High Mobility Group B1 Research Articles

Insulin infusion suppresses while glucose infusion induces Toll-like receptors and high-mobility group-B1 protein expression in mononuclear cells of type 1 diabetes patients

The purpose of this study was to determine whether an insulin infusion exerts an anti-inflammatory effect and whether the infusion of small amounts of glucose results in oxidative and inflammatory stress in patients with type 1 diabetes. Ten patients with type 1 diabetes were infused with either 2 U/h of insulin with 100 ml 5% dextrose/h to or just dextrose (100 ml/h) or physiological saline (100 ml/h) for 4 h after an overnight fast on three separate days. Blood samples were collected at 0, 2, 4, and 6 h. Insulin with glucose infusion led to the maintenance of euglycemia and a significant suppression of reactive oxygen species (ROS) generation, p47(phox) expression, Toll-like receptor (TLR)-4, TLR-2, TLR-1, CD14, high-mobility group-B1 (HMGB1), p38 mitogen-activated protein (MAP) kinase, c-Jun NH2-terminal kinase (JNK)-1, and platelet/endothelial cell adhesion molecule expression and a fall in serum concentrations of C-reactive protein, HMGB1, and rapid upon activation T cell expressed and secreted. Glucose infusion led to an increase in plasma glucose concentration from 115 (fasting) to 215 (at 4 and 6 h) mg/dl and to an increase in ROS generation, the expression of TLR-4, TLR-2, TLR-1, HMGB1, p38 MAP kinase, and JNK-1, and plasma concentrations of HMGB1. While insulin reduces indexes of oxidative and inflammatory stress in patients with type 1 diabetes, even small amounts of glucose (20 g over 4 h) induce oxidative and inflammatory stress. These effects are reflected in TLR, p38 MAP kinase, and HMGB1 expression. The induction of significant oxidative and inflammatory stress by small amounts of glucose in patients with type 1 diabetes may have important pathophysiological and therapeutic implications.

Inhaled aerosolized insulin ameliorates hyperglycemia-induced inflammatory responses in the lungs in an experimental model of acute lung injury

IntroductionPrevious studies have shown that patients with diabetes mellitus appear to have a lower prevalence of acute lung injury. We assumed that insulin prescribed to patients with diabetes has an anti-inflammatory property and pulmonary administration of insulin might exert beneficial effects much more than intravenous administration.MethodsTwenty-eight mechanically ventilated rabbits underwent lung injury by saline lavage, and then the animals were allocated into a normoglycemia group (NG), a hyperglycemia group (HG), an HG treated with intravenous insulin (HG-VI) group or an HG treated with aerosolized insulin (HG-AI) group with continuous infusion of different fluid solutions and treatments: normal saline, 50% glucose, 50% glucose with intravenous insulin, or 50% glucose with inhaled aerosolized insulin, respectively. After four hours of treatment, the lungs and heart were excised en bloc, and then high-mobility group B1 concentration in bronchoalveolar lavage fluid, interleukin-8 and toll-like receptor 4 mRNA expression in bronchoalveolar lavage fluid cells, and lung myeloperoxidase activity were measured.ResultsTreatment with both aerosolized insulin and intravenous insulin attenuated toll-like receptor 4 mRNA expressions in the bronchoalveolar lavage fluid cells. Interleukin-8 and toll-like receptor 4 mRNA expression was significantly lower in the HG-AI group than in the HG-IV group. The lung myeloperoxidase activity in the normal healthy group showed significantly lower levels compared to the NG group but not different compared to those of the HG, HG-VI and HG-AI groups.ConclusionsThe results suggest that insulin attenuates inflammatory responses in the lungs augmented by hyperglycemia in acute lung injury and the insulin's efficacy may be better when administered by aerosol.

High mobility group B1 levels in sepsis and Disseminated Intravascular Coagulation.

