Higher High Mobility Group Box 1 Levels Research Articles

Alarmin High Mobility Group Box 1 (HMGB1) Activates Alternative NFkB Signaling in Bone Marrow Macrophages in the Leukemia Microenvironment

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that signals through pattern recognition receptors on monocyte/macrophages to recruit them to sites of tissue injury. In models of sepsis and autoimmunity, HMGB1 can have both immune-activating and regulatory effects depending on the receptor system engaged and the downstream pathway activated. Although chemotherapy-stressed tumor cells are known to release HMGB1, little is known about the effects of tumor-derived HMGB1 on macrophages in the tumor microenvironment.We have previously shown that primary human acute lymphoblastic leukemia cells release high levels of HMGB1 after treatment with anthracycline chemotherapy, and that leukemia-associated HMGB1 induces expression of the IL-1 converting enzyme (caspase-1), consistent with activation of the inflammasome. Paradoxically, inflammatory cytokine production (including IL-1beta) is reduced in this setting. In the present study, we test the hypothesis leukemia-associated HMGB1 activates alternative NFkB signaling through the repressor protein RelB which has been recently shown to silence inflammatory cytokine production via epigenetic effects in sepsis models.To establish proof-of-principle, we first treated primary human bone marrow macrophages (BMM) and THP1 monocytes with 200 ng/ml recombinant HMGB1 (r-HMGB1) in the presence or absence of bacterial lipopolysaccharide (LPS). As predicted, LPS alone increased baseline expression of total NFkB, total p65/RelA and phosphorylated p65/RelA mRNA (20.6 + 7.9-fold by qPCR ddCT, p<0.0001) and protein (measured by Western blot, in BMM and THP1. In contrast, r-HMGB1 treatment produced a significant increase in the expression of RelB mRNA (2.5 + 0.7 fold increase p<0.05) and protein without a significant change in the expression of tNFkB or p65/RelA. Treatment with both r-HMGB1 and LPS maintained this pattern of increased RelB expression without increased expression of p65/RelA, suggesting HMGB1 activity as the dominant effect. Increased RelB expression was unaffected by the addition of an antibody to TLR4, the primary receptor for LPS and a candidate receptor for HMGB1.Next, we determined whether induction of RelB expression also occurs in response to leukemia-derived HMGB1. Immunomagnetically selected ALL cells from the bone marrow of 3 pediatric patients and NALM 6 ALL cells were treated with 20 nM doxorubicin, washed, and placed in culture for 24 hours prior to the addition of BMM in direct coculture. In our model, ALL cells typically release 100-450 ng/ml HMGB1 under these conditions. Again, significantly increased expression of RelB mRNA (6.7 + 2.9 fold, p<0.001) and protein was observed in BMM cultured with doxorubicin-treated ALL cells compared to ALL cells treated with media control, with a corresponding decrease in phospho-IkBa protein expression and unchanged total NFkB, p65/RelA and phosphorylated p65/RelA. Pre-treatment of ALL cells with quercetin, a commercially available flavonoid antioxidant shown to inhibit HMGB1 release, inhibited protein expression of RelB in BMM in a dose-dependent fashion.These data indicate that tumor-derived HMGB1 activates alternative NFkB signaling and is capable of reprogramming the inflammatory response to LPS. Studies are ongoing to the determine downstream effects on macrophage antitumor function to optimize therapeutic reversal of HMGB1 effects and improve the potential for bone marrow macrophages to eradicate residual leukemia that remains after chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

The relationship between serum high-mobility group box-1 and prognosis of coronary heart disease in old patients

Objective To investigate the correlation of serum high-mobility group box-1 (HMGB1) with the severity of lesion of coronary artery disease (CAD) and its prognosis in elderly patients. Methods A total of 180 CAD patients with coronary stenosis exceeding 50 percent by coronary angiography were divided into three groups: one branch stenosis; two branches stenosis and three branches stenosis. The control group included 50 patients without coronary stenosis. The degrees of coronary stenosis were diagnosed as mild stenosis, moderate stenosis and severe stenosis based on improved Gensini scores. The severity of decrements of left ventricular ejection fraction (LVEF) by echocardiogram were divided into three groups: mild, moderate and severe LVEF. Levels of HMGB1, hs-CRP and glucose were measured in all the patients. According to whether there was a complication of type 2 diabetic mellitus (T2DM), the 180 patients were classified as two groups. The patients were also divided into two groups according to whether there were adverse events. Results The HMGB1 levels of the CAD group were increased along with the number of affected vessels〔three bunch group (40.5±6.0) ng/ml, double bunch group (33.1±4.9)μg/L, single bunch group (20.5±3.3)μg/L and control group (6.2±1.4)μg/L (all P<0.05)〕. And the HMGB1 levels of the CAD group were increased along with the degrees of CAD stenosis〔severe stenosis group (43.0±5.8)μg/L, medium stenosis group (32.1±4.5)μg/L, mild stenosis group(19.3±2.0)μg/L〕(all P<0.05). Meanwhile, the levels of HMGB1 were increased along with the decrement of left ventricular ejection fraction〔left ventricular severe dysfunction group(41.0±5.5)μg/L, medium dysfunction group(33.1±4.3)μg/L, mild dysfunction group (21.3±2.0)μg/L〕 (all P<0.05). CAD with T2DM had a higher HMGB1 level than non-T2DM group〔(35.7±5.0) Ĉ/L vs. (23.3±3.0) Ĉ/L, P<0.05〕. The adverse events group had a higher HMGB1 level than non- adverse events group〔(38.7±5.5) Ĉ/L vs. (25.3±3.3)μg/L, P<0.05〕. Besides, HMGB1 had a positive correlation with levels of hs-CRP and glucose(r=0.680, 0.571, P<0.05). Conclusions Serum HMGB1 change is closely related to morbid change degree of elderly CAD patients as well as prognosis. As a new type of inflammatory factor, HMGB1 may serve as a new target for disease treatment. Key words: High mobility proteins; Coronary disease; Prognosis

