in the sivelestat group compared to the LPS group. Serum and pulmonary HMGB1 levels were lower over time among sivelestat-treated animals. Furthermore, inhibition of NF-kappaB activity was observed with the administration of sivelestat. These results suggest that sivelestat reduces LPS-induced lung injury at least partially by inhibiting inflammation and NF-kappaB activity.

High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

HMGB1 (high mobility group box 1) is a multifunctional, ubiquitous protein located inside and outside cells that plays a critical role in various physiological and pathological processes including cell development, differentiation, inflammation, immunity, metastasis, metabolism, and death. Increasing evidence demonstrates that HMGB1-dependent autophagy promotes chemotherapy resistance, sustains tumor metabolism requirements and T cell survival, prevents polyglutamine aggregates and excitotoxicity, and protects against endotoxemia, bacterial infection, and ischemia-reperfusion injury in vitro or in vivo. In contrast, HMGB1 may not be required for autophagy in some organs such as the liver and heart. Understanding HMGB1-dependent and -independent autophagy in more detail will provide insight into the integrated stress response and guide HMGB1-based therapeutic intervention.

HMGB1 is a nuclear protein that signals tissue damage, as it is released by cells dying traumatically or secreted by activated innate immunity cells. Extracellular HMGB1 elicits the migration to the site of tissue damage of several cell types, including inflammatory cells and stem cells. The identity of the signaling pathways activated by extracellular HMGB1 is not known completely: We reported previously that ERK and NF-kappaB pathways are involved, and we report here that Src is also activated. The ablation of Src or inhibition with the kinase inhibitor PP2 blocks migration toward HMGB1. Src associates to and mediates the phosphorylation of FAK and the formation of focal adhesions.

BACKGROUND

Acetaminophen (APAP) overdose induces massive hepatocyte necrosis. Necrotic tissue releases high mobility group B1 (HMGB1), and HMGB1 contributes to liver injury. Even though blockade of HMGB1 does not protect against APAP-induced acute liver injury (ALI) at 9 h time point, the later time points are not studied and the role of HMGB1 in APAP overdose is unknown, it is possible that neutralization of HMGB1 might improve hepatocyte regeneration. This study aims to test whether blockade of HMGB1 improves hepatocyte regeneration after APAP overdose.

METHODS

Male C57BL/6 mice were treated with a single dose of APAP (350 mg/kg). 2 hrs after APAP administration, the APAP challenged mice were randomized to receive treatment with either anti-HMGB1 antibody (400 μg per dose) or non-immune (sham) IgG every 24 hours for a total of 2 doses.

RESULTS

24 hrs after APAP injection, anti-HMGB1 therapy instead of sham IgG therapy significantly improved hepatocyte regeneration microscopically; 48 hrs after APAP challenge, the sham IgG treated mice showed 14.6% hepatic necrosis; in contrast, blockade of HMGB1 significantly decreased serum transaminases (ALT and AST), markedly reduced the number of hepatic inflammatory cells infiltration and restored liver structure to nearly normal; this beneficial effect was associated with enhanced hepatic NF-κB DNA binding and increased the expression of cyclin D1, two important factors related to hepatocyte regeneration.

CONCLUSIONS

HMGB1 impairs hepatocyte regeneration after APAP overdose; Blockade of HMGB1 enhances liver recovery and may present a novel therapy to treat APAP overdose.

High mobility group box protein 1 (HMGB1) was previously considered a strict nuclear protein, but lately data are accumulating on its extranuclear functions. In addition to its potent proinflammatory capacities, HMGB1 has a prominent role in a number of processes of specific interest for the placenta. Our overall aim was to investigate the expression of HMGB1 in human term placenta and elucidate a potential difference in HMGB1 expression comparing vaginal deliveries with elective Caesarean sections. In addition, placentas from normal pregnancies were compared with placentas from pregnancies complicated by pre-eclampsia. Twenty-five placentas, 12 from normal term pregnancies and 13 from pregnancies complicated by pre-eclampsia were analysed with immunohistochemistry for HMGB1 and its putative receptors; receptor for advanced glycation end-products (RAGE), Toll-like receptor 2 (TLR2) and TLR4. We present the novel finding that in addition to a strong nuclear HMGB1 expression in almost all cells in investigated placentas, an individual variation of cytoplasmic HMGB1 expression was detected in the syncytiotrophoblast covering the peripheral chorionic villi, by cells in the decidua and in amnion. Production of HMGB1 was confirmed by in situ hybridization. Although labour can be described as a controlled inflammatory-like process no differences in HMGB1 expression could be observed comparing active labour and elective Caesarean sections. However, a tendency towards a higher expression of cytoplasmic HMGB1 in the decidua from women with pre-eclampsia was demonstrated. The abundant expression of the receptors RAGE, TLR2 and TLR4 implicates a local capability to respond to HMGB1, although the precise role in the placenta remains to be elucidated.