Cytokines trigger coagulant and fibrinolytic systems in sepsis to result in Disseminated Intravascular Coagulation (DIC) that is an important complication and leads to disseminated hemorrhages and multi-organ failure. High Mobility Group B1 DNA Binding (HMGB1) protein is a cytokine taking part in systemic inflammatory response. The objective of this study was to investigate HMGB1 levels in groups of septic patients with and without DIC.Twenty-one septic patients without DIC and 12 septic patients with DIC from the Intensive Care Unit (ICU) were included in the study. In addition, 20 patients admitted to the ICU without sepsis or DIC and 20 healthy volunteers served as controls. Levels of HMGB1, prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimer, protein C, protein S, anti-thrombin III (ATIII), platelet (thrombocyte) and leukocyte count were determined. Levels of fibrinogen, protein C, ATIII and platelet count were significantly lower and D-dimer was significantly higher in the group with sepsis plus DIC compared to the group with sepsis without DIC. Levels of HMGB1 were higher in the group with sepsis and DIC compared to the group with sepsis; however, the difference was not statistically significant and the levels of HGMB1 of both groups were significantly higher compared to ICU and healthy control groups. HMGB1 levels were not significantly different in survivor and non survivor patients. HMGB1 levels did not differ in lower respiratory tract infection (LRTI) and urinary tract infection (UTI) in regard to the etiology of sepsis.

HMGB1-Facilitated p53 DNA Binding Occurs via HMG-Box/p53 Transactivation Domain Interaction, Regulated by the Acidic Tail

Facilitated binding of p53 to DNA by high mobility group B1 (HMGB1) may involve interaction between the N-terminal region of p53 and the high mobility group (HMG) boxes, as well as HMG-induced bending of the DNA. Intramolecular shielding of the boxes by the HMGB1 acidic tail results in an unstable complex with p53 until the tail is truncated to half its length, at which point the A box, proposed to be the preferred binding site for p53(1-93), is exposed, leaving the B box to bind and bend DNA. The A box interacts with residues 38-61 (TAD2) of the p53 transactivation domain. Residues 19-26 (TAD1) bind weakly, but only in the context of p53(1-93) and not as a free TAD1 peptide. We have solved the structure of the A-box/p53(1-93) complex by nuclear magnetic resonance spectroscopy. The incipient amphipathic helix in TAD2 recognizes the concave DNA-binding face of the A box and may be acting as a single-stranded DNA mimic.

Damage Associated Molecular Pattern Molecule-Induced microRNAs (DAMPmiRs) in Human Peripheral Blood Mononuclear Cells

Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10−4 and p<3.7×10−3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1+/+) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1−/− MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic “PAMPmiR”. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.

A Historical Perspective on Sepsis

In North America, approximately 700,000 cases of sepsis occur each year, with mortality ranging between 30% and 50%. The American Journal of Pathology has featured numerous articles on the topic, revealing mechanistic insights gleaned from both experimental rodent models and human sepsis. Nonetheless, there remains urgent need to determine the basis for sepsis-related complications and how they can be avoided, as well as how they can be most effectively treated once recognized. This historical perspective reviews what we currently understand about the mechanisms of sepsis, as well as the barriers that remain in our treatment strategies.

High mobility group B1 impairs hepatocyte regeneration in acetaminophen hepatotoxicity