HMGB1 overexpression correlates with poor prognosis in early-stage squamous cervical cancer.

High mobility group box 1 (HMGB1) is associated with tumor progression and a poor prognosis; microtubule-associated protein 1 light chain 3 (LC3) plays a critical role in autophagy. However, the roles of HMGB1 and LC3 in squamous cervical cancer (SCC) remain unclear. An array of 166 early-stage SCC, 62 cervical intraepithelial neoplasia (CIN), and 50 normal cervical tissue samples was assessed. HMGB1 and LC3 protein levels were examined by immunohistochemistry, and the associations of HMGB1 and LC3 levels with clinicopathological characteristics evaluated, to assess their prognosis significance. High nuclear HMGB1 levels were detected in 72.9% SCC cases; 16% cases showed cytoplasmic expression of HMGB1 in cancer cells with low nuclear expression. Interestingly, HMGB1 levels in SCC samples were significantly higher than CIN and control specimens, while lower LC3 expression was found in SCC samples (P < 0.001). Nuclear HMGB1 expression was weakly negatively correlated to LC3 amounts (r = -0.254, P = 0.001). High nuclear HMGB1 levels were associated with vascular metastasis (P < 0.05). In addition, cytoplasmic HMGB1 expression was associated with lymph node metastasis (P < 0.05). Furthermore, high nuclear HMGB1 levels and cytoplasmic HMGB1 expression predicted poor overall survival (OS) and disease-free survival (DFS). Meanwhile, high LC3 expression was associated with favorable prognosis. Multivariate analysis showed that both nuclear and cytoplasmic HMGB1 expressions were independent prognostic factors for overall- and disease-free survival, along with nodule metastasis. HMGB1 overexpression plays a significant role in SCC progression. Both nuclear and cytoplasmic HMGB1 are independent factors for poor prognosis in early-stage SCC.

High mobility group box 1 levels in large vessel vasculitis are not associated with disease activity but are influenced by age and statins.

IntroductionTakayasu arteritis (TA) and giant cell arteritis (GCA) are large vessel vasculitides (LVV) that usually present as granulomatous inflammation in arterial walls. High mobility group box 1 (HMGB1) is a nuclear protein that acts as an alarmin when released by dying or activated cells. This study aims to evaluate whether serum HMGB1 can be used as a biomarker in LVV.MethodsTwenty-nine consecutive TA patients with 29 healthy controls (HC) were evaluated in a cross-sectional study. Eighteen consecutive GCA patients with 16 HC were evaluated at the onset of disease and some of them during follow-up. Serum HMGB1 levels were measured by enzyme-linked immunosorbent assay.ResultsIn GCA patients at disease onset mean serum HMGB1 levels did not differ from HC (5.74 ± 4.19 ng/ml vs. 4.17 ± 3.14 ng/ml; p = 0.230). No differences in HMGB1 levels were found between GCA patients with and without polymyalgia rheumatica (p = 0.167), ischemic manifestations (p = 0.873), systemic manifestations (p = 0.474) or relapsing disease (p = 0.608). During follow-up, no significant fluctuations on serum HMGB1 levels were observed from baseline to 3 months (n = 13) (p = 0.075), 12 months (n = 6) (p = 0.093) and at the first relapse (n = 4) (p = 0.202). Serum HMGB1 levels did not differ between TA patients and HC [1.19 (0.45–2.10) ng/ml vs. 1.46 (0.89–3.34) ng/ml; p = 0.181] and no difference was found between TA patients with active disease and in remission [1.31 (0.63–2.16) ng/ml vs. 0.75 (0.39–2.05) ng/ml; p = 0.281]. HMGB1 levels were significantly lower in 16 TA patients on statins compared with 13 patients without statins [0.59 (0.29–1.46) ng/ml vs. 1.93 (0.88–3.34) ng/ml; p = 0.019]. Age was independently associated with higher HMGB1 levels regardless of LVV or control status.ConclusionsPatients with TA and GCA present similar serum HMGB1 levels compared with HC. Serum HMGB1 is not useful to discriminate between active disease and remission. In TA, use of statins was associated with lower HMGB1 levels. HMGB1 is not a biomarker for LVV.