Granulomatous nephritis can be triggered by diverse factors and results in kidney failure. However, despite accumulating data about granulomatous inflammation, pathogenetic mechanisms in nephritis remain unclear. The DNA-binding high-mobility group box-1 protein (HMGB1) initiates and propagates inflammation when released by activated macrophages, and functions as an 'alarm cytokine' signaling tissue damage. In this study, we showed elevated HMGB1 expression in renal granulomas in rats with crystal-induced granulomatous nephritis caused by feeding an adenine-rich diet. HMGB1 levels were also raised in urine and serum, as well as in monocyte chemoattractant protein-1 (MCP-1), a mediator of granulomatous inflammation. Injection of HMGB1 worsened renal function and upregulated MCP-1 in rats with crystal-induced granulomatous nephritis. HMGB1 also induced MCP-1 secretion through mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) pathways in rat renal tubular epithelial cells in vitro. Hmgb1(+/-) mice with crystal-induced nephritis displayed reduced MCP-1 expression in the kidneys and in urine and the number of macrophages in the kidneys was significantly decreased. We conclude that HMGB1 is a new mediator involved in crystal-induced nephritis that amplifies granulomatous inflammation in a cycle where MCP-1 attracts activated macrophages, resulting in excessive and sustained HMGB1 release. HMGB1 could be a novel target for inhibiting chronic granulomatous diseases.

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.

'Alarmins' are a group of proteins or molecules that are released from cells during cellular demise to alert the host immune system. Two of them, Interleukin-33 (IL-33) and high-mobility group box-1 (HMGB1), share many similarities of cellular localization, functions and involvement in various inflammatory pathologies including hepatitis. The expressions of IL-33 and HMGB1, and their receptors ST2 and receptor for advanced glycation end products (RAGE), are substantially up-regulated during acute and chronic hepatitis. Recent data evidence a possible protective role of IL-33/ST2 axis during liver injury. A contrast in expression of IL-33 and HMGB1 alarmins were associated with type of hepatocellular death mediated by immune cells or hepato-toxic agents. The massive release of active form of IL-33 from hepatocytes may affect the recruitment and activation of its ST2-positive target immune cells in the liver to confer its alarmin functions. This review highlights the emerging roles of alarmin proteins in various liver pathologies, by focusing on classical HMGB1 and a newly discovered alarmin, the IL-33.

OBJECTIVE

To examine how high-mobility group box 1 (HMGB1) regulates hepatocyte apoptosis and, furthermore, to determine whether glycyrrhizin (GL), a known HMGB1 inhibitor, prevents HMGB1-induced hepatocyte apoptosis.

METHODS

A human hepatocellular carcinoma cell line stably transfected with a bile acid transporter (Huh-BAT cells), were used in this study. Apoptosis was quantified using 4',6-diamidino-2-phenylindole dihydrochloride staining and the APO Percentage apoptosis assay, and its signaling cascades were explored by immunoblot analysis. Kinase signaling was evaluated by immunoblotting and by using selective inhibitors. It is also tried to identify hepatocyte apoptosis affected by the HMGB1 inhibitor, GL.

RESULTS

HMGB1 increased cellular apoptosis in Huh-BAT cells. HMGB1 led to increased cytochrome c release from mitochondria into the cytosol, and induced the cleavage of procaspase 3. However, it did not affect the activation of caspase 8. HMGB1-induced caspase 3 activation was significantly attenuated by the p38 inhibitor SB203580. GL significantly attenuated HMGB1-induced hepatocyte apoptosis. GL also prevented HMGB1-induced cytochrome c release and p38 activation in Huh-BAT cells.

CONCLUSIONS

The present study demonstrated that HMGB1 promoted hepatocyte apoptosis through a p38-dependent mitochondrial pathway. In addition, GL had an anti-apoptotic effect on HMGB1-treated hepatocytes.

