Radioembolization therapy (RE) is an efficient locoregional treatment for liver metastases from colorectal cancer. Serum biomarkers involved in immunogenic cell death are potentially valuable for early predicting therapy response and estimating prognosis. In a prospective observation study, blood samples were taken from 49 consecutive colorectal cancer patients with extensive hepatic metastases before, 24 and 48 hr after RE. Serum levels of high mobility group box 1 (HMGB1), receptor of glycation end products (RAGE) and activity of desoxyribonuclease were compared with response to therapy regularly determined radiologically 3 months after therapy and with overall survival. Serum levels of HMGB1 were increased already 24 hr after RE, while RAGE levels were decreased and DNAse remained unchanged. In radiological staging, 35 patients demonstrated disease progression while 14 patients had stable disease or remission. Serum HMGB1 levels 24 hr after RE were significantly higher in progressive than in nonprogressive patients while for RAGE and DNAse no difference was observed between the response groups. Concerning overall survival, high pretherapeutic (0 hr) and 24 hr levels of HMGB1 were associated with poor outcome. Multivariate analysis including HMGB1, tumor, liver and inflammation markers revealed HMGB1 and CRP as independent prognostic parameters. HMGB1 is a valuable serum biomarker for early estimation of therapy response and prognosis in colorectal cancer patients with liver metastases undergoing RE therapy.

High-mobility group box 1 (HMGB1) was recently established as a proinflammatory mediator of sepsis, and its potential role in the pathogenesis of sepsis remains elusive. In the present study, we determined whether HMGB1 increases the permeability of the endothelial cell monolayer in sepsis. Permeability was measured from fluorescein isothiocyanate (FITC)-dextran 40-kDa flux across the endothelial cell monolayer at control and after HMGB1 administration. We found that HMGB1 increased human umbilical vein endothelial cell permeability to FITC-dextran 40 kDa in a time- and concentration-dependent manner. HMGB1 induced the mRNA transcription and protein expression of receptor for advanced glycation end products (RAGE). Blockade of cell surface receptors RAGE with specific neutralizing antibodies and RAGE siRNA or blockade of Src family tyrosine kinase with inhibitor PP2 significantly reduced HMGB1-induced hyperpermeability of endothelial cell monolayer. Our data demonstrate that (1) HMGB1 increases permeability of endothelial cell monolayer in a time- and concentration-dependent manner and (2) HMGB1-induced hyperpermeability is mediated through RAGE and Src family tyrosine kinase signaling pathway. These findings may have implications for therapeutic interventions in patients with sepsis.

OBJECTIVE

Free cholesterol (FC) accumulates in non-alcoholic steatohepatitis (NASH) but not in simple steatosis. We sought to establish how FC causes hepatocyte injury.

METHODS

In NASH-affected livers from diabetic mice, subcellular FC distribution (filipin fluorescence) was established by subcellular marker co-localization. We loaded murine hepatocytes with FC by incubation with low-density lipoprotein (LDL) and studied the effects of FC on JNK1 activation, mitochondrial injury and cell death and on the amplifying roles of the high-mobility-group-box 1 (HMGB1) protein and the Toll-like receptor 4 (TLR4).

RESULTS

In NASH, FC localized to hepatocyte plasma membrane, mitochondria and ER. This was reproduced in FC-loaded hepatocytes. At 40 μM LDL, hepatocyte FC increased to cause LDH leakage, apoptosis and necrosis associated with JNK1 activation (c-Jun phosphorylation), mitochondrial membrane pore transition, cytochrome c release, oxidative stress (GSSG:GSH ratio) and ATP depletion. Mitochondrial swelling and crystae disarray were evident by electron microscopy. Jnk1(-/-) and Tlr4(-/-) hepatocytes were refractory to FC lipotoxicity; JNK inhibitors (1-2 μM CC-401, CC-930) blocked apoptosis and necrosis. Cyclosporine A and caspase-3 inhibitors protected FC-loaded hepatocytes, confirming mitochondrial cell death pathways; in contrast, 4-phenylbutyric acid, which improves ER folding capacity did not protect FC-loaded hepatocytes. HMGB1 was released into the culture medium of FC-loaded wild type (WT) but not Jnk1(-/-) or Tlr4(-/-) hepatocytes, while anti-HMGB1 anti-serum prevented JNK activation and FC lipotoxicity in WT hepatocytes.