Increased HMGB1 and cleaved caspase-3 stimulate the proliferation of tumor cells and are correlated with the poor prognosis in colorectal cancer.

BackgroundDying tumor cells after irradiation could promote the proliferation of living tumor cells might cause tumor relapse and treatment failure. Our previous study showed that activated caspase-3 after irradiation probably participates in tumor repopulation. In this study, we investigated whether high mobility group box 1(HMGB1) is also involved in tumor repopulation.MethodsColorectal tumor cells were irradiated. The cleaved caspase-3 (CC3) in irradiated tumor cells and HMGB1 in the supernatant of irradiated tumor cells were detected by Western blot. A large number of irradiated colorectal tumor cells (feeder cells) were then co-cultured with a small number of luciferase-labeled living colorectal tumor cells (reporter cells) and proliferation of reporter cells was measured by bioluminescence imaging. The CC3 and HMGB1 protein expression in colorectal tumor and peritumoral tissues were detected by immunohistochemistry and their correlation with prognosis were analyzed.ResultsThe irradiated colorectal tumor cells underwent apoptosis and necrosis and produced CC3 in tumor cells and HMGB1 in the supernatant of cultured cells. The increased expression of secretory HMGB1 correlated with CC3 level and proliferating cell nuclear antigen (PCNA) after irradiation in vitro. The irradiated dying cells remarkably stimulated living tumor cell proliferation. Interestedly, immunohistochemistry staining showed that positive HMGB1, CC3, and Ki67 expression were significantly higher in colorectal tumor tissues than in peritumoral tissues (p <0.01). The Kaplan-Meier survival analysis revealed that high HMGB1, CC3, and Ki67 levels were significantly associated with poor prognosis (p <0.05, p <0.01). Multivariate analysis using Cox proportional hazards model showed that TNM staging and HMGB1 were independent prognostic factors in patients with colorectal cancer (CRC) (p <0.01, p <0.001).ConclusionBoth apoptotic and necrotic cells could stimulate proliferation of living tumor cells, and the increased expression of CC3 and HMGB1 in tumor cells could be new markers for poor prognosis in colorectal cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0166-1) contains supplementary material, which is available to authorized users.

High mobility group box-1 and its clinical value in breast cancer.

BackgroundHigh mobility group box-1 (HMGB1) is a factor regulating malignant tumorigenesis, proliferation, and metastasis, and is associated with poor clinical pathology in various human cancers. We investigated the differential concentrations of HMGB1 in tissues and sera, and their clinical value for diagnosis in patients with breast cancer, benign breast disease, and healthy individuals.MethodsHMGB1 levels in tumor tissues, adjacent normal tissues, and benign breast disease tissues was detected via immunohistochemistry. Serum HMGB1 was measured using an enzyme-linked immunosorbent assay in 56 patients with breast cancer, 25 patients with benign breast disease, and 30 healthy control subjects. The clinicopathological features of the patients were compared. Tissues were evaluated histopathologically by pathologists.ResultsHMGB1 levels in the tissues and sera of patients with breast cancer were significantly higher than those in patients with benign breast disease or normal individuals. The 56 cancer patients were classified as having high tissue HMGB1 levels (n=41) or low tissue HMGB1 levels (n=15), but the corresponsive serum HMGB1 in these two groups was not significantly different. HMGB1 levels in breast cancer tissues significantly correlated with differentiation grade, lymphatic metastasis, and tumor-node-metastasis stage, but not patient age, tumor size, or HER-2/neu expression; no association between serum HMGB1 levels and these clinicopathological parameters was found. The sensitivity and specificity of tissue HMGB1 levels for the diagnosis of breast cancer were 73.21% and 84.00%, respectively, while positive and negative predictive values were 91.11% and 58.33%.ConclusionHMGB1 might be involved in the development and progression of breast cancer and could be a supportive diagnostic marker for breast cancer. Serum HMGB1 could be a useful serological biomarker for diagnosis and screening of breast cancer.

Low-intensity aerobic exercise training attenuates airway inflammation and remodeling in a rat model of steroid-resistant asthma