BackgroundAcetaminophen (APAP) overdose induces massive hepatocyte necrosis. Necrotic tissue releases high mobility group B1 (HMGB1), and HMGB1 contributes to liver injury. Even though blockade of HMGB1 does not protect against APAP-induced acute liver injury (ALI) at 9 h time point, the later time points are not studied and the role of HMGB1 in APAP overdose is unknown, it is possible that neutralization of HMGB1 might improve hepatocyte regeneration. This study aims to test whether blockade of HMGB1 improves hepatocyte regeneration after APAP overdose.MethodsMale C57BL/6 mice were treated with a single dose of APAP (350 mg/kg). 2 hrs after APAP administration, the APAP challenged mice were randomized to receive treatment with either anti-HMGB1 antibody (400 μg per dose) or non-immune (sham) IgG every 24 hours for a total of 2 doses.Results24 hrs after APAP injection, anti-HMGB1 therapy instead of sham IgG therapy significantly improved hepatocyte regeneration microscopically; 48 hrs after APAP challenge, the sham IgG treated mice showed 14.6% hepatic necrosis; in contrast, blockade of HMGB1 significantly decreased serum transaminases (ALT and AST), markedly reduced the number of hepatic inflammatory cells infiltration and restored liver structure to nearly normal; this beneficial effect was associated with enhanced hepatic NF-κB DNA binding and increased the expression of cyclin D1, two important factors related to hepatocyte regeneration.ConclusionHMGB1 impairs hepatocyte regeneration after APAP overdose; Blockade of HMGB1 enhances liver recovery and may present a novel therapy to treat APAP overdose.

Preservation of pancreatic islets in cold UW solution before transplantation

Culture of islets prior to transplantation needs to be revisited for maintaining functional islet capacity. This study was conducted to compare cold UW (University of Wisconsin) preservation with conventional culture based on insulin secretory capacity in vitro and in vivo. Islets isolated from Wistar rats were either cultured for 24 h at 37°C in RPMI1640 medium or DMEM containing various concentrations of glucose or preserved for the same period in UW solution or in DMEM solution at 4°C. The islet yield in UW group, but not in other groups, was maintained as comparable with that of fresh islets. Insulin secretory capacity in response to glucose was maintained only in the islets of UW group, but not in other groups. SCID mice given 300 IEQ islets of UW group showed gradual restoration of normoglycemia as found in the mice given freshly isolated islets. Meanwhile, those mice given cultured islets for 24 h at 37°C in RPMI1640 medium showed rapid decrease of blood glucose levels on day 1 followed by relatively elevated levels on day 2, suggesting unstable insulin secretory capacity of islets. Morphological staining with anti-HMGB1 (high mobility group B1) antibody revealed central damage of islets in all culture groups regardless of glucose concentration and in islets of cold DMEM group, whereas those in the UW group were quite intact. These results suggest that cold preservation in UW solution is simple and beneficial in protecting islets morphologically and functionally before transplantation.

Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation

BackgroundThe murine air pouch membrane represents an easily accessible tissue for studies on gene regulation in acute inflammation. Considering that acute inflammation may affect expression of molecular reference genes, we evaluated the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prolylpeptidyl isomerase A (PPIA) in the air pouch membrane during a complete time course of urate crystal inflammation and correlated the results with expression of interleukin (IL)-1β and hypoxia inducible factor (HIF)-1α. In addition, we aimed to identify alternate potential reference genes.MethodsUsing custom microfluidic real-time PCR arrays, the expression of 96 genes including GAPDH, PPIA, IL-1β, and HIF-1α was determined in dissected air pouch membranes 1, 4, 9, 18, 27, and 50 hours (h) after injecting monosodium urate (MSU) crystals into the pouch. One-way ANOVA was used to detect differential gene expression throughout the time course. Using the genes on these arrays as a convenience sample, alternate candidate reference genes were sought (1) with a biostatistical approach and (2) using the geNorm software tool.ResultsPouch leukocytes peaked at t = 9h and declined toward t = 50h. PPIA expression was not differentially regulated (p = 0.52, ANOVA). In contrast, GAPDH mRNA increased steadily after crystal injection, reaching a maximal 2.8-fold increase at t = 18h (p = 0.0006, t test), which followed a marked induction of IL-1β (max., 208-fold at t = 4h, p = 8.4 × 10-5, t test) and HIF-1α (max., 6.6-fold at t = 4h, p = 0.00025, t test). Fifteen genes were artifactually identified as "significantly regulated" when Ct values were normalized against GAPDH expression. The biostatistical approach and the geNorm analysis identified overlapping sets of candidate reference genes. Both ranked PPIA as the best candidate, followed by defender against cell death 1 (DAD1) and high-mobility group B1 (HMGB1).ConclusionsGAPDH mRNA expression is up-regulated in urate crystal inflammation, possibly due to inflammation-associated hypoxia. Using GAPDH mRNA for molecular normalization resulted in significant artifacts in the calculated expression of the target mRNAs. PPIA and other stably expressed genes promise to be more appropriate reference genes in this model.

