Serum High-mobility Group Box Protein 1 Research Articles

Dynamic changes and clinic significance of serum high mobility group box 1 in patients with total hip arthroplasty.

Deeper understanding on the risk factors and seeking potential predicted biomarkers for prognosis of total hip arthroplasty (THA) patients are of great significance. Limited researches focused the correlation between high mobility group box protein-1 (HMGB1) and the prognosis of THA patients. The objective of this study was to investigate the role of HMGB1 and inflammatory factors in patients underwent total hip arthroplasty (THA). The present prospective study enrolled 208 THA patients who went to our hospital during January 2020 to January 2022. Serum levels of HMGB1, C-reactive protein (CRP), interleukin-1b (IL-1β), and IL-6 were detected at the admission, 1 day, 3 days, 7 days, 30 days, and 90 days after surgery. The levels of Harris score, Fugl-Meyer, 36-item short-form health survey (SF-36), and Pittsburgh sleep quality index (PSQI) were detected on 90 days after surgery in two groups. Receiver operating characteristic curve (ROC) was performed for analyzing the diagnostic value of HMGB1 and logistic regression model was used for identifying the risk factor for poor prognosis of THA patients. Serum levels of HMGB1 and inflammatory factors increased after surgery compared with the baselines. A positive correlation was found between HMGB1 and CRP on 1 day after surgery, and positive correlations were found among HMGB1, IL-1β, and IL-6 on 3 day after surgery. Besides, low HMGB1 reduced the incidence of post-operative complications and improved prognosis of THA patients. Serum HMGB1 was correlated with inflammatory factors and the prognosis of THA patients.

High mobility group box protein 1 (HMGB1) serum and urinary levels and gene polymorphism in Egyptian patients with systemic lupus erythematosus: A possible relation to lupus nephritis.

The aim of this study was to evaluate the effects of the high mobility group box protein 1 (HMGB1) serum and urinary levels and gene polymorphisms on systemic lupus erythematosus (SLE) development and investigate their link to lupus nephritis (LN). We enrolled 120 Egyptian SLE patients and 120 healthy controls. Thorough medical and clinical evaluation were carried out, and SLE disease activity index (SLEDAI) was assessed. Lupus patients were divided into two groups according to the presence of LN. Measurement of HMGB1 serum and urinary levels was done using ELISA and genotyping for HMGB1 (rs1045411) was performed. There were statistically significantly higher HMGB1 serum and urinary levels in SLE patients (p < 0.001). There was a marginally significant association between lupus and alleles (p = 0.059, φ = -0.086). 'C' allele was marginally significant risk allele for SLE. After classifying SLE patients based on the presence or absence of LN, there was no significant difference as regard sex (p = 0.387), age (p = 0.208) and disease duration (p = 0.094).However, there was a significant difference between the 2 groups in regard to the frequency of musculoskeletal manifestations (p = 0.035), SLEDAI score (p < 0.001), both serum (p < 0.001) and urinary HMGB1 levels (p < 0.001) in addition to the frequency of HMGB1 genotypes (p = 0.003). Lupus patients with C/T-T/T HMGB1 genotypes had 3.5-times higher odds to exhibit LN. Serum and urine HMGB1 measurements are helpful in the diagnosis of SLE and the prediction of LN. There is a link between HMGB1 gene variations and the risk of SLE, with evidence that the C/T-T/T HMGB1 genotype is linked to a significantly greater risk of LN in the Egyptian population.

LPS-induced macrophage HMGB1-loaded extracellular vesicles trigger hepatocyte pyroptosis by activating the NLRP3 inflammasome

