Exosomes in Sepsis.

Sepsis is a severe state of infection with high mortality. Pathogen-associated molecular patterns and damage-associated molecular patterns (DAMPs) initiate dysregulated systemic inflammation upon binding to pattern recognition receptors. Exosomes are endosome-derived vesicles, which carry proteins, lipids and nucleic acids, and facilitate intercellular communications. Studies have shown altered contents and function of exosomes during sepsis. In sepsis, exosomes carry increased levels of cytokines and DAMPs to induce inflammation. Exosomal DAMPs include, but are not limited to, high mobility group box 1, heat shock proteins, histones, adenosine triphosphate, and extracellular RNA. Exosomes released during sepsis have impact on multiple organs, including the lungs, kidneys, liver, cardiovascular system, and central nervous system. Here, we review the mechanisms of inflammation caused by exosomes, and their contribution to multiple organ dysfunction in sepsis.

Serum high-mobility group box 1 as a predictive marker for cytotoxic chemotherapy-induced lung injury in patients with lung cancer and interstitial lung disease.

BACKGROUND: High-mobility group box 1 (HMGB1) is a pro-inflammatory protein, that is associated with tumorigenesis, interstitial lung disease (ILD), and acute lung injury. Chemotherapy-induced lung injury is a common and serious adverse event in patients with lung cancer and ILD, but its pathogenesis and predictive biomarkers are not known. This study aimed to investigate the predictive potential of serum HMGB1 levels for cytotoxic chemotherapy-induced lung injury in these patients. METHODS: From 743 patients with advanced lung cancer, we enrolled 83 consecutive patients with ILD and background-matched 83 patients without ILD. Additionally, 83 healthy subjects were included. After measuring baseline levels of serum HMGB1 in three groups, we evaluated the predictive values of baseline HMGB1 levels for cytotoxic chemotherapy-induced lung injury in patients with lung cancer and ILD. RESULTS: Higher levels of serum HMGB1 were independently associated with higher tumor burden, as assessed by total tumor size, and the presence of ILD. Twenty-five (30.1%) of patients with lung cancer and ILD experienced cytotoxic chemotherapy-induced lung injury within one year. Univariate Cox proportional hazards model showed that higher levels of HMGB1 and higher tumor burden were associated with disease onset. Moreover, multivariate analysis revealed that only HMGB1 was independently associated with this severe complication in patients with lung cancer and ILD. CONCLUSIONS: HMGB1 is a potential predictive blood biomarker for cytotoxic chemotherapy-induced lung injury in patients with lung cancer and ILD. This study also suggests a potential pathogenesis of this serious adverse event that tumor- and ILD-derived HMGB1 accelerates lung injury.

Atractylenolide III Enhances Energy Metabolism by Increasing the SIRT-1 and PGC1α Expression with AMPK Phosphorylation in C2C12 Mouse Skeletal Muscle Cells.

Targeting energy expenditure provides a potential alternative strategy for achieving energy balance to combat obesity and the development of type 2 diabetes mellitus (T2DM). In the present study, we investigated whether atractylenolide III (AIII) regulates energy metabolism in skeletal muscle cells. Differentiated C2C12 myotubes were treated with AIII (10, 20, or 50 µM) or metformin (2.5 mM) for indicated times. The levels of glucose uptake, the expressions of key mitochondrial biogenesis-related factors and their target genes were measured in C2C12 myotubes. AIII significantly increased the glucose uptake levels, and significantly increased the expressions of peroxisome proliferator-activated receptor coactivator-1α (PGC1α) and mitochondrial biogenesis-related markers, such as, nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM) and mitochondrial mass and total ATP contents. In addition, AIII significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of sirtuin1 (SIRT1). These results suggest that AIII may have beneficial effects on obesity and T2DM by improving energy metabolism in skeletal muscle.

Effects of hemoadsorption with a novel adsorbent on sepsis: in vivo and in vitro study.

BACKGROUND/AIMS: Hemoadsorption may improve outcomes for sepsis by removing circulating cytokines. We tested a new sorbent used for hemoadsorption. METHODS: CTR sorbent beads were filled into columns of three sizes: CTR0.5 (0.5 ml), CTR1 (1.0 ml) and CTR2 (2.0 ml) and tested using IL-6 capture in vitro. Next, rats were subjected to cecal ligation and puncture and randomly assigned to hemoadsorption with CTR0.5, CTR1, CTR2 or sham treatment. Plasma biomarkers were measured. RESULTS: In vitro, IL-6 removal was accelerated with increasing bead mass. In vivo, TNF, IL-6, IL-10, high mobility group box1, and cystatin C were significantly lower 24 h after CTR2 treatment. Seven-day survival rate was 50, 64, 63, and 73% for the sham, CTR0.5, CTR1, CTR2, respectively. CONCLUSION: CTR appeared to have a favorable effect on kidney function despite no immediate effects on cytokine removal. However, CTR2 beads did result in a late decrease of cytokines.