High mobility group B1 protein interacts with its receptor RAGE in tumor cells but not in normal tissues

The high mobility group box 1 (HMGB1) protein is an abundant non-histone component of chromatin well known for its two DNA binding domains, HMG box A and HMG box B. The main characteristics of the HMGB1 protein as an 'architectural' factor are its ability to recognize and bind with high affinity to distorted DNA and its ability to induce kinks in linear DNA fragments. The HMGB1 protein has been correlated to cancer progression. An elevated expression of HMGB1 occurred in certain types of primary tumor, including melanoma and colon, prostate, pancreatic and breast cancers, and in the majority of cases HMGB1 is associated with invasion and metastasis. The main signaling pathway is activated through the interaction of HMGB1 with its Receptor for Advanced Glycation End products (RAGE). Certain data indicate that an elevated expression of RAGE and HMGB1 is not always a prerequisite of poor prognosis of tumor development. The cellular localization of the ligand/receptor pair also requires consideration. The data concerning the expression of HMGB1 protein and its receptor RAGE in various tissues and tumor cells reflect the overall production of the proteins. However, they do not refer to their cellular localization and there is no direct evidence for the formation of a stable complex between them. In the present study, we investigated the subcellular distribution of HMGB1 and its receptor RAGE in various rat organs compared to Guerin ascites tumor cells. In the normal tissues the proteins exist in their soluble form, whereas in the tumor cells they are insoluble and membrane-bound. HMGB1 forms a stable complex with RAGE only in the protein extract derived from the cancer cells predominantly in the membrane fraction.

Elevated levels of circulating IL-7 and IL-15 in patients with early stage prostate cancer

BackgroundChronic inflammation has been suggested to favour prostate cancer (PCA) development. Interleukins (IL) represent essential inflammation mediators. IL-2, IL-7, IL-15 and IL-21, sharing a common receptor γ chain (c-γ), control T lymphocyte homeostasis and proliferation and play major roles in regulating cancer-immune system interactions. We evaluated local IL-2, IL-7, IL-15 and IL-21 gene expression in prostate tissues from patients with early stage PCA or benign prostatic hyperplasia (BPH). As control, we used IL-6 gene, encoding an IL involved in PCA progression. IL-6, IL-7 and IL-15 titres were also measured in patients' sera.MethodsEighty patients with BPH and 79 with early (1 to 2c) stage PCA were enrolled. Gene expression in prostate tissues was analyzed by quantitative real-time PCR (qRT-PCR). Serum IL concentrations and acute phase protein titres were evaluated by ELISA. Mann-Whitney, Wilcoxon and χ2 tests were used to compare IL gene expression and serum titers in the two groups of patients. Receiver operating characteristic (ROC) curves were constructed to evaluate the possibility to distinguish sera from different groups of patients based on IL titers.ResultsIL-2 and IL-21 gene expression was comparably detectable, with low frequency and at low extents, in PCA and BPH tissues. In contrast, IL-6, IL-7 and IL-15 genes were expressed more frequently (p < 0.0001, p = 0.0047 and p = 0.0085, respectively) and to significantly higher extents (p = 0.0051, p = 0.0310 and p = 0.0205, respectively) in early stage PCA than in BPH tissues. Corresponding proteins could be detected to significantly higher amounts in sera from patients with localized PCA, than in those from patients with BPH (p = 0.0153, p = 0.0174 and p = 0.0064, respectively). Analysis of ROC curves indicates that IL-7 (p = 0.0039), but not IL-6 (p = 0.2938) or IL-15 (p = 0.1804) titres were able to distinguish sera from patients with malignancy from those from patients with benign disease. Serum titres of C reactive (CRP), high mobility group B1 (HMGB1) and serum amyloid A (SAA) acute phase proteins were similar in both groups of patients.ConclusionsExpression IL-7 and IL-15 genes in prostate tissues and corresponding serum titres are significantly increased in patients with early stage PCA as compared with patients with BPH.