Extracellular vesicles (EVs) have emerged as important vectors of intercellular dialogue. High mobility group box protein 1 (HMGB1) is a typical damage-associated molecular pattern (DAMP) molecule, which is cytotoxic and leads to cell death and tissue injury. Whether EVs are involved in the release of HMGB1 in lipopolysaccharide (LPS)-induced acute liver injuries need more investigation. EVs were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. The co-localization of HMGB1, RAGE (receptor for advanced glycation end-products), EEA1, Rab5, Rab7, Lamp1 and transferrin were detected by confocal microscopy. The interaction of HMGB1 and RAGE were investigated by co-immunoprecipitation. EVs were labeled with the PKH67 and used for uptake experiments. The pyroptotic cell death was determined by FLICA 660-YVAD-FMK. The expression of NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasomes were analyzed by western-blot or immunohistochemistry. Serum HMGB1, ALT (alanine aminotransferase), AST (aspartate aminotransferase), LDH (lactate dehydrogenase) and MPO (myeloperoxidase) were measured using a commercial kit. The extracellular vesicle HMGB1 was detected in the serums of sepsis patients. Macrophages were found to contribute to HMGB1 release through the EVs. HMGB1-RAGE interactions participated in the loading of HMGB1 into the EVs. These EVs shuttled HMGB1 to target cells by transferrin-mediated endocytosis leading to hepatocyte pyroptosis by the activation of NLRP3 inflammasomes. Moreover, a positive correlation was verified between the sepsis serum EVs-HMGB1 level and clinical liver damage. This finding provides insights for the development of novel diagnostic and therapeutic strategies for acute liver injuries.

Possible role of the HMGB1 and RAGE inflammatory pathway in primary sclerosing cholangitis

BackgroundActivation of the receptor for advanced glycation end products (RAGE) and its ligand High Mobility Group Box Protein 1 (HMGB1), a nuclear protein with proinflammatory properties, has been implicated in several inflammatory disorders. ObjectiveTo analyse the influence of RAGE and HMGB1 signalling in patients with primary sclerosing cholangitis (PSC). MethodsLevels of HMGB1 and bile acid in serum and bile samples of 69 PSC patients and 32 controls were measured. Additionally, 640 patients with PSC and other liver diseases were analysed for the gain-of-function RAGE G82S SNP by PCR. Laboratory and clinical parameters were retrieved by chart review. ResultsELISA analysis showed significantly higher biliary HMGB1 concentrations in PSC patients (n=69, median 124,1 ng/ml) than in the control group (n=32, median 6,85 ng/ml, p<0,001). Median serum HMGB1 (n=22, median 2,4 ng/ml) was significantly lower than median biliary HMGB1 of the concomitant bile samples (n=22, median 151 ng/ml, p =0,001). There was no correlation of biliary HMGB1 levels with laboratory parameters or clinical end points. Analysis of the gain-of-function G82SSNP RAGE SNP in PSC patients showed 8 patients with heterozygote mutant alleles (8/324, 2,5%). Patients carrying the mutation developed more often dominant strictures of the large bile ducts (100.0% vs. 61.3%; p=0.04) and had reduced transplantation-free survival in the mutant allele group (p<0.001). ConclusionsBiliary HMGB1 levels are elevated in PSC patients compared to controls and a gain-of-function SNP in RAGE is associated with development of dominant strictures and reduced survival in PSC patients.

Aberrant Expression of High Mobility Group Box Protein 1 in the Idiopathic Inflammatory Myopathies.

IntroductionHigh Mobility Group Box Protein 1 (HMGB1) is a DNA-binding protein that exerts inflammatory or pro-repair effects upon translocation from the nucleus. We postulate aberrant HMGB1 expression in immune-mediated necrotising myopathy (IMNM).MethodsHerein, we compare HMGB1 expression (serological and sarcoplasmic) in patients with IMNM with that of other myositis subtypes using immunohistochemistry and ELISA.ResultsIMNM (n = 62) and inclusion body myositis (IBM, n = 14) patients had increased sarcoplasmic HMGB1 compared with other myositis patients (n = 46). Sarcoplasmic HMGB1 expression correlated with muscle weakness and histological myonecrosis, inflammation, regeneration and autophagy. Serum HMGB1 levels were elevated in patients with IMNM, dermatomyositis and polymositis, and those myositis patients with extramuscular inflammatory features.DiscussionAberrant HMGB1 expression occurs in myositis patients and correlates with weakness. A unique expression profile of elevated sarcoplasmic and serum HMGB1 was detected in IMNM.

Expression of HMGB1 and TLR4 in neuropsychiatric systemic lupus erythematosus patients with seizure disorders.