HMGB1 in renal ischemic injury.

Factors that initiate cellular damage and trigger the inflammatory response cascade and renal injury are not completely understood after renal ischemia-reperfusion injury (IRI). High-mobility group box-1 protein (HMGB1) is a damage-associated molecular pattern molecule that binds to chromatin, but upon signaling undergoes nuclear-cytoplasmic translocation and release from cells. Immunohistochemical and Western blot analysis identified HMGB1 nuclear-cytoplasmic translocation and release from renal cells (particularly vascular and tubular cells) into the venous circulation after IRI. Time course analysis indicated HMGB1 release into the venous circulation progressively increased parallel to increased renal ischemic duration. Ethyl pyruvate (EP) treatment blocked H(2)O(2) (oxidative stress)-induced HMGB1 release from human umbilical vein endothelial cells in vitro, and in vivo resulted in nuclear retention and significant blunting of HMGB1 release into the circulation after IRI. EP treatment before IRI improved short-term serum creatinine and albuminuria, proinflammatory cyto-/chemokine release, and long-term albuminuria and fibrosis. The renoprotective effect of EP was abolished when exogenous HMGB1 was injected, suggesting EP's therapeutic efficacy is mediated by blocking HMGB1 translocation and release. To determine the independent effects of circulating HMGB1 after injury, exogenous HMGB1 was administered to healthy animals at pathophysiological dose. HMGB1 administration induced a rapid surge in systemic circulating cyto-/chemokines (including TNF-α, eotaxin, G-CSF, IFN-γ, IL-10, IL-1α, IL-6, IP-10, and KC) and led to mobilization of bone marrow CD34+Flk1+ cells into the circulation. Our results indicate that increased ischemic duration causes progressively enhanced HMGB1 release into the circulation triggering damage/repair signaling, an effect inhibited by EP because of its ability to block HMGB1 nuclear-cytoplasmic translocation.

High-dose immunoglobulin preparations improve survival in a CLP-induced rat model of sepsis.

PURPOSE: The efficacy of intravenous immunoglobulin G in the treatment of patients with severe sepsis or septic shock is still being debated. We investigated the impact of high-dose immunoglobulin administration on the survival rate and serum high-mobility group box chromosomal protein 1 (HMGB1) level in a rat model of sepsis created by cecal ligation and puncture (CLP). METHODS: Rats received either CLP-induced sepsis or had additional immunoglobulin treatment in 1,500 or 300 mg/kg. After induction of sepsis and respective treatment conditions, pulmonary and renal tissues were examined histologically for pathological changes at postoperative hour (POH) 4, and serum cytokine and HMGB1 levels were measured at POH 4, 8, 20, and 44. Using other rats, we also observed the survival rate after CLP for 7 days. RESULTS: Treatment with immunoglobulin significantly improved survival rate at postoperative day 7 (73% in the high-dose group vs. 33% in the control group; p = 0.037). The serum lactate dehydrogenase, endotoxin, creatinine, and blood urea nitrogen levels were significantly lower in the high-dose group than in the other groups. The serum HMGB1 level had increased at 4 h postoperatively in the control group (10.2 ± 3.3 ng/mL) and low-dose group (10.3 ± 4.0 ng/mL), but it was significantly reduced in the high-dose group (4.2 ± 0.8 ng/mL) compared with the control group (p = 0.03). CONCLUSIONS: Our results suggest that high-dose immunoglobulin therapy may improve the serum endotoxin and HMGB1 levels and overall survival rate in sepsis by inhibiting the inflammation.

Ethyl pyruvate improves survival and ameliorates distant organ injury in rats with severe acute pancreatitis.

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.

Anti-high-mobility group box chromosomal protein 1 antibodies improve survival of rats with sepsis.