CONCLUSIONS

These novel findings show that mitochondrial FC deposition causes hepatocyte apoptosis and necrosis by activating JNK1; inhibition of which could be a novel therapeutic approach in NASH. Further, there is a tight link between JNK1-dependent HMGB1 secretion from lipotoxic hepatocytes and a paracrine cytolytic effect on neighbouring cholesterol-loaded hepatocytes operating via TLR4.

Previous studies proved that melatonin protected against secondary brain damage by modulating oxidative stress after experimental subarachnoid hemorrhage (SAH), but it has not been evaluated yet about its effects on inflammatory pathway and secondary cognitive dysfunction in SAH model. This study was undertaken to evaluate the influence of melatonin on toll-like receptor 4 (TLR4) signaling pathway and neurobehavioral tests after SAH. Adult SD rats were divided into four groups: control group (n = 20), SAH group (n = 20), SAH+vehicle group (n = 20), and SAH+melatonin group (n = 20). The rat SAH model was induced by injection of 0.3 mL fresh arterial, nonheparinized blood into the prechiasmatic cistern in 20 s. In SAH+melatonin group, melatonin was administered i.p. at 150 mg/kg at 2 and 24 hr after the induction of SAH. Cognitive and memory changes were investigated in the Morris water maze. Treatment with melatonin markedly decreased the expressions of TLR4 pathway-related agents, such as high-mobility group box 1 (HMGB1), TLR4, nuclear factor-κB (NF-κB), myeloid differentiation factor 88 (MyD88), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS). Administration of melatonin following SAH significantly ameliorated spatial learning and memory deficits in this prechiasmatic blood injection model. Staining of apoptosis and necrosis indicated that fewer positive cells appeared in melatonin-treated group than SAH+vehicle group. In conclusion, melatonin may attenuate neurobehavioral dysfunction in this SAH model, and melatonin exhibits neuroprotection possibly not only through anti-oxidative pathway but also anti-inflammatory signaling after experimental SAH.

The adolescent brain undergoes important dynamic and plastic cell changes, including overproduction of axons and synapses, followed by rapid pruning along with ongoing axon myelination. These developmental changes make the adolescent brain particularly vulnerable to neurotoxic and behavioral effects of alcohol. Although the mechanisms of these effects are largely unknown, we demonstrated that ethanol by activating innate immune receptors toll-like receptor 4 (TLR4), induces neuroinflammation and brain damage in adult mice. The present study aims to evaluate whether intermittent ethanol treatment in adolescence promotes TLR4-dependent pro-inflammatory processes, leading to myelin and synaptic dysfunctions, and long-term cognitive impairments. Using wild-type (WT) and TLR4-deficient (TLR4-KO) adolescent mice treated intermittently with ethanol (3.0g/kg) for 2weeks, we show that binge-like ethanol treatment activates TLR4 signaling pathways (MAPK, NFκB) leading to the up-regulation of cytokines and pro-inflammatory mediators (COX-2, iNOS, HMGB1), impairing synaptic and myelin protein levels and causing ultrastructural alterations. These changes were associated with long-lasting cognitive dysfunctions in young adult mice, as demonstrated with the object recognition, passive avoidance and olfactory behavior tests. Notably, elimination of TLR4 receptors prevented neuroinflammation along with synaptic and myelin derangements, as well as long-term cognitive alterations. These results support the role of the neuroimmune response and TLR4 signaling in the neurotoxic and behavioral effects of ethanol in adolescence.

High mobility group box 1 (HMGB1) as a novel inflammatory molecule has been shown to be involved in a variety of cell physiological and pathological behaviors including immune response, inflammation and cancer. Evidence suggests that HMGB1 plays a critical role in the development and progression of multiple malignancies. However, the underlying molecular mechanisms for the HMGB1-mediated growth and invasion of gastric cancer have not yet been elucidated. The present study investigated the expression of HMGB1 in gastric adenocarcinoma (GAC) and the mechanisms by which it contributes to tumor growth and invasion. The correlation between HMGB1 expression and clinicopathological characteristics of GAC patients was assessed by immunohistochemical assay through tissue microarray procedures. The RNA and protein expressions of HMGB1 and downstream factors were detected by quantitative PCR and western blot assays; cell proliferation and invasion were determined by MTT, wound-healing and 3D-Matregel assays, subcutaneous SGC-7901 tumor models were established to verify tumor growth in vivo. We demonstrated that, the expression of HMGB1 was significantly increased in the nucleus of GAC tissues compared with that in adjacent non-cancer tissues (88.6 vs.70.5%, P<0.001), and correlated with the metastatic lymph node of GAC (P=0.018). Furthermore, knockdown of HMGB1 by shRNA inhibited cell proliferative activities and invasive potential, and downregulated the expression of NF-κB p65, PCNA and MMP-9 in GAC cells (SGC-7901 and AGS). The tumor volumes in SGC7901 subcutaneous nude mouse models treated with Lv-shHMGB1 was significantly smaller than those of the nonsense sequence group. Taken together, these findings suggest that increased expression of HMGB1 is associated with tumor metastasis of GAC, and knockdown of HMGB1 suppresses growth and invasion of GAC cells through the NF-κB pathway in vitro and in vivo, suggesting that HMGB1 may serve as a potential therapeutic target for GAC.