Background Aerobic exercise can improve symptoms, reduce airway inflammation, and even ameliorate airway remodeling in asthmatic animals and patients. However, previous studies have focused mainly on the effect of aerobic exercise on steroid-sensitive asthma (SSA). The goals of this study were to determine the effect of low-intensity aerobic exercise training on airway hyperresponsiveness, inflammation, and remodeling in a rat model of steroid-resistant asthma (SRA) and to identify the potential mechanisms underlying these effects. Methods Endotoxin-free ovalbumin with or without lipopolysaccharide were applied to establish rat models of SRA and SSA, respectively. Airway hyperresponsiveness, inflammation, remodeling, expression of interleukin (IL)-25, IL-33, thymic stromal lymphopoietin (TSLP), high mobility group box-1 (HMGB1), and IL-17 in bronchoalveolar lavage fluid (BALF), and the role of dexamethasone (DXM) were compared between these two asthmatic rat models. The effect of low-intensity aerobic exercise training and anti-HMGB1 treatment on airway hyperresponsiveness, inflammation, and remodeling in SRA rats also was evaluated. Results SRA rats developed neutrophil-dominated airway inflammation ((29.5±4.1)% of the total cell numbers in BALF), whereas SSA rats developed eosinophil-dominated airway inflammation ((24.0±6.1)% of the total cell numbers in BALF). Compared with SSA rats, SRA rats had more severe airway hyperresponsiveness, lower levels of IL-25 ((33.6±10.3) vs. (104.8±24.9) pg/ml), IL-33 ((87.5±25.0) vs. (226.6±40.7) pg/ml), and TSLP ((1 933.2±899.5) vs. (7 224.0±992.1) pg/ml), and higher levels of HMGB1 ((21.2±4.5) vs. (5.4±1.6) ng/ml) and IL-17 ((780.5±261.7) vs. (291.4±76.4) pg/ml) in BALF (all P &lt;0.05). However, there was no significant difference in goblet cell hyperplasia, subepithelial collagen thickness, and airway smooth muscle remodeling between the two groups. Compared with control SSA rats, airway hyperresponsiveness, inflammation, and remodeling in SRA rats were less sensitive to DXM treatment. Anti-HMGB1 treatment attenuated airway hyperresponsiveness, inflammation, and remodeling in SRA rats to a certain extent and was accompanied by lower levels of IL-17 ((369.2±126.7) vs. (780.5±261.7) pg/ml in control SRA rats) in BALF (P &lt;0.05). Low-intensity aerobic exercise training decreased the expression of both HMGB1 ((14.1±2.9) vs. (21.2±4.5) ng/ml in control SRA rats) and IL-17 ((545.3±148.6) vs. (780.5±261.7) pg/ml in control SRA rats) in BALF (all P &lt;0.05) and was accompanied by improved airway hyperresponsiveness, inflammation, and remodeling in SRA rats (all P &lt;0.05). Conclusions Low-intensity aerobic exercise training attenuated airway hyperresponsiveness, inflammation, and remodeling in a rat model of SRA. Decreased HMGB1 and IL-17 levels in BALF by aerobic exercise training at least partly contributed to the improvements of SRA.

High-mobility group box 1 exacerbates CCl₄-induced acute liver injury in mice.

High-mobility group box 1 (HMGB1) is a nuclear factor that can also serve as an imflammatory mediator once released into extracellular milieu. Therefore, HMGB1 has been recognized to play a pivotal role in inflammatory diseases such as sepsis, acute lung injury, ischemia reperfusion injury and type 1 diabetes. Nevertheless, its impact on carbon tetrachloride (CCl4)-induced hepatic injury is yet to be elucidated. In the present report, we demonstrated evidence indicating that high levels of HMGB1 were not only present in the necrotic area of liver but also in the serum after CCl4 challenge. In line with these observations, administration of exogenous recombinant HMGB1 exacerbated CCl4-induced hepatic injury, while HMGB1 blocking antibody provided protection for mice against CCl4-induced acute liver injury as evidenced by the decrease of serum transaminase and reduction of hepatic tissues necrosis. Mechanistic studies revealed that blockade of HMGB1 attenuated CCl4-induced MDA accumulation along with improved SOD and GSH activity. Treatment of mice with HMGB1 neutralizing antibody also significantly inhibited the production of proinflammatory mediators TNF-α and IL-6 along with attenuated HMGB1 expression and its extracellular release. Together, our data suggest an essential role for HMGB1 in CCl4-induced acute liver injury, while HMGB1 neutralizing antibody could be served as an effective regimen for preventing CCl4-induced acute liver injury.

Abundant expression of HMGB1 in human T-cell lymphotropic virus type I-infected T-cell lines and high plasma levels of HMGB1 in patients with adult T-cell leukemia.

High mobility group box 1 (HMGB1) functions as a chromatin-associated nuclear protein and an extracellular signaling molecule. The concentration of HMGB1 protein and the expression of HMGB1 mRNA were analyzed by ELISA and polymerase chain reaction (PCR), respectively. The present study reports high plasma HMGB1 levels in patients with adult T-cell leukemia [ATL; which is caused by infection with human T-cell lymphotropic virus type I (HTLV-I)] compared with normal controls. In addition, HMGB1 was highly expressed in HTLV-I-infected T-cell lines compared with uninfected T-cell lines. The HTLV-I oncoprotein, Tax, induced extracellular release of HMGB1 in T cells. The results suggest that HMGB1 is a potential biomarker and a therapeutic target for ATL.

