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.

Use of a gene score of multiple low-modest effect size variants can predict the risk of obesity better than the individual SNPs.

BACKGROUND: Obesity is a complex disorder, the development of which is modulated by a multitude of environmental, behavioral and genetic factors. The common forms of obesity are polygenic in nature which means that many variants in the same or different genes act synergistically and affect the body weight quantitatively. The aim of the current study was to use information from many common variants previously identified to affect body weight to construct a gene score and observe whether it improves the associations observed. The SNPs selected were G2548A in leptin (LEP) gene, Gln223Arg in leptin receptor (LEPR) gene, Ala54Thr in fatty acid binding protein 2 (FABP2) gene, rs1121980 in fat mass and obesity associated (FTO) gene, rs3923113 in Growth Factor Receptor Bound Protein 14 (GRB14), rs16861329 in Beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), rs1802295 in Vacuolar protein sorting-associated protein 26A (VPS26A), rs7178572 in high mobility group 20A (HMG20A), rs2028299 in adaptor-related protein complex 3 (AP3S2), and rs4812829 in Hepatocyte Nuclear Factor 4 Alpha (HNF4A). METHODS: A total of 475 subjects were genotyped for the selected SNPs in different genes using different genotyping techniques. The study subjects' age, weight, height, BMI, waist and hip circumference, serum total cholesterol, triglycerides, LDL and HDL were measured. A summation term, genetic risk score (GRS), was calculated using SPSS. RESULTS: The results showed a significantly higher mean gene score in obese cases than in non-obese controls (9.1 ± 2.26 vs 8.35 ± 2.07, p = 2 × 10- 4). Among the traits tested for association, gene score appeared to significantly affect BMI, waist circumference, and all lipid traits. CONCLUSION: In conclusion, the use of gene score is a better way to calculate the overall genetic risk from common variants rather than individual risk variants.

Serum high mobility group box 1 protein levels are not associated with either histological severity or treatment response in children and adults with nonalcoholic fatty liver disease.

AIM: Serum high mobility group box 1 protein (HMGB1) is a proinflammatory molecule that could potentially serve as a biomarker for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) due to its correlation with degree of liver fibrosis. The aim of the current study was to examine the cross-sectional and longitudinal relationships between serum HMGB1 levels and liver histology in adults and children with NAFLD participating in two large randomized controlled trials. METHODS: Serum HMGB1 levels were measured at various time points in adults and children with NAFLD, who participated in PIVENS and TONIC clinical trials respectively. PIVENS trial compared vitamin E or pioglitazone to placebo in adults whereas TONIC trial compared vitamin E or metformin to placebo in children. Participants had liver biopsies at baseline and the end of treatment (96 weeks), and liver histology was reviewed by a central committee of study pathologists. RESULTS: In the cross-sectional analyses (n = 205 for PIVENS and 109 for TONIC), there was no significant relationship between serum HMGB1 levels and histological features such as steatosis, ballooning, inflammation, fibrosis, or presence of steatohepatitis in either adults or children. Serum HMGB1 levels did not change significantly during treatment either with placebo, vitamin E therapy (P = 0.81) or pioglitazone (P = 0.09) in the PIVENS trial. Similarly, serum HMGB1 levels did not change significantly during treatment either with placebo, metformin (P = 0.15) or vitamin E (P = 0.23) in the TONIC trial. In the longitudinal analyses (n = 105 for PIVENS and 109 for TONIC), changes in serum HMGB1 levels did not correlate with histologic improvement or resolution of NASH in either adults or children. There was no relationship between serum HMGB1 and ALT levels in either adults or children with NAFLD. CONCLUSION: Serum HMGB1 levels were not associated with histological severity or treatment response in either children or adults with NAFLD.

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.

[Novel therapeutic target in neuromyelitis optica and multiple sclerosis: high mobility group box 1 (HMGB1)].

High mobility group box 1 (HMGB1) is a nuclear protein, and released from necrotic cells. Recently, It has been known that HMGB1 is released from monocyte/macrophage, neurons, and endothelial cells, and that HMGB1 is involved in sepsis, brain infarction, etc. We have reported that HMGB1 concentrations were elevated in cerebrospinal fluid (CSF) from patients with neuromyelitis optica (NMO) and multiple sclerosis (MS) and that the elevation was significant in CSF from NMO patients. Moreover, we have also reported that experimental autoimmune encephalitis (EAE) induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide (35-55) showed decrease of clinical and pathological severity by treatment with monoclonal anti-HMGB1 antibody. Thus, we think that HMGB1 is associated with pathophysiology in central nervous system in NMO and MS (especially in NMO), and that HMGB1 can be a target molecule for NMO and MS.