The inflammatory mediator high-mobility group box 1 (HMGB1) plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the regulation of HMGB1 in NAFLD, particularly through sirtuin 1 (SIRT1), remains unclear. In this study, we investigated the role of SIRT1-mediated inhibition of HMGB1 release in NAFLD and the effect of salvianolic acid B (SalB), which is a water-soluble phenolic acid extracted from Radix Salvia miltiorrhiza, on NAFLD through SIRT1/HMGB1 signaling. In vivo, SalB treatment significantly attenuated high-fat diet (HFD)-induced liver damage, hepatic steatosis, and inflammation. Importantly, SalB significantly inhibited HMGB1 nuclear translocation and release, accompanied by SIRT1 elevation. In HepG2 cells, palmitic acid (PA)-induced pro-inflammatory cytokines release were blocked by HMGB1 small interfering RNA (siRNA) transfection. Moreover, pharmacological SIRT1 inhibition by Ex527 induced HMGB1 translocation and release, whereas SIRT1 activation by resveratrol or SalB reversed this trend. SIRT1 siRNA abrogated the SalB-mediated inhibition of HMGB1 acetylation and release, suggesting that SalB-mediated protection occurs by SIRT1 targeting HMGB1 for deacetylation. We are the first to demonstrate that the SIRT1/HMGB1 pathway is a key therapeutic target for controlling NAFLD inflammation and that SalB confers protection against HFD- and PA-induced hepatic steatosis and inflammation through SIRT1-mediated HMGB1 deacetylation.

This review aimed to take stock of the current status of research on damage-associated molecular pattern (DAMP) protein. We discuss the Janus-faced role of DAMP molecules in inflammation, cancer, and tissue repair. The high-mobility group box (HMGB)-1 and adenosine triphosphate proteins are well-known DAMP molecules and have been primarily associated with inflammation. However, as we shall see, recent data have linked these molecules to tissue repair. HMGB1 is associated with cancer-related inflammation. It activates nuclear factor kB, which is involved in cancer regulation via its receptor for advanced glycation end-products (RAGE), Toll-like receptors 2 and 4. Proinflammatory activity and tissue repair may lead to pharmacologic intervention, by blocking DAMP RAGE and Toll like receptor 2 and 4 role in inflammation and by increasing their concentration in tissue repair, respectively.

We conducted a MEDLINE search for articles pertaining to the various issues related to DAMP, and we discuss the most relevant articles especially (ie, not only those published in journals with a higher impact factor).

A cluster of remarkable articles on DAMP have appeared in the literature in recent years. Regarding inflammation, several strategies have been proposed to target HMGB1, from antibodies to recombinant box A, which interacts with RAGE, competing with the full molecule. In tissue repair, it was reported that the overexpression of HMGB1 or the administration of exogenous HMGB1 significantly increased the number of vessels and promoted recovery in skin-wound, ischemic injury.

Due to the bivalent nature of DAMP, it is often difficult to explain the relative role of DAMP in inflammation versus its role in tissue repair. However, this point is crucial as DAMP-related treatments move into clinical practice.