Cyclophilin A release as a biomarker of necrotic cell death

Necroptosis is a form of programmed necrotic cell death mediated by the activity of receptor-interacting protein 1 (RIP1) kinase (1). This type of cell death is caspase-independent and marked by early membrane permeabilization and does not exhibit any other hallmarks of apoptotic cell death such as phosphatidylserine externalization or DNA strand breaks (1). Thus, the markers available to characterize apoptotic cell death cannot be used to analyze necroptosis. Only one marker of necrosis has been identified, and that is the release of the chromatin protein high mobility group B1 (HMGB1) (2). However, we have found the release of HMGB1 to occur at a late stage of death. In this study we have found that cyclophilin A (CypA), a cytosolic peptidyl-prolyl cis-trans isomerase, is released early in necroptosis. We propose that the release of CypA may be used as a biomarker for necroptosis and other cell death processes when the integrity of the plasma membrane is compromised. Since necroptotic cells exhibit early permeabilization of the plasma membrane (1), we hypothesized that the release of intracellular protein(s) may be used as a biomarker for necroptosis. Treatment of L929 cells with zVAD.fmk induces necroptosis, which can be inhibited by treatment with the RIP1 kinase inhibitor, necrostatin-1 (Nec-1) (1). We observed the appearance of a strong band around 17 kD by SDS-PAGE in the supernatant of zVAD-treated L929 cells at an early stage of cell death (Supplemental Figure 1, top panel). Mass spectrometry analysis identified this band as cyclophilin A (CypA), a soluble cytosolic protein that catalyzes the cis-trans isomerization of proline bonds. The release of CypA was further confirmed by western blot (Supplemental Figure 1, bottom panel). In contrast to the necrosis marker HMGB1, the timing of CypA release precedes or occurs around the same time as that of HMGB1 (Figure 1A). Nec-1 blocks the release of CypA into the supernatant (Figure 1B). Figure 1 Cyclophilin A is released from necroptotic cells as a result of cytoplasmic membrane permeabilization. A, L929 cells were treated with 100μM zVAD.fmk for indicated hrs. The supernatants were collected, concentrated, and analyzed by SDS-PAGE followed ... To determine if the release of CypA from the cells was specific to necroptosis, we tested the supernatant of apoptotic cells. We were unable to detect CypA released from Jurkat cells treated with TNFα and CHX to induce apoptosis, even though intracellular CypA appears to be degraded at 24h when cell death was completed (Figure 1C). However, FADD-deficient Jurkat cells, which undergo necroptosis in response to TNFα, did release CypA (Figure 1C). We also tested different inducers of apoptosis for the ability to release CypA. Treatment of doxorubicin and staurosporine caused a small amount of CypA release at later stages of cell death, hours after caspase-3 activation (Supplemental Figures 2-3). Thus, CypA may also be released after permeabilization of the plasma membrane in late phase of apoptotic cell death. However, since apoptotic cells are efficiently engulfed in vivo, the release of CypA by apoptotic cells may not occur in vivo. To examine if CypA could be released by permeabilization of the cells, we treated cells with the detergent digitonin, which led to cell membrane permeabilization. We found that digitonin permeabilized cells also release CypA (Supplemental Figure 4). This may explain why CypA is released during necroptosis, which exhibit early permeabilization of the plasma membrane (1). Elevated serum CypA levels have been found in inflammatory diseases such as rheumatoid arthritis and CypA is secreted in vivo by progressive solid tumors (3, 4). CypA release from necrotic cells may play a role in the effects of necrosis on surrounding tissue. Necrosis is typically associated with inflammation. Necroptosis occurs in models of neuronal ischemia and traumatic brain injury (1, 5). Treatment with Nec-1 significantly reduces cell death and inhibits the recruitment of neutrophils to the injured area (5). Extracellular CypA has been shown to induce a rapid inflammatory response upon injection in vivo and to recruit neutrophils. This recruitment is dependent on the CypA receptor, CD147 (EMMPRIN) (3). Signaling of CypA through CD147 is proposed to contribute to the migration of neutrophils into the joints in rheumatoid arthritis and possibly in the destruction of bone and cartilage in the disease (6). We propose that CypA may be used as a biomarker for necroptosis. The release of CypA, in combination with Nec-1, a specific inhibitor of necroptosis, may be used as an assay for the activation of necroptosis, both in vitro and in vivo.