Previous studies show that the high-mobility group box protein 1 (HMGB1) and the toll-like receptor 4 (TLR4) participate in systemic lupus erythematosus (SLE). The two molecules contribute to the occurrence and persistence of seizures in various disease conditions, such as epilepsy. Since seizures are one of the most severe complications associated with neuropsychiatric SLE (NPSLE), the current study aimed at investigating whether HMGB1 and TLR4 play any role in NPSLE related seizures. Data from 291 SLE patients and 100 healthy controls (HC) were prospectively collected from 2013 to 2018. The ELISA test was used to determine serum levels of HMGB1 for all patients and HC and cerebrospinal fluid (CSF) levels of NPSLE patients. The expression levels of TLR4 by the peripheral blood monocytes (PBMCs) were determined by real-time PCR of TLR4 mRNA. Binary logistic regression and ROC curve analysis were used to predict NPSLE. Among the 291 SLE patients, 188 had active disease and were grouped into two, NPSLE (N=86) and Non-NPSLE (N=102) groups. Among the NPSLE patients, 21 had seizure disorders. Serum HMGB1 levels were increased in NPSLE (8.73±0.29 ng/mL) and were associated with disease activity (r=0.6527, P=0.000). Both serum and CSF HMGB1 levels in NPSLE patients with seizure disorders (9.59±0.63 and 2.90±2.29 ng/mL, respectively) were higher than in patients with other neuropsychiatric symptoms (8.45±0.33 and 2.56±1.70 ng/mL, respectively), though without significance. The gene expression of mRNA TLR4 in PBMCs was similar to serum HMGB1 in the investigated groups. Independent predictors of NPSLE were SLEDAI-2k (OR 1.25; 95% CI: 1.155-1.353), serum HMGB1 (OR 1.659; 95% CI: 1.266-2.175), and anti-Rib-P Ab (OR 3.296; 95% CI: 1.013-10.725). ROC curves for the above predictors had a large AUC (95% CI) of 0.936 (0.900-0.971), indicating a good prediction of NPSLE occurrence. The expression of HMGB1 and TLR4 was increased in NPSLE, but HMGB1 and TLR4 had minimal effect on NPSLE related seizures. The serum levels of HMGB1 were positively correlated with disease activity, and could, therefore, be a potential biomarker of NPSLE for use in future clinical practice.

Serum high mobility group box protein 1 (HMGB1) levels reflect clinical features of childhood hemophagocytic lymphohistiocytosis.

PurposeHemophagocytic lymphohistiocytosis (HLH) is a potentially lethal hyperinflammatory disorder. For further understanding of the pathogenesis of HLH, we examined serum levels of high mobility group box protein 1 (HMGB1) in children with HLH.Patients and methodsSerum HMGB1 levels were measured in 28 patients with HLH and 6 normal controls using a quantitative enzyme-linked immunosorbent assay. The patients were 21 boys and 7 girls, aged from 10 days to 21 years, with a median age of 8.5 years. The underlying conditions of HLH were infection-associated HLH in 18 patients, malignancy-associated HLH in 7 patients, and genetic HLH in 3 patients. The relations between serum HMGB1 levels and clinical symptoms and laboratory parameters were analyzed.ResultsSerum HMGB1 levels were significantly higher in patients with HLH than in normal controls (median, 6.5 ng/mL, interquartile range, 4.25–13.1). The serial serum HMGB1 levels in one patient fell to reflect the disease activity. Serum HMGB1 levels were significantly higher in patients with disseminated intravascular coagulation (DIC) than in patients without DIC (p<0.001) and were also significantly higher in patients with central nervous system (CNS) complications than in patients without CNS complications (p<0.01). Serum HMGB1 levels were positively correlated with aspartate aminotransferase (rs =0.48, p<0.01, Spearman’s rank correlation coefficient) and negatively correlated with fibrinogen (rs = −0.475, p=0.011) and hemoglobin (rs = −0.465, p=0.013).ConclusionSerum HMGB1 levels reflect clinical features of childhood HLH. HMGB1 is a potential mediator involved in the pathogenesis and determining the clinical findings of HLH.