BACKGROUND: High-mobility group box chromosomal protein 1 (HMGB1) has recently been shown to be an important late mediator of endotoxin shock, intraabdominal sepsis, and acute lung injury, and a promising therapeutic target of severe sepsis. We sought to investigate the effect of antibodies to HMGB1 on severe sepsis in a rat cecal ligation and puncture (CLP) model. METHODS: Adult male Sprague-Dawley rats underwent CLP and then were randomly divided into two groups: treatment with anti-HMGB1 polyclonal antibodies, and non-immune IgG-treated controls. The serum HMGB1 concentrations were measured at ten time points (preoperatively, and postoperatively at 4, 8, 20, 32, and 48 h and at 3, 4, 5, and 6 days). Hematoxylin-eosin staining, elastica-Masson staining, and immunohistochemical staining for HMGB1 were performed on the cecum and the lung to assess pathological changes 24 h after the CLP procedure. RESULTS: Treatment with anti-HMGB1 antibodies significantly increased survival [55% (anti-HMGB1) vs. 9% (controls); P< 0.01]. The serum HMGB1 concentrations at postoperative hours 20 and 32 of the anti-HMGB1 antibody-treated animals were significantly lower than those of the controls (P < 0.05). Treatment with anti-HMGB1 antibodies markedly diminished the pathological changes and the number of HMGB1-positive cells in the cecum and the lung. CONCLUSIONS: The present study demonstrates that anti-HMGB1 antibodies are effective in the treatment of severe sepsis in a rat model, thereby supporting the relevance of HMGB1 eradication therapy for severe sepsis.

HMG-1 rediscovered as a cytokine.

High-mobility group-1 (HMG-1), an abundant, highly conserved cellular protein, is widely known as a nuclear DNA-binding protein that stabilizes nucleosome formation, facilitates gene transcription, and regulates the activity of steroid hormone receptors. We discovered that HMG-1 is a late mediator of delayed endotoxin lethality. When released by activated monocytes, it participates in the development of lethality and it activates downstream cytokine release. This review covers the general features of HMG-1 and its newly appreciated role as a cytokine.

Detection of HMGI-C in the peripheral blood of breast cancer patients.

The human high mobility group (HMG) protein (HMGI-C) belongs to the HMG family of architectural transcription factors which are expressed only during embryonic development, and not in normal adult tissues. Considerable interest has recently been shown in HMGI-C and its expression in a variety of neoplastic tissues, whereas no expression could be found in normal tissue adjacent to the tumour. So far, no data is available on the expression of HMGI-C in the peripheral blood of patients with solid tumours. In this study we analysed the expression of HMGI-C in peripheral blood samples of 61 patients with breast cancer and 35 healthy donors using a haemi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) technique. No HMGI-C could be detected in any of the healthy donors' samples. In the three prognostic groups according to the Nottingham Prognostic Score, the proportion of patients expressing HMGI-C differed significantly (P=0.001). The worse the prognosis was, the more patients expressed HMGI-C. This is the first report on the expression of HMGI-C in the peripheral blood of patients with breast cancer and our data suggest that this expression is correlated with a poor prognosis.

[Certain biochemical features of leukocytes in blast crisis of chronic myelogenous leukemia]. / Nekotorye biokhimicheskie osobennosti leikotsitov pri blastnom krize khronicheskogo mieloleikoza.

The content of nuclear high mobility group (HMG) proteins, activities of ornithine decarboxylase (ODC), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) and also glycosaminoglycan (GAG) content and composition were studied in leukocytes of patients with chronic myelogenous leukemia in the phase of blast crisis (BC CML). Myeloid and lymphoid cytochemical variants of BC CML differ by biochemical parameters. It is suggested, that the content of HMG-proteins, activities of ODC and PNP, and electrophoretic patterns of GAGs could be used in diagnostics of two main variants of BC CML.

Elevated levels of the chromosomal protein HMG 17 in chronic myelogenic leukemia.

The High Mobility Group protein HMG 17 has been isolated from human leukemia cells obtained from patients with chronic myelogenic leukemia (CML). The level of expression of HMG 17 was investigated Human leukemia cells have three times more HMG 17 than normal human leukocytes. Three other malignant tissues were also compared. Two of these breast adenocarcinoma and other intestine- also exhibit higher amounts of HMG 17. The elevated expression of HMG 17 suggests that the level of the protein may be associated with rates of cellular proliferation.

Differential expression of nuclear HMG1, HMG2 proteins and H1(zero) histone in various blood cells.

Changes in the levels of chromosomal high-mobility group proteins HMG1, HMG2 and histone H1 zero were investigated in blood cells of various types, proliferation activity and stage of differentiation. The relative amounts of proteins HMG1, HMG2 and histone H1 zero were evaluated densitometrically by SDS-PAGE of 5 per cent w/v perchloric acid extracts of blood cells. Concerning the HMG1 and HMG2, the main conclusions were: the expression of these HMG proteins was higher in malignant cells, namely leukemia cell lines, then in lymphocytes or granulocytes and the distribution of HMG1 and HMG2 was highly cell-specific. In comparison with lymphoid cells, the levels of HMG1/2 were higher in myeloid cells. The results revealed that in myeloid cells HMG2 prevails over HMG1. There was no direct correlation between HMG1/2 expression and proliferation activity. The levels of HMG1/2 did not depend on the transcription of chromatin either. However, there was some connection between irreversibly differentiated nonproliferating cells and a loss of HMG1/2 proteins. Reversibly differentiated leukemic cells retain their HMG1/2 levels. Similarly to HMG1/2,H1 zero showed a strong cell specificity. The level of H1 zero was different in the various blood cell types. As compared with lymphoid cells, the level of H1 zero was several-fold higher in myeloid cells, regardless of whether they were normal or malignant. Moreover, there was an accumulation of H1 zero in differentiating HL-60 cells accompanied by only a slight decline in cell proliferation; this agrees with the idea that H1 zero expression is not directly associated with the inhibition of cell growth. Rather higher expression of H1 zero is related to changes during cell differentiation.