Insulin Suppresses Endotoxin-Induced Oxidative, Nitrosative, and Inflammatory Stress in Humans

OBJECTIVETo investigate whether insulin reduces the magnitude of oxidative, nitrosative, and inflammatory stress and tissue damage responses induced by endotoxin (lipopolysaccharide [LPS]).RESEARCH DESIGN AND METHODSNine normal subjects were injected intravenously with 2 ng/kg LPS prepared from Escherichia coli. Ten others were infused with insulin (2 units/h) for 6 h in addition to the LPS injection along with 100 ml/h of 5% dextrose to maintain normoglycemia.RESULTSLPS injection induced a rapid increase in plasma concentrations of nitric oxide metabolites, nitrite and nitrate (NOM), and thiobarbituric acid–reacting substances (TBARS), an increase in reactive oxygen species (ROS) generation by polymorphonuclear leukocytes (PMNLs), and marked increases in plasma free fatty acids, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibition factor (MIF), C-reactive protein, resistin, visfatin, lipopolysaccharide binding protein (LBP), high mobility group-B1 (HMG-B1), and myoglobin concentrations. The coinfusion of insulin led to a total elimination of the increase in NOM, free fatty acids, and TBARS and a significant reduction in ROS generation by PMNLs and plasma MIF, visfatin, and myoglobin concentrations. Insulin did not affect TNF-α, MCP-1, IL-6, LBP, resistin, and HMG-B1 increases induced by the LPS.CONCLUSIONSInsulin reduces significantly several key mediators of oxidative, nitrosative, and inflammatory stress and tissue damage induced by LPS. These effects of insulin require further investigation for its potential use as anti-inflammatory therapy for endotoxemia.

High-mobility group B1 proteins in canine lymphoma: prognostic value of initial and sequential serum levels in treatment outcome following combination chemotherapy

Elevated high-mobility group box 1 (HMGB1) levels have been demonstrated in different human neoplasias. Information on serum HMGB1 before and during chemotherapy is lacking, as is data pertaining to its prognostic significance. The aim of this study was to characterize serum HMGB1 level in dogs with lymphoma and to assess its influence on the outcome following chemotherapy. Serum HMGB1 concentrations were measured in 16 dogs with lymphoma before treatment (W1) and on weeks 2 (W2), 6 (W6) and 12 (W12) of treatment with chemotherapy. Initial serum HMGB1 levels were significantly higher than HMGB1concentrations in control dogs and the levels in W2, W6 and W12. HMGB1-W1 concentrations were lower in dogs achieving complete remission than that in the single dog with partial remission. The ratio W12/W6 exhibited significant influence on remission duration. In these dogs with lymphoma, serum HMGB1 was elevated in comparison with that in controls. Initial serum HMGB1 level and its modulation during treatment may possess prognostic value.