HMGB1 in ANCA-associated vasculitis: A longitudinal study

Background High mobility group box 1 (HMGB1) is a non-histone DNA binding protein that is passively released by dying cells or actively secreted by immunologic competent cells. Higher levels of HMGB1 have been found in ANCA-associated vasculitis (AAV), especially in patients with granulomatosis with polyangiitis (GPA) and active disease. HMGB1 has been associated with granulomatous manifestations of GPA and with biopsy-proven renal involvement in AAV. Objectives To measure HMGB1 levels longitudinally in a cohort of patients with AAV in order to evaluate the association with disease activity, remission, relapses, and specific organ involvement. Methods HMGB1 levels were tested by ELISA at diagnosis, at least twice during the remission period and during a disease flare. Disease activity was ascertained using the Birmingham Vasculitis Activity Score (BVAS). Results 50 patients with AAV and 35 age and sex matched healthy controls (HC) (58.5±14.1 years vs. 55.1±11.7 years at baseline; P=0.256), frequency of females (44.0% vs. 51.4%; P =0.499) were included. The frequency of each AAV subgroup was as follows: GPA (62%), microscopic polyangiitis (MPA) (20%), isolated necrotizing glomerulonephritis (iNGN) (16%) and Churg-Strauss syndrome (CSS) (2%). Localized disease was found in 16% and renal involvement in 74% at disease onset. Patients and HC had similar mean levels of HMGB1 at baseline (2.61±1.83 vs. 2.34±0.94 ng/mL; P=0.389). Patients with localized disease presented higher median levels of HMGB1 than those with generalized disease but this difference was not significant [3.14 (0.50-9.03) vs. 1.97 (0.46-7.54) ng/mL; P=0.233] whereas patients with renal involvement had lower levels of HMGB1 in comparison to those without renal involvement at baseline (2.29±1.48 vs. 3.52±2.41 ng/mL; P=0.035). Negative correlations were found between serum HMGB1 and 24 hours proteinuria levels (ρ = -0.352; P=0.038) and between HMGB1 and serum creatinine at baseline (ρ = -0.287; P=0.045). The mean follow-up period was 53.4 months and at least one relapse was observed in 34.0% of patients. Among relapsing patients, at least one relapse was found in 64.7% and 2 relapses in 35.3%. At baseline, relapsing patients presented significantly higher mean BVAS when compared to non-relapsing patients (20.2±8.8 vs. 15.3±5.8; P=0.038) while other baseline features were similar between relapsing and non-relapsing patients including age, gender, ANCA specificity, HMGB1 and C-reactive protein (CRP) levels. Median HMGB1 levels were as follows: 2.12 ng/mL at baseline, 1.44 ng/mL and 1.90 ng/mL in patients in remission 3 and 11 months after commencing therapy (P=0.029). Median HMGB1 levels were 2.26 ng/mL and 2.04 ng/mL in the first and in the second relapse, respectively. Conclusions Serum HMGB1 levels were similar between patients with AAV and controls at baseline and renal involvement may account for unexpectedly lower levels of HMGB1 in patients with AAV at baseline, possibly due to urinary loss in patients with proteinuria. No clear relation between HMGB1 levels and disease activity could be found. Nonetheless, a significant decrease in HMGB1 levels was observed in AAV patients from baseline to remission.

Urinary HMGB1 levels are associated with CD4+ T-cells in urine in patients with ANCA-associated vasculitis and active nephritis

Background High mobility group box 1 (HMGB1) is a nuclear non-histone protein that acts as an alarmin when released in the extra-cellular medium by necrotic or activated cells. High serum HMGB1 levels have been found in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and active nephritis as well as in patients with granulomatosis with polyangiitis (GPA) and predominant granulomatous manifestations. High serum and urinary HMGB1 levels have also been found in patients with systemic lupus erythematosus (SLE) and active nephritis. Objectives To evaluate serum and urinary levels of HMGB1, CD4+ T-cells and effector memory T-cells (CD4+ T EM )in peripheral blood (PB) and urine from patients with AAV and active nephritis. Methods 24 AAV patients with active nephritis and 10 healthy controls (HC) were evaluated in a cross-sectional study. Clinical diagnosis of GPA or microscopic polyangiitis (MPA) was made according to the European Medicines Agency algorithm whereas patients with isolated renal involvement, ANCA positivity and/or biopsy-proven pauci immune necrotizing glomerulonephritis were classified as renal limited vasculitis (RLV). Serum and urinary levels of HMGB1 were measured by Western blot while CD4+ T-cells and CD4+ T EM cells (CD4+CD45+RO+CCR7-) in urine were analyzed by flow cytometry. Disease activity was evaluated according to the Birmingham Vasculitis Activity Score (BVAS). Results Median urinary intensity of HMGB1 was significantly higher in AAV patients when compared to HC [5.15 (IQR 3.52-9.25) vs. 2.58 (1.88-4.24); P =0.006] whereas no difference was found among different AAV subsets regarding median urinary intensity of HMGB1 [GPA: 4.1 (2.6-16.4) vs. MPA: 5.1 (4.4-5.6) vs. RLV 5.7 (1.4-13.6); P =0.951] and HMGB1/creatinine ratio [GPA: 0.93 (0.29-3.91) vs. MPA: 1.29 (0.90-1.89) vs. RLV 0.54 (0.14-1.28); P =0.186]. In urine of AAV patients, median CD4+ T-cells was 125.00 (58.93-429.28) cells/ml and CD4+ T EM cells was 93.24 (43.21-343.37) cells/ml. Urine levels of HMGB1 were positively correlated with CD4+ T-cell counts in urine (rho: 0.454; P =0.014) and with CD4+ T EM cells in urine (rho: 0.378; P =0.049) while a tendency for a negative correlation between urinary HMGB1 and CD4+ T-cells in PB (rho: -0.307; P =0.083). No correlation could be found between urine levels of HMGB1 and 24 hour proteinuria (rho: 0.065; P =0.390), creatinine clearance (rho: -0.049; P =0.832), BVAS (rho: -0.203; P =0.171) and serum HMGB1 (rho: -0.046; P =0.419). Conclusions Urine HMGB1 levels are increased in AAV patients with active nephritis when compared to HC and are associated with CD4+ T-cells in urine suggesting that urine HMGB1 is a biomarker for renal activity in AAV.