Glycyrrhizin, a High-Mobility Group Box 1 Inhibitor, Improves Lipid Metabolism and Suppresses Vascular Inflammation in Apolipoprotein E Knockout Mice

Background: High-mobility group box protein 1 (HMGB1) is known to have proinflammatory properties; however, the mechanisms by which HMGB1 influences immune responses during atherosclerosis (AS) development are not well understood. Thus, this study investigated the relationship between HMGB1 and vascular inflammation in Apoe^–/– mice and whether glycyrrhizin (GLY), a small inhibitor of HMGB1, could have atheroprotective effects in AS. Methods: Apoe^–/– mice on a high-fat diet were treated with GLY (50 mg/kg) or vehicle by gavage once daily for 12 weeks, respectively. Results: The GLY group exhibited significantly decreased serum lipid levels, atherosclerotic plaque deposition, and serum HMGB1 levels, as well as an increased Treg/Th17 ratio. The GLY group displayed increased interleukin-10 (IL-10) and IL-2 expression and decreased IL-17A and IL-6 expression. Furthermore, the GA treatment significantly reduced STAT3 phosphorylation in Th17 cells and increased STAT5 phosphorylation in Treg cells. Conclusions: Our findings indicate that the attenuation of atherosclerotic lesions in Apoe^–/– mice by GLY might be associated with the amelioration of lipid metabolism abnormalities, inhibition of HMGB1 expression, and alterations in the Treg/Th17 ratio.

High expression of the HMGB1-TLR4 axis and its downstream signaling factors in patients with Parkinson's disease and the relationship of pathological staging.

ObjectiveTo detect the expression of high‐mobility group box protein 1 (HMGB1) and toll‐like receptor 4 (TLR4) and their downstream signaling factors—myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF‐κB), and tumor necrosis factor alpha (TNF‐α)—in the sera of patients with Parkinson's disease (PD) in order to evaluate the relationship of the HMGB1–TLR4 axis with PD development and progression.MethodsThe serum HMGB1 and TLR4 protein levels of 120 patients with PD and 100 healthy volunteers were measured using double‐antibody sandwich ELISA, and their correlations with PD staging, disease duration, drug treatment effectiveness, and clinical classification were analyzed. In addition, their correlations with the key downstream factors of the HMGB1–TLR4 axis (MyD88, NF‐κB, and TNF‐α) were analyzed.ResultsHMGB1 and TLR4 expressions were higher in the peripheral blood of patients with PD than in healthy volunteers. PD patients with poor drug treatment outcomes had significantly higher HMGB1 and TLR4 expressions than PD patients with stable drug treatment outcomes. Higher HMGB1 and TLR4 expressions were found in patients at higher PD stages, and patients with >4‐year disease duration had significantly higher HMGB1 and TLR4 expressions than patients with <4‐year disease duration. No significant difference in HMGB1 and TLR4 expressions was found among patients with tremor‐dominant, akinetic‐rigid, and mixed subtypes of PD. NF‐κB and TNF‐α expressions were positively correlated with high expression of the HMGB1–TLR4 axis.ConclusionHigh expression of the HMGB1–TLR4 axis is closely associated with PD development, progression, drug treatment effectiveness, staging, and disease duration and has great significance for PD diagnosis and treatment.

Alarmin HMGB1 induces systemic and brain inflammatory exacerbation in post-stroke infection rat model

Post-stroke infection (PSI) is known to worsen functional outcomes of stroke patients and accounts to one-third of stroke-related deaths in hospital. In our previous reports, we demonstrated that massive release of high-mobility group box protein 1 (HMGB1), an endogenous danger signal molecule, is promoted by N-methyl-d-aspartic acid-induced acute damage in the postischemic brain, exacerbating neuronal damage by triggering delayed inflammatory processes. Moreover, augmentation of proinflammatory function of lipopolysaccharides (LPS) by HMGB1 via direct interaction has been reported. The aim of this study was to investigate the role of HMGB1 in aggravating inflammation in the PSI by exacerbating the function of LPS. PSI animal model was produced by administrating a low-dose LPS at 24 h post-middle cerebral artery occlusion (MCAO). Profound aggravations of inflammation, deterioration of behavioral outcomes, and infarct expansion were observed in LPS-injected MCAO animals, in which serum HMGB1 surge, especially disulfide type, occurred immediately after LPS administration and aggravated brain and systemic inflammations probably by acting in synergy with LPS. Importantly, blockage of HMGB1 function by delayed administrations of therapeutic peptides known to inhibit HMGB1 (HMGB1 A box, HPep1) or by treatment with LPS after preincubation with HMGB1 A box significantly ameliorated damages observed in the rat PSI model, demonstrating that HMGB1 plays a crucial role. Furthermore, administration of Rhodobacter sphaeroides LPS, a selective toll-like receptor 4 antagonist not only failed to exert these effects but blocked the effects of LPS, indicating its TLR4 dependence. Together, these results indicated that alarmin HMGB1 mediates potentiation of LPS function, exacerbating TLR4-dependent systemic and brain inflammation in a rat PSI model and there is a positive-feedback loop between augmentation of LPS function by HMGB1 and subsequent HMGB1 release/serum. Therefore, HMGB1 might be a valuable therapeutic target for preventing post-stroke infection.

Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

The aim of the present study was to investigate the increase in serum and urine levels of high mobility group box protein 1 (HMGB1) during sepsis and the effect of blood purification treatments on HMGB1 levels and patient prognosis. A total of 40 intensive care patients with sepsis were randomly assigned to different groups (n=10 per group): A control group (sepsis group), a continuous renal replacement treatment (CRRT) group, a hemoperfusion (HP) group and an HP+CRRT group. The blood purification treatments using HP and/or CRRT were performed immediately after the diagnosis of sepsis. HMGB1 levels were measured using ELISA, and Acute Physiology and Chronic Health Evaluation (APACHE) II scores and 30-day survival rates were evaluated. Relative to a healthy control group (n=10), HMGB1 levels were observed to be significantly upregulated during sepsis (P<0.05). Following the initiation of sepsis, serum HMGB1 continued to increase in the sepsis group and was significantly elevated at 24 h (P<0.05), whereas urine HMGB1 levels decreased significantly at 12 and 24 h (P<0.05). Serum HMGB1 levels were significantly positively correlated with APACHE II scores (r=0.7284, P<0.01) and significantly negatively correlated with urine HMGB1 levels (r=−0.5103, P=0.026). Serum HMGB1 levels were significantly reduced in the HP and HP+CRRT groups by 12 and 24 h following the initiation of treatment (both P<0.05). Changes in the urine HMGB1 level differed in each group. Relative to the sepsis group, the APACHE II scores of all blood purification groups were significantly reduced (P<0.05) and the 30-day survival rate of the HP+CRRT group was significantly increased (P=0.0107). The results of the present study indicate that blood purification initiated at the point of diagnosis in patients with sepsis may attenuate serum HMGB1 upregulation, promote urinary excretion of HMGB1 and improve the prognosis of sepsis.

OA22.03 HMGB1, a Target for Mesothelioma Therapy and a Biomarker to Detect Asbestos Exposure and to Identify Mesothelioma Patients

Millions of people have been potentially exposed to asbestos, the primary cause of malignant mesothelioma (MM). Presently, no reliable biomarkers are available to identify among potentially exposed people, those individuals who have actually been exposed and who are at high risk of MM. High Mobility Group Box Protein-1 (HMGB1) is a key mediator of asbestos-induced inflammation and MM pathogenesis. Recently, HMGB1 hyper-acetylation has been functionally associated to its active release by inflammatory cells. Here, we compared the serum levels of total and hyper-acetylated HMGB1 in individuals professionally exposed to asbestos, MM patients and healthy unexposed controls. We compared the serum levels of total and hyper-acetylated HMGB1 in individuals professionally exposed to asbestos, MM patients and healthy unexposed controls. Previously proposed MM biomarkers fibulin-3, osteopontin, and mesothelin were also blindly measured in blood collected from participants to the study. HMGB1 serum levels reliably distinguished asbestos-exposed individuals and MM patients from unexposed controls. Moreover, the levels of total and hyper-acetylated HMGB1 were significantly higher in MM patients compared to asbestos-exposed individuals, and did not vary with tumor stage, suggesting that early lesions are also associated to increased HMGB1 levels. At a cutoff value of 2.00 ng/mL, the sensitivity and specificity of hyper-acetylated serum HMGB1 in differentiating MM patients from asbestos-exposed individuals was 100%, outperforming, in parallel experiments, other previously proposed biomarkers: osteopontin, fibulin-3, and mesothelin. When comparing MM patients to patients with other non-MM cytologically benign or malignant pleural effusion, the combination of two biomarkers, HMGB1 and fibulin-3, provided the highest sensitivity and specificity in differentiating these two groups. Moreover, we found that HMGB1 drives MM development and sustains MM progression, and we demonstrated that targeting HMGB1 inhibits MM cell growth and motility in vitro, reduced tumor growth in vivo and prolonged survival. Despite the relatively small size of our cohorts, our results are of exceptional significance and clinical relevance as they provide the first biomarker of asbestos exposure and indicate that hyper-acetylated HMGB1 is a very sensitive and specific biomarker to differentiate MM patients from individuals occupationally exposed to asbestos and unexposed controls. Moreover, our results on HMGB1 inhibitors indicate that HMGB1 targeting hampers the malignant phenotype of MM, offering a novel potential therapeutic approach to patients afflicted with this dismal disease.