HMGB1: A Dangerous Player in Lupus Pathogenesis

In an article in this issue of The Journal , Li, et al 1 provide compelling evidence for a role of HMGB1 (high mobility group B1) in the pathogenesis of systemic lupus erythematosus (SLE) and highlight the potential of this molecule as both a biomarker and a target of new therapy. HMGB1 is a non-histone nuclear protein that has emerged as the prototypic alarmin2. While the terminology and classification of alarmins are in flux, alarmins can be considered as cellular components that stimulate the immune system when they leave their usual intracellular location during either cell activation or cell death. In other terminology, alarmins are danger signals, or DAMP (damage/death associated molecular patterns). Like PAMP (pathogen associated molecular patterns), DAMP can stimulate Toll-like receptors (TLR) to activate innate immunity3. The importance of HMGB1 in lupus is not unexpected in view of the mechanisms of its release from activated or dying cells and its surprisingly powerful immunological activity. These activities were initially discovered in seminal studies to identify immune mediators that could be targeted in sepsis more effectively than cytokines such as tumor necrosis factor-α (TNF-α)4. While TNF blockade is successful in animal models, it did not work in human trials of this often fatal condition. The failure could reflect species differences or the transient production of TNF-α after PAMP stimulation. When infection became clinically obvious, TNF-α may have already left the scene, nullifying the action of a TNF blocker. Thus, the therapy of sepsis may require a target whose action still persists when the patient comes to medical attention5. As shown in studies by Wang, et al , stimulation of macrophages by lipopolysaccharide (LPS) leads to the production of an immunologically active protein that importantly shows delayed production in in vitro and … Address correspondence to Dr. D. Pisetsky, Department of Medicine and Immunology, Medical Research Service, Durham VA Hospital, 151G Durham VAMC, 508 Fulton Street, Durham, NC 27705. E-mail: dpiset001{at}mc.duke.edu

Orexigenic hormone ghrelin ameliorates gut barrier dysfunction in sepsis in rats*

We have recently shown that ghrelin, a novel orexigenic hormone, is reduced in sepsis. Ghrelin treatment, mediated through ghrelin receptors in the brain, attenuates sepsis-induced inflammation and mortality. Gut barrier dysfunction is common in sepsis. High-mobility group B1 (HMGB1) increases gut permeability both in vitro and in vivo. However, it remains unknown whether ghrelin has any effects on HMGB1 and gut barrier function in sepsis. We hypothesized that ghrelin decreases HMGB1 release and attenuates sepsis-induced gut barrier dysfunction through central ghrelin receptors. Prospective, controlled, and randomized animal study. A research institute laboratory. Male adult Sprague-Dawley rats (275-325 g). Cecal ligation and puncture (CLP) followed by injection/infusion of ghrelin. Five hours after CLP, a bolus intravenous injection of 2 nmol of ghrelin was followed by a continuous infusion of 12 nmol of ghrelin via an osmotic mini-pump for 15 hrs. Twenty hours after CLP, brain ghrelin levels, serum HMGB1 levels, ileal mucosal permeability to fluorescein isothiocyanate dextran, bacterial counts in the mesenteric lymph nodes complex, and gut water content were determined. In additional groups of animals, bilateral trunk vagotomy was performed at 5 hrs after CLP before ghrelin injection. Furthermore, to confirm the role of central ghrelin receptors in ghrelin's effect, ghrelin (1 nmol) was administered through intracerebroventricular injection at 5 hrs after CLP. Our results showed that brain levels of ghrelin decreased by 34% at 20 hrs after CLP. Intravenous administration of ghrelin completely restored brain levels of ghrelin, significantly reduced the elevated HMGB1 levels, and attenuated gut barrier dysfunction. Vagotomy eliminated ghrelin's inhibition of HMGB1 and attenuation of gut barrier dysfunction. Intracerebroventricular injection of ghrelin decreased serum HMGB1 levels and ameliorated gut barrier dysfunction. Ghrelin reduces serum HMGB1 levels and ameliorates gut barrier dysfunction in sepsis by vagus nerve activation via central ghrelin receptors. Ghrelin can be further developed as a novel agent to protect gut barrier function in sepsis.