[Nuclear proteins of human leukocytes in health and in chronic leukemias]. / Iadernye belki leikotsitov cheloveka v norme i pri khronicheskikh leikozakh.

The levels and fractional composition of nuclear proteins of human leukocytes (histones, total non-histone proteins and HMG-proteins) have been studied in healthy subjects and cases of leukemia. It has been shown that the levels of histones and total non-histone proteins in normal myeloid cells are, on the average, 1.5 times those in lymphoid cells. Leukemia has been shown to involve elevated concentrations of high-molecular components in the histone and HMG-protein fraction of white blood cells as well as a slight decrease in both high- and low-molecular components in the fraction of total non-histone proteins. This points to disturbances in the epigenomic regulation of the genome involved in leukemia.

cis-Diamminedichloroplatinum(II) selectively cross-links high mobility group proteins 1 and 2 to DNA in micrococcal nuclease accessible regions of chromatin.

The reaction of cis-diamminedichloroplatinum(II) with chicken erythrocyte nuclei produces covalent cross-linking of HMG proteins 1, 2 and E to DNA, in addition to cross-links amongst LMG proteins. This is supported by and consistent with the observations that all cross-links are chemically reversed by NaCN treatment, while only the cross-links involving the HMG proteins 1,2 and E are eliminated after a limited digestion with micrococcal nuclease. Having identified the subset of proteins selectively cross-linked to DNA by the bifunctional cis-(NH3)2PtCl2, a tentative model is proposed for the interactions between DNA and HMG proteins 1 and 2 in bulk chromatin. In addition, possible modes of action for this anti-neoplastic drug are suggested in light of these findings.

Kinetics of 125I-ubiquitin conjugation with and liberation from rabbit reticulocyte stroma.

The breakdown of mitochondria-containing stroma of rabbit reticulocytes is an ATP- and ubiquitin-dependent process and there is no evidence for an ATP-dependent but ubiquitin-independent proteolysis in these cells. The ubiquitin conjugate formation with heat-denatured stroma proteins is about one-fifth of that with native stroma. In reticulocytes there exist two mechanisms of ubiquitin liberation from its conjugates with stroma proteins: an ATP-dependent and hemin-resistant release of ubiquitin, which is assumed to be the first step in the degradation of ubiquitin conjugates by the protease system, and a release of ubiquitin catalyzed by an isopeptidase activity.

The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates.

ATP, ubiquitin-dependent proteolysis proceeds through covalent intermediates between target proteins destined for degradation and the 8,600-Da polypeptide ubiquitin. The ubiquitin moiety therefore represents a sensitive immunological marker for the specificity and function of this novel post-translational modification. Methods are described for the immunochemical detection of ubiquitin conjugates immobilized on nitrocellulose filters following electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. A further modification allows quantitation of conjugated ubiquitin to the exclusion of free polypeptide. Comparisons of conjugate pools in rabbit reticulocytes and erythrocytes demonstrate that 83 +/- 3% and 31 +/- 0.2%, respectively, of total intracellular ubiquitin exists covalently bound to target proteins. Similar large proportions of conjugated ubiquitin were found in three tissue culture cell lines. Subcellular fractionation revealed that 25% of total ubiquitin conjugates of reticulocytes sediment with the 22,000 X g stromal fraction with the remainder found in the 100,000 X g supernatant. In contrast, significant levels of erythrocyte ubiquitin conjugates occur only in the 100,000 X g supernatant, suggesting ubiquitin-mediated proteolysis actively degrades stromal components lost during terminal maturation. Reticulocytes retain their full complement of active ubiquitin during maturation indicating the concomitant decline in energy-dependent proteolysis does not result from ubiquitin inactivation. That the lower level of ubiquitin conjugates and the accompanying rate of energy-dependent proteolysis in erythrocytes is a consequence of limited substrate availability is suggested by observed increases in conjugate pools and induction of specific ubiquitin-protein adducts on incubation with either phenylhydrazine or sodium nitrite.