Human Mesenchymal Stem Cells Migrate toward Colon Cancer Partially Regulated by HMGB1

Although Mesenchymal Stem Cells (MSC) have been explored as a new clinically relevant cell type to repair injured tissue, a number of studies have highlighted the important aspect of MSC therapy. Studies have shown that systemically administered MSCs migrate to sites of malignant tumor. The focus of this study was to identify the mechanism of migration of human MSCs into cancerous tissue. First, the effect of cultured medium from cancer cell lines on modulating the migration of MSCs was evaluated, using seven different human colon cancer cell lines. Interestingly, the secretion level of High Mobility Group Box 1 (HMGB1) protein from each cell line affected the migration capacity of MSCs. In addition, recombinant human HMGB1 increased MSC’s migration capacity in a dose-dependent manner. Finally, 1×106 human MSCs were injected subcutaneously into mice (n=14) with colon cancer tumors that secreted high levels of HMGB1. Bioluminescence live image analysis showed that MSCs surrounded the tumors after injection into these mice through day 6. Immunohistochemical analysis using CD90 as a specific antibody revealed the existence of MSCs in and around the tumors as well as the secretion of local HMGB1 from the tumors detected by anti-HMGB1 antibody. These findings are critical in understanding the role of MSCs in development of solid tumors and further, they offer insight that may be useful in therapeutic application of MSCs in the treatment of malignant tumors.

Local and systemic occurrences of HMGB1 in gnotobiotic piglets infected with E. coli O55 are related to bacterial translocation and inflammatory cytokines

High mobility group box 1 (HMGB1), a nuclear protein, can be secreted by stimulated cells or released from damaged cells. It is recognized as a late mediator of sepsis, but its extracellular occurrence has primarily been studied on the systemic level. Acute and chronic diseases of the gastrointestinal tract, however, have usually been connected with immediate local cell damage. We present local and systemic findings of HMGB1 in Escherichia coli O55-caused infection, in relation to inflammatory cytokines, using a pig gnotobiotic infection model. High levels of HMGB1 were detected in the intestine of those piglets that suffered from infection (fever, anorexia, and diarrhea), as compared to their E. coli 055-infected counterparts that thrived. These local changes were also reflected at the systemic level and related to inflammatory cytokines.Based on our findings of high levels of HMGB1 in the intestinal content of the infection-suffering gnotobiotic piglets, its concurrent presence with inflammatory cytokines, and the published literature, we propose that the detection and analysis of HMGB1 levels in feces can be a non-invasive method of clinical evaluation of the severity of enteric infections.

Serum high mobility group box‐1 is a powerful diagnostic and prognostic biomarker for pancreatic ductal adenocarcinoma

Extracellular high mobility group box-1 (HMGB1) contributes to tumor growth and invasiveness. We evaluated the diagnostic and prognostic ability of serum HMGB1 for pancreatic ductal adenocarcinoma (PDAC). Serum HMGB1 measured by enzyme-linked immunosorbent assay (ELISA) were compared among normal, chronic pancreatitis, PDAC group in both training (n = 25, each group) and independent validation set (n = 45, each group). To determine the usability of serum HMGB1 as a diagnostic predictor of PDAC, receiver operating characteristic (ROC) curves with sensitivity/specificity and logistic regression were evaluated. To assess the HMGB1-associated prognosis of PDAC, Kaplan-Meier survival and Cox proportional-hazards regression were applied. Serum HMGB1 was correlated with presence and advanced-stage of PDAC. Logistic regression exhibited serum HMGB1 was a remarkable biomarker to predict PDAC as a single or multiple-markers; sensitivity/specificity of serum HMGB1 were superior to carbohydrate antigen (CA) 19-9 or carcinoembryonic antigen (CEA) in both training and independent datasets. Kaplan-Meier survival analysis showed PDAC patients with high serum HMGB1 levels (>30 ng/mL; median survival, 192 days) had a worse prognosis than patients with low HMGB1 levels (≤30 ng/mL; 514 days) by log-rank (P = 0.017). Cox proportional-hazards model showed the relative hazard ratios in high-serum HMGB1 group was 3.077 compared with the low-serum HMGB1 group. In conclusion, serum HMGB1 is a desirable diagnostic and prognostic biomarker for PDAC compared with pre-existing PDAC biomarkers, CA19-9 and CEA.