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

Role of high mobility group protein B1 in cancer

As a nonhistone chromosomal protein,high mobility group B1 ( HMGB1 ) is involed in a va-riety of biological processes, including transcription, DNA repair, V (D) J recombination, differentiation, develop-ment,and extracellular signaling. Increased expression of HMGB1 is associated with the proliferation, invasion and metastasis of many tumor types. Furthermore, HMGB1 overexpression inhibits apoptosis of cancer cells and tumor suppressor genes,resuhing in the genesis and development of tumor. HMGB1 plays roles in activation of plasminogen and matrix metalloproteases,reorganization of cytoskeleton. HMGB1 can also be involved in DNA damage repair,preventing cell carcinogenesis. HMGB1 can increase the sensitivity of breast cancer cells to γ-ray radiation. Key words: HMGB1 protein; Neoplasm metastasis; Radiobiology

Anchoring Fusion Thrombomodulin to the Endothelial Lumen Protects against Injury-induced Lung Thrombosis and Inflammation

Endothelial thrombomodulin (TM) regulates thrombosis and inflammation. Diverse forms of pulmonary and vascular injury are accompanied by down-regulation of TM, which aggravates tissue injury. We postulated that anchoring TM to the endothelial surface would restore its protective functions. To design an effective and safe strategy to treat pulmonary thrombotic and inflammatory injury. We synthesized a fusion protein, designated scFv/TM, by linking the extracellular domain of mouse TM to a single-chain variable fragment of an antibody to platelet endothelial cell adhesion molecule-1 (PECAM-1). The targeting and protective functions of scFv/TM were tested in mouse models of lung ischemia-reperfusion and acute lung injury (ALI) caused by intratracheal endotoxin and hyperoxia, both of which caused approximately 50% reduction in the endogenous expression of TM. Biochemical assays showed that scFv/TM accelerated protein C activation by thrombin and bound mouse PECAM-1 and cytokine high mobility group-B1. After intravenous injection, scFv/TM preferentially accumulated in the mouse pulmonary vasculature. In a lung model of ischemia-reperfusion injury, scFv/TM attenuated elevation of early growth response-1, inhibited pulmonary deposition of fibrin and leukocyte infiltration, and preserved blood oxygenation more effectively than soluble TM. In an ALI model, scFv/TM, but not soluble TM, suppressed activation of nuclear factor-kappaB, inflammation and edema in the lung and reduced mortality without causing hemorrhage. Targeting TM to the endothelium using an scFv anchor enhances its antithrombotic and antiinflammatory effectiveness in models of ALI.

High mobility group B2 is secreted by myeloid cells and has mitogenic and chemoattractant activities similar to high mobility group B1

High mobility group B box (HMGB) proteins are a family of chromatin proteins made up of two basic DNA binding domains, HMG box A and B, and a C-terminal acidic tail. HMGB have a highly conserved sequence, but different expression pattern: HMGB1 is almost ubiquitous, whereas the others are highly expressed in only a few tissues in adults. We previously demonstrated that HMGB1 is released by necrotic cells and has chemoattractant activity for inflammatory and stem cells, via binding to receptor for advanced glycation endproducts (RAGE). HMGB1 can be actively secreted by inflammatory cells. Here, we report that also HMGB2 can be secreted by THP-1 cells, and promotes proliferation and migration of endothelial cells. These functions of HMGB2 are exerted via engagement of RAGE, whose blockade completely abrogates cell responses. Since extracellular HMGB2 has been detected in the blood and other biological fluids, it might be necessary to target HMGB2 at the same time as HMGB1 for therapeutical efficacy.