High mobility group box protein 1-A prognostic marker for structural joint damage in 10-year follow-up of patients with juvenile idiopathic arthritis.

High mobility group box protein 1 (HMGB1) is an important pro-inflammatory mediator in adult rheumatoid arthritis. The diagnostic utility of HMGB1 in Juvenile Idiopathic Arthritis (JIA) is still unclear. The aim was to examine whether serum HMGB1 levels are associated with inflammation, radiological disease progression, and long-term prognosis in JIA. We included 131 children with JIA from a population-based prevalence study; 38 of them were prospectively followed up for 10 years. Clinical and laboratory disease characteristics at study entry and after 10 years as well as radiological progression over 10 years were recorded. HMGB1 levels were analyzed by an ELISA. The HMGB1 levels were similar in children with different JIA subgroups and in children with established (53%) or newly diagnosed (47%) disease. HMGB1 levels did not differ between groups at entry into the study or at 10 years, by sex, or by the presence or absence of RF or ANA antibodies. HMGB1 levels at the study entry correlated with HMGB1 levels at 10 years and with blood neutrophil count. Most importantly, children with destructive arthritis at 10 years had a tendency toward higher HMGB1 levels at study entry (median 1.2 vs 0.6ng/ml, ns) and displayed 4-fold higher circulating HMGB1 levels (median 3.4 vs 0.8ng/ml, p = 0.0014) than children without radiological destructions. Our results suggest that HMGB1 is a marker of inflammatory activity in children with JIA. Higher serum HMGB1 levels are related to more destructive JIA and could be used as a negative prognostic marker at the disease start. Clinicaltrials.gov NCT01905319. Registered July 16, 2013.

HMGB1 and thrombin mediate the blood-brain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer's disease.

BackgroundThe blood-brain barrier (BBB) dysfunction represents an early feature of Alzheimer’s disease (AD) that precedes the hallmarks of amyloid beta (amyloid β) plaque deposition and neuronal neurofibrillary tangle (NFT) formation. A damaged BBB correlates directly with neuroinflammation involving microglial activation and reactive astrogliosis, which is associated with increased expression and/or release of high-mobility group box protein 1 (HMGB1) and thrombin. However, the link between the presence of these molecules, BBB damage, and progression to neurodegeneration in AD is still elusive. Therefore, we aimed to profile and validate non-invasive clinical biomarkers of BBB dysfunction and neuroinflammation to assess the progression to neurodegeneration in mild cognitive impairment (MCI) and AD patients.MethodsWe determined the serum levels of various proinflammatory damage-associated molecules in aged control subjects and patients with MCI or AD using validated ELISA kits. We then assessed the specific and direct effects of such molecules on BBB integrity in vitro using human primary brain microvascular endothelial cells or a cell line.ResultsWe observed a significant increase in serum HMGB1 and soluble receptor for advanced glycation end products (sRAGE) that correlated well with amyloid beta levels in AD patients (vs. control subjects). Interestingly, serum HMGB1 levels were significantly elevated in MCI patients compared to controls or AD patients. In addition, as a marker of BBB damage, soluble thrombomodulin (sTM) antigen, and activity were significantly (and distinctly) increased in MCI and AD patients. Direct in vitro BBB integrity assessment further revealed a significant and concentration-dependent increase in paracellular permeability to dextrans by HMGB1 or α-thrombin, possibly through disruption of zona occludins-1 bands. Pre-treatment with anti-HMGB1 monoclonal antibody blocked HMGB1 effects and leaving BBB integrity intact.ConclusionsOur current studies indicate that thrombin and HMGB1 are causal proximate proinflammatory mediators of BBB dysfunction, while sTM levels may indicate BBB endothelial damage; HMGB1 and sRAGE might serve as clinical biomarkers for progression and/or therapeutic efficacy along the AD spectrum.