Abstract LB-483: p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

Abstract Cellular senescence irreversibly arrests the proliferation of cells at risk for malignant transformation in part through activities of the transcriptional regulator and tumor suppressor p53. Cells that senesce owing to DNA damage also secrete many biologically active factors, including inflammatory cytokines such as IL-6. The High Mobility Group Box 1 (HMGB1) protein is unusual in having two distinct functions. Intracellularly, it binds chromatin and modulates transcription, including stimulating p53 activity. In addition, necrosis or microbial infection causes HMGB1 leakage or active secretion, respectively, whereupon it functions as an extracellular Alarmin to signal tissue damage and promote tissue regeneration, stem cell recruitment and immune activation. We show that HMGB1 is largely nuclear in non-senescent human and mouse fibroblasts and epithelial cells, but is actively exported from the nucleus and secreted by senescent cells. In culture and in vivo, HMGB1 re-localization occurred prior to the appearance of other hallmarks of senescence, and depended on the function of p53, but not the upstream p53 activator ATM. Old mice contained significantly higher levels of HMGB1 in serum compared to young animals. Disruption of HMGB1 stoichiometry, either by overexpression or depletion, induced a p53-dependent senescence growth arrest, but only HMGB1 overexpression, not HMGB1 depletion, promoted IL-6 secretion. Senescence-associated secretion required endogenous and secreted HMGB1 because deletion of endogenous HMGB1 – or addition of an HMGB1 blocking antibody - attenuated IL-6 secretion. Recombinant HMGB1 protein induced IL-6 secretion in cells depleted, but not harboring, endogenous HMGB1. Depletion of endogenous HMGB1 promoted NF-κ B transcriptional activity in cells cultured with recombinant HMGB1. Our findings identify a novel biological setting (senescence), independent of necrosis or microbial infection, in which HMGB1 secretion occurs, and link senescence-dependent HMGB1 redistribution to p53 activity and inflammatory cytokine secretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-483. doi:1538-7445.AM2012-LB-483

Anti-inflammatory activities of oleanolic acid on HMGB1 activated HUVECs

As a late mediator of inflammation, high mobility group box 1 (HMGB1) protein up-regulates pro-inflammatory cytokines in several inflammatory diseases. Further, high plasma levels of HMGB1 correlate with poor prognosis and increased mortality in patients with severe inflammation. Oleanolic acid (OA), a triterpenoid known for its anti-inflammatory and anti-cancer properties, is commonly present in several medicinal plants but the effects of OA on HMGB1-mediated pro-inflammatory responses of human endothelial cells is not well-studied. In this study, we investigated this question by monitoring the effect of OA on lipopolysaccharide (LPS)-mediated release of HMGB1 and the HMGB1-mediated modulation of inflammatory responses in human umbilical vein endothelial cells (HUVECs). OA potently inhibited the release of HMGB1 by HUVECs as well as down-regulated HMGB1-dependent adhesion and migration of the monocytic cell line THP-1 to activated HUVECs. OA also down-regulated the cell surface expression of the receptor of HMGB1, thereby inhibiting HMGB1-dependent pro-inflammatory responses by inhibiting activation of nuclear factor-κB (NF-κB) and production of tumor necrosis factor-α (TNF-α) by HMGB1. Given these results, OA showed anti-inflammatory activities and could be a candidate as a therapeutic agent for various inflammatory diseases through the inhibition of the HMGB1 signaling pathway.

Increased serum levels of high-mobility group box 1 (HMGB1) in primary Sjögren's syndrome

Objective: To determine serum levels of high-mobility group box 1 (HMGB1) in patients with primary Sjögren’s syndrome (pSS) as compared to healthy volunteers and patients with sicca symptoms, and to determine whether serum HMGB1 levels are correlated with disease activity in pSS.Methods: Serum HMGB1 levels were determined by enzyme-linked immunosorbent assay (ELISA) in 101 patients with pSS, 13 patients with sicca symptoms, and 40 healthy volunteers. Clinical and laboratory variables were also analysed and serum HMGB1 levels were correlated with the Sjögren’s Syndrome Disease Activity Index (SSDAI).Results: The serum levels of HMGB1 were significantly increased in pSS patients as compared to patients with sicca symptoms and healthy controls (p = 0.04 and p = 0.01, respectively). In the subgroups of patients with anti-SSA autoantibodies, the serum levels of HMGB1 were significantly higher than those in the subgroup of pSS patients who were anti-SSA negative and in healthy controls and patients with sicca symptoms (p < 0.001, p < 0.001, and p = 0.004, respectively). There was no significant correlation between serum HMGB1 levels (in pSS patients with anti-SSA autoantibodies) and SSDAI score (r = 0.03, p = 0.83). Patients with active disease had higher HMGB1 levels than patients with low disease activity (p = 0.04), but HMGB1 levels did not correlate with the SSDAI.Conclusion: Serum HMGB1 levels are increased in pSS patients and more specifically in patients with SSA autoantibodies. There was, however, no correlation of HMGB1 with the SSDAI.