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.

New high mobility group box 1 assay system.

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.

[Diagnostic role of SOX13 antibody in latent autoimmune diabetes of adults].

OBJECTIVE: To explore the diagnostic role of SOX13 antibody in latent autoimmune diabetes of adults (LADA). METHODS: Sera of 328 patients with slow-onset diabetes and 120 sex and age-matched healthy controls underwent radiochemical test to detect the positive rates of SOX13-Ab, glutamic acid decarboxylace antibody (GAD-Ab) and carboxypeptidase H antibody (CPH-Ab). According to the GAD-Ab and CPH-Ab status the patients were divided into autoimmune (GAD-Ab and/or CPH-Ab positive, n = 130) and non-autoimmune (GAD-Ab and CPH-Ab negative, n = 198) diabetic subgroups. Then according to SOX13-Ab, GAD-Ab and CPH-Ab status, the diabetic patients were divided into 4 groups: SOX13-Ab positive, GAD-Ab positive, CPH-Ab positive, and antibody-negative group to compare their clinical characteristics. The effect of SOX13-Ab on islet beta-cell function was evaluated by comparison of C-peptide among the four subgroups. RESULTS: The positive rates of SOX13-Ab in the slow-onset diabetic patients (10.4%), autoimmune subgroup patients (13.1%), and non-autoimmune subgroup patients (8.6%) were all higher than that in the healthy controls (2.5%, all P < 0.05). Thirteen patients were positive for both SOX13-Ab and CPH-Ab (13/328, 4.0%), but only 2 were positive for both SOX13-Ab and GAD-Ab (2/328, 0.6%). The highest prevalence of SOX13-Ab was observed in the patients with the duration of disease ranging from 16 to 20 years. There were no differences in the clinical parameters between the SOX13-Ab positive and antibody-negative diabetic patients. CONCLUSION: SOX13-Ab testing helps improve the sensitivity of screening for LADA. SOX13-Ab positive patients tend to have a longer course of disease and varied clinical manifestations.

Prevalence of ICA-12 and other autoantibodies in north Indian patients with early-onset diabetes.

This study attempts to assess the prevalence of various autoantibodies in early-onset diabetics in northern India, with emphasis on antibodies against glutamic acid decarboxylase (GAD65), IA-2, ICA-12, 21-hydroxylase (21-OH), and tissue transglutaminase (TTG). GAD65 and IA-2 antibodies were found to be present in approximately 26% of cases of type 1 diabetes. A subset of patients clinically diagnosed to have MMDM appears to have an autoimmune etiology, with more than 20% showing serpositivity for IA-2 antibodies. Antibodies against ICA-12 were prevalent in both type 1 diabetes and MMDM. Approximately one of seven patients with type 1 diabetes showed erological evidence of celiac disease.

Increased autoantibodies to SOX13 in Swedish patients with type 1 diabetes.

This article aims to estimate the prevalence of SOX13 antibodies in Swedish patients with type 1 diabetes and healthy controls. The patients (n = 102; median age 35 years [range, 9-89]) were newly diagnosed with type 1 diabetes in a defined area in southern Sweden during 1995-1998. Islet cell antibodies (ICA) were analyzed with immunofluorescence, while glutamic acid decarboxylase antibodies (GADA), tyrosine phosphatase antibodies (IA-2A), and antibodies against the transcription factor SOX13 (SOX13Ab) were analyzed with radioimmunoprecipitating assays. SOX13Ab were found in 9.8% (10/102) of type 1 patients compared to 2.0% (2/99) in healthy controls (P = 0.033). At least one of the four autoantibodies (ICA, GADA, IA-2A or SOX13Ab) were identified in 67% (68/102) of the patients. Samples positive for IA-2A were only in one case positive also for SOX13Ab. IA-2A-positive patients were often positive also for ICA and GADA (19/27), and the same combination was also common for SOX13Ab-positive patients (6/10). Only 2.0% (2/102) were positive for SOX13Ab alone. ICA, GADA and IA-2A were more frequent in younger patients (

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.

Increased serum concentrations of high-mobility-group protein 1 in haemorrhagic shock.

Serum concentrations of high-mobility-group protein 1 (HMG1) were increased during an episode of haemorrhagic shock in a patient who had undergone repair of an abdominal aortic aneurysm. HMG1 may be involved in the pathogenesis of human haemorrhagic shock.