Abstract 3112: HMGB1 and its isoform are sensitive and specific biomarkers to detect asbestos exposure and to identify mesothelioma patients

Abstract Millions of people have been potentially exposed to asbestos, the primary cause of malignant mesothelioma (MM). Presently, no reliable biomarkers are available to identify among potentially exposed people, those individuals who have actually been exposed and who are at high risk of MM. High Mobility Group Box Protein-1 (HMGB1) is a key mediator of asbestos-induced inflammation and MM pathogenesis. Recently, HMGB1 hyper-acetylation has been functionally associated to its active release by inflammatory cells. Here, we compared the serum levels of total and hyper-acetylated HMGB1 in individuals professionally exposed to asbestos, MM patients and healthy unexposed controls. HMGB1 serum levels reliably distinguished asbestos-exposed individuals and MM patients from unexposed controls. Moreover, the levels of total and hyper-acetylated HMGB1 were significantly higher in MM patients compared to asbestos-exposed individuals, and did not vary with tumor stage, suggesting that early lesions are also associated to increased HMGB1 levels. At a cutoff value of 2.00 ng/mL, the sensitivity and specificity of hyper-acetylated serum HMGB1 in differentiating MM patients from asbestos-exposed individuals was 100%, outperforming, in parallel experiments, other previously proposed biomarkers: osteopontin, fibulin-3, and mesothelin. When comparing MM patients to patients with other non-MM cytologically benign or malignant pleural effusion, the combination of two biomarkers, HMGB1 and fibulin-3, provided the highest sensitivity and specificity in differentiating these two groups. We propose total and hyper-acetylated HMGB1 as valuable biomarkers to differentiate MM patients from individuals professionally exposed to asbestos and from unexposed people. Citation Format: Andrea Napolitano, Daniel J. Antoine, Laura Pellegrini, Francine Baumann, Ian Pagano, Sandra Pastorino, Chandra M. Goparaju, Kirill Prokrym, Claudia Canino, Harvey I. Pass, Michele Carbone, Haining Yang. HMGB1 and its isoform are sensitive and specific biomarkers to detect asbestos exposure and to identify mesothelioma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3112.

Levo-1-methyl tryptophan aggravates the effects of mouse hepatitis virus (MHV-A59) infection.

Mice infected with mouse hepatitis virus A59 (MHV-A59) develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH) with a concomitant enhancement of transaminases and release of alarmins such as uric acid and high-mobility group box protein 1 (HMGB1). Tryptophan catabolism is an endogenous mechanism that restricts excessive immune responses, thereby preventing immunopathology. Since indoleamine-2,3-dioxygenase (IDO) is the key and rate-limiting enzyme of tryptophan catabolism, the aim of this work was to explore whether specific inhibition of IDO by Levo-1-methyl tryptophan (MT) could affect MHV actions. Results showed that MT strongly enhanced the hypergammaglobulinemia induced by the virus, as well as anti-MHV Ab and uric acid release. Moreover, infected mice treated with MT did express anti-FAH autoAb and high levels of serum HMGB1. Survival of MHV-infected animals treated with MT was severely reduced compared with that of MHV-infected mice or controls only treated with MT. Furthermore, histological liver examination indicated that MT induced fibrosis in MHV-infected animals, whereas MT itself increased uric acid levels without shortening the animal life Thus, under our experimental conditions, results indicated an exacerbated response to MHV infection when IDO was blocked by MT.

Analysis of the expression of HMGB-1, CXCL16, miRNA-30a, and TGF-β1 in primary nephritic syndrome patients and its significance.

We investigated the expression levels of high-mobility group box protein 1 (HMGB-1), CXC chemokine ligand 16 (CXCL16), microRNA (miRNA)-30a and transforming growth factor ß1 (TGF-ß1) in primary nephritic syndrome (PNS) patients and the clinical significance of this expression. A total of 56 patients with PNS were included in the PNS group, while 50 healthy subjects formed the normal control group. Serum levels of HMGB-1, CXCL16, miRNA-30a, and urinary TGF-ß1 concentrations were quantified along with other biochemical indices, including serum albumin, triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein, and urinary proteins. The correlation between levels of HMGB-1, CXCL16, miRNA-30a, and TGF-ß1 and biochemical indexes was further analyzed. PNS group patients had significantly higher levels of HMGB-1, CXCL16, miRNA- 30a, and TGF-ß1 compared to the control group (P < 0.05). PNS patients also had higher 24-h urinary protein, TG, TC, and LDL levels but lower serum albumin compared to subjects in the control group (P < 0.05). Serum HMGB-1, CXCL16, miRNA-30a, and urinary TGF-ß1 levels were all negatively correlated with serum albumin levels, but were positively correlated with TG, TC, LDL, and 24-h urinary protein (P < 0.05 in all cases). Additionally, a positive correlation existed among serum HMGB-1, CXCL16, miRNA-30a, and urinary TGF-ß1 levels (P < 0.01). HMGB-1, CXCL16, miRNA-30a, and urinary TGF-ß1 were highly expressed in PNS patients and may play important roles in the pathogenesis and development of PNS.