Osteopontin and Other Regulators of Angiogenesis and Fibrogenesis in the Vitreous from Patients with Proliferative Vitreoretinal Disorders

The aim of this study was to determine the levels of the angiogenic and fibrogenic factors osteopontin (OPN), high-mobility group box-1 (HMGB1), and connective tissue growth factor (CTGF) and the antiangiogenic and antifibrogenic pigment epithelium-derived factor (PEDF) in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD). Vitreous samples from 48 PDR, 17 PVR and 30 RD patients were studied by enzyme-linked immunosorbent assay. OPN, HMGB1, CTGF, and PEDF levels were significantly higher in PDR patients than in RD patients (P < 0.001; 0.002; <0.001; <0.001, resp.). CTGF and PEDF levels were significantly higher in PVR patients than in RD patients (P < 0.001; 0.004, resp.). Exploratory logistic regression analysis identified significant associations between PDR and high levels of HMGB1, CTGF and PEDF, between PDR with active neovascularization and high levels of CTGF and PEDF, and between PDR with traction retinal detachment and high levels of HMGB1. In patients with PDR, there were significant correlations between the levels of PEDF and the levels of OPN (r = 0.544, P = 0.001), HMGB1 (r = 0.719, P < 0.001), and CTGF (r = 0.715, P < 0.001). In patients with PVR, there were significant correlations between the levels of OPN and the levels of HMGB1 (r = 0.484, P = 0.049) and PEDF (r = 0.559, P = 0.02). Our findings suggest that OPN, HMGB1, and CTGF contribute to the pathogenesis of proliferative vitreoretinal disorders and that increased levels of PEDF may be a response to counterbalance the activity of angiogenic and fibrogenic factors in PDR and PVR.

Plasma High-Mobility Group Box 1 Levels Predict Mortality After ST-Segment Elevation Myocardial Infarction

We evaluated the potential association between plasma high-mobility group box 1 (HMGB1) levels and outcome in patients with ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention. The positive effect of reperfusion after STEMI may be compromised by ischemic/reperfusion injury. HMGB1 is released by necrotic cells and, in pre-clinical studies, has been implicated to play a role in myocardial ischemic/reperfusion injury. The study included 141 STEMI patients, with acute occlusion of the left anterior descending coronary artery successfully treated with percutaneous coronary intervention. Plasma HMGB1 levels were measured by enzyme-linked immunoadsorbent assay at admission. Forty-two healthy individuals served as control subjects. After a median of 10 months of follow-up, 13 STEMI patients died. There were no significant differences with regard to baseline variables between the group of patients who survived and those who died. Baseline HMGB1 levels were increased in STEMI patients when compared with control subjects. Furthermore, the STEMI patients who died had higher HMGB1 levels than those who survived. After adjusting for age, sex, troponin I, and creatine kinase-myocardial band, we found that a doubling of HMGB1 concentrations increased the risk of mortality by 75% (hazard ratio: 1.75; 95% confidence interval: 1.1 to 2.8). Plasma HMGB1 levels are elevated in STEMI patients compared with healthy control subjects. Furthermore, after a follow-up period of 10 months, plasma HMGB1 levels are shown to be independently associated with increased mortality in STEMI patients treated with PCI. These data suggest that plasma HMGB1 may be used as a new prognostic biomarker in STEMI patients.

High-mobility group box 1 exacerbates concanavalin A-induced hepatic injury in mice

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as an early endogenous alarmin of inflammation following injury and as a late mediator of lethality in sepsis. Although HMGB1 has been implicated in acute lung injury, rheumatoid arthritis, and allograft rejection, its role in T-cell mediated hepatitis remains obscure. Here, we investigated the role and the underlying mechanisms of HMGB1 in concanavalin A (Con A) induced hepatic injury. We demonstrate that high levels of HMGB1 were detected in the necrotic area and in the cytoplasm of hepatocytes after Con A treatment. Administration of exogenous recombinant HMGB1 enhanced Con A-induced hepatitis, while blockade of HMGB1 protected animals from T cell-mediated hepatitis as evidenced by decreased serum transaminase, associated with reduced hepatic necrosis and mortality. Blockade of HMGB1 by a neutralizing antibody inhibited proinflammatory cytokine production, NFκB activity, and the late stage of T/NKT cell activation. These finding thus suggest a pivotal factor of HMGB1 in Con A-induced hepatitis. Blockage of extracellular HMGB1 may represent a novel therapeutic strategy to prevent hepatic injury in T cell-mediated hepatitis.