DNA-based vaccines, while highly immunogenic in mice, generate significantly weaker responses in primates. Therefore, current efforts are aimed at increasing their immunogenicity, which include optimizing the plasmid/gene, the vaccine formulation and method of delivery. For example, co-immunization with molecular adjuvants encoding an immunomodulatory protein has been shown to improve the antigen (Ag)-specific immune response. Thus, the incorporation of enhancing elements, such as these, may be particularly important in the influenza model in which high titered antibody (Ab) responses are critical for protection. In this regard, we compared the ability of plasmid-encoded high-mobility group box 1 protein (HMGB1), a novel cytokine in which we have previously mutated in order to increase DNA vaccine immunogenicity, with boost Ag-specific immune responses during DNA vaccination with influenza A/PR/8/34 nucleoprotein or the hemagglutinin of A novel H1N1/09. We show that the HMGB1 adjuvant is capable of enhancing adaptive effector and memory immune responses. Although Ag-specific antibodies were detected in all vaccinated animals, a greater neutralizing Ab response was associated with the HMGB1 adjuvant. Furthermore, these responses improved CD8 T+-cell effector and memory responses and provided protection against a lethal mucosal influenza A/PR/8/34 challenge. Thus, co-immunization with HMGB1 has strong in vivo adjuvant activity during the development of immunity against plasmid-encoded Ag.

HMGB1 is a chromosome-binding protein that also acts as a damage-associated molecular pattern molecule. It has potent proinflammatory effects and is one of key mediators of organ injury. Evidence from research has revealed its involvement in the signaling mechanisms of Toll-like receptors and the receptor for advanced glycation end-products in organ injury. HMGB1-mediated organ injuries are acute damage including ischemic, mechanical, allograft rejection and toxicity, and chronic diseases of the heart, kidneys, lungs, and brain. Strategies against HMGB1 and its associated cellular signal pathways need to be developed and may have preventive and therapeutic potentials in organ injury.

Kupffer cells (KCs) were a significant source of cytokine release during the early stage of severe burns. High mobility group box protein 1 (HMGB1) was recently identified as a new type of proinflammatory cytokine. The ability of HMGB1 to generate inflammatory responses after burn trauma has not been well characterized. KCs were isolated from sham animals and rats with a 30% full-thickness burn, and then were stimulated with increasing concentrations of HMGB1. The levels of Tumor necrosis factor (TNF)-α and interleukin (IL)-1β in culture supernatant were measured by enzyme-linked immunosorbent assay. Northern blot analysis was performed to detect the expressions of TNF-α and IL-1β mRNAs. The activities of p38 MAPK and JNK (by Western blot analysis) as well as NF-κB (by EMSA) in KCs were also examined. As a result, HMGB1 in vitro upregulated expressions of TNF-α and IL-1β of KCs in a dose-dependent manner, and HMGB1 promoted KCs from burn rats to produce significantly more TNF-α and IL-1β proteins than those from sham animals. After harvested from burn rats, KCs were pre-incubated with anti-TLR2 or anti-TLR4 antibody prior to HMGB1 administration. HMGB1 exposure not only significantly increased expressions of TNF-α and IL-1β mRNAs in KCs from burn rats, but also enhanced activities of p38 MAPK, JNK and NF-κB. However, these upregulation events were all reduced by pre-incubation with anti-TLR2 or anti-TLR4 antibody. These results indicate that HMGB1 induces proinflammatory cytokines production of KCs after sever burn injury, and this process might be largely dependent on TLRs-dependent MAPKs/NF-κB signal pathway.

OBJECTIVE

To determine whether cerebrospinal fluid (CSF) levels of high mobility group box 1 (HMGB1) or heat shock protein 72 (Hsp72) are elevated in patients with meningitis.

METHODS

Prospective study of four cohorts of patients.

METHODS

Intensive care unit and infectious disease clinic of pediatrics at the Xiangya Hospital.

METHODS

A total of 104 children (13 with bacterial meningitis, 38 with aseptic meningitis, 7 with tuberculous meningitis, and 46 without meningitis).

METHODS

None.

RESULTS

At the time of admission, CSF samples were obtained from 104 patients with suspected meningitis and examined for the presence of invading pathogens, changes in CSF white blood cell counts, and protein and/or glucose concentrations. Based on CSF parameters, 13, 38, and 7 patients were diagnosed as having bacterial, aseptic, and tuberculous meningitis, respectively. All CSF samples were assayed for HMGB1 or Hsp72 using semiquantitative Western blot analysis. CSF levels of HMGB1 were elevated in patients with bacterial meningitis or aseptic meningitis but were four times higher in patients with bacterial meningitis vs. aseptic meningitis. There was a significant correlation between CSF HMGB1 levels and CSF white blood cell counts and glucose levels in patients with bacterial meningitis. Similarly, CSF levels of Hsp72 were significantly elevated in patients with bacterial meningitis or tuberculous meningitis and correlated well with CSF white blood cell counts in patients with bacterial meningitis or tuberculous meningitis.

CONCLUSIONS

CSF levels of HMGB1 and Hsp72 were significantly higher in patients with bacterial meningitis than those with aseptic meningitis and correlated well with CSF white blood cell counts in patients with bacterial (but not aseptic) meningitis.