TLR4 as receptor for HMGB1-mediated acute lung injury after liver ischemia/reperfusion injury

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

Uric acid and HMGB1 are involved in the induction of autoantibodies elicited in mice infected with mouse hepatitis virus A59

We have shown that mice infected with mouse hepatitis virus A59 develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH). Because it has been proposed that the immune system is stimulated by alarm signals called damage-associated molecular patterns or alarmins, we investigated the participation of uric acid and high-mobility group box protein 1 (HMGB1) in the autoimmune response elicited by mouse hepatitis virus (MHV). Mice subjected to MHV infection had increased plasmatic uric acid concentration that significantly decreased after 20 days of daily treatment with allopurinol and, simultaneously, autoAb to FAH were undetected. Furthermore, this autoAb disappeared after 30 days of treatment with ethyl pyruvate, along with a substantial reduction in serum HMGB1 concentration. Both results indicated a remarkable relationship between the autoimmune process induced by the virus and uric acid and HMGB1 liberation. Unexpectedly, it was found that allopurinol and ethyl pyruvate inhibited the release of both uric acid and HMGB1. Because HMGB1 is activated through binding to interleukin 1β, and that this cytokine is produced by the NLRP3 inflammasome that could be stimulated by uric acid, we propose that both alarmins could be acting in concert with the induction of the autoAb to FAH in MHV-infected mice.

Mitochondrial antiviral signaling protein defect links impaired antiviral response and liver injury in steatohepatitis in mice

Mitochondrial dysfunction is a pathogenic feature of nonalcoholic steatohepatitis (NASH). NASH complicates hepatotropic viral disease. The mitochondrial antiviral signaling protein (MAVS) is the adapter of helicase receptors involved in sensing double-stranded RNA (dsRNA). We hypothesized that impaired MAVS function may contribute to insufficient antiviral response and liver damage in steatohepatitis. We identified reduced MAVS protein levels and increased MAVS association with the proteasome subunit alpha type 7 (PSMA7) in livers from mice given a methionine-choline-deficient (MCD) diet. Decreased association of MAVS with mitochondria and increased cytosolic cytochrome c indicated mitochondrial damage in steatohepatitis. In vivo administration of the synthetic dsRNA polyinosinic:polycytidylic acid [poly(I:C)], but not lipopolysaccharide or cytidine-phosphate-guanosine-rich DNA, resulted in impaired induction of type I interferons (IFNs) and proinflammatory cytokines in steatohepatitis. Consistent with a defect in helicase receptor-induced signaling, there was loss of poly(I:C)-induced translocation of MAVS to the cytosol and decreased IFN regulatory factor 3 phosphorylation. Caspases 1 and 8, both of which cleave MAVS, were increased in MCD diet-fed mice. At baseline, steatohepatitis was associated with increased serum alanine aminotransferase (ALT), apoptosis and caspase 3 activation compared with controls. In contrast to apoptosis in controls, necrosis was induced by poly(I:C) stimulation in steatohepatitis. Hepatocyte necrosis was indicated by elevated serum high-mobility group box protein-1 and ALT and was correlated with increased expression of receptor-interacting protein 3 (RIP3), a master regulator of necrosis. Increased expression of MAVS, PSMA7, and RIP3 messenger RNA was also present in human NASH livers. Our novel findings suggest that mitochondrial damage in steatohepatitis extends to MAVS, an adapter of helicase receptors, resulting in inefficient type I IFN and inflammatory cytokine response but increased hepatocyte necrosis and RIP3 induction in response to a dsRNA viral challenge. These mechanisms may contribute to progressive liver damage and impaired viral clearance in NASH.