The link between platelet activation and vascular injury in Systemic Sclerosis (SSc) is poorly characterized. Here we report that platelet activation results in i) the translocation from the cytoplasm to the surface of HMGB1, a prototypical DAMP signal associated with tissue regeneration and ii) the release of platelet derived microparticles (PDμP) expressing HMGB1. Decreased HMGB1 content (334.6 ± 21.2 vs 587.1 ± 11.1 AUF, P < 0.001) and HMGB1 translocation to the outer leaflet of the plasma membrane (17.8 ± 3.5 vs 4.5 ± 0.5%, P < 0.001) characterize circulating platelets of SSc patients (n = 29) when compared with age-matched healthy controls (HC, n = 20). Conversely, a significantly higher fraction of PDμP in the blood of SSc patients, but not of HC, consistently expose (HMGB1 (MFI 62.8 ± 3.95 vs 4.3 ± 0.7). Platelet HMGB1 depletion is significantly associated in SSc patients with degranulation and with expression of P-selectin and of tissue factor as well as with fibrinogen binding to their plasma membrane. These findings indicate that platelets represent a source of HMGB1, an ancestral signal of necrosis, in the vasculature of SSc patients, possible contributing to persistent microvascular injury and endothelial cell activation.

Recent studies indicate that the release of high mobility group box 1 (HMGB1) following nerve injury may play a central role in the pathogenesis of neuropathic pain. HMGB1 is known to influence cellular responses within the nervous system via two distinct receptor families; the Receptor for Advanced Glycation End-products (RAGE) and Toll-like receptors (TLRs). The degree to which HMGB1 activates a receptor is thought to be dependent upon the oxidative state of the ligand, resulting in the functional isoforms of all-thiol HMGB1 (at-HMGB1) acting through RAGE, and disufide HMGB1 (ds-HMGB1) interacting with TLR4. Though it is known that dorsal root ganglia (DRG) sensory neurons exposed to HMGB1 and TLR4 agonists can influence excitation, the degree to which at-HMGB1 signaling through neuronal RAGE contributes to neuropathic pain is unknown. Here we demonstrate that at-HMGB1 activation of nociceptive neurons is dependent on RAGE and not TLR4. To distinguish the possible role of RAGE on neuropathic pain, we characterized the changes in RAGE mRNA expression up to one month after tibial nerve injury (TNI). RAGE mRNA expression in lumbar dorsal root ganglion (DRG) is substantially increased by post-injury day (PID) 28 when compared with sham injured rodents. Protein expression at PID28 confirms this injury-induced event in the DRG. Moreover, a single exposure to monoclonal antibody to RAGE (RAGE Ab) failed to abrogate pain behavior at PID 7, 14 and 21. However, RAGE Ab administration produced reversal of mechanical hyperalgesia on PID28. Thus, at-HMGB1 activation through RAGE may be responsible for sensory neuron sensitization and mechanical hyperalgesia associated with chronic neuropathic pain states.

High mobility group box-1 protein (HMGB1, formerly known as HMG-1), a highly conserved ubiquitous protein, has been for a long time described as a nuclear DNA-binding protein involved in nucleosome stabilization and gene transcription. Recent discoveries indicate that HMGB1 is released from activated innate immune cells or necrotic cells and functions as an important mediator of endotoxemia, sepsis, arthritis, and local inflammation. Therapeutic agents that inhibit HMGB1 release or action confer significant protection against endotoxemia, sepsis, and arthritis in animal models and thus hold potential for the clinical management of various inflammatory diseases.

Emerging studies have suggested that the Hippo pathway is involved in the tumorigenesis of hepatocellular carcinoma (HCC). However, the key regulator of the Hippo pathway in liver tumor metabolic reprogramming remains elusive. Here, we provide evidence that high mobility group box 1 (HMGB1), a chromosomal protein, plays a role in the regulation of the Hippo pathway during liver tumorigenesis. Cre/loxP recombination-mediated HMGB1 depletion in hepatocytes blocks diethylnitrosamine-induced liver cancer initiation in mice, whereas short hairpin RNA-mediated gene silencing of HMGB1 inhibits HCC cell proliferation. Mechanistically, the binding of HMGB1 to GA-binding protein alpha promotes the expression of yes-associated protein (YAP), a major downstream effector of the Hippo pathway that contributes to liver tumorigenesis by inducing hypoxia-inducible factor 1α (HIF1α)-dependent aerobic glycolysis. Like wild-type YAP-complementary DNA, YAP-5SA-S94A can restore HIF1α DNA binding activity, glycolysis-associated gene expression, and HIF1α-YAP complex formation in YAP-knockdown HCC cell lines. In contrast, verteporfin, a reagent targeting the interface between YAP and TEA domain transcription factor, has the ability to block YAP-HIF1α complex formation. Notably, genetic or pharmacologic inhibition of the HMGB1-YAP-HIF1α pathway confers protection against excessive glycolysis and tumor growth in mice.

These findings demonstrate that HMGB1 plays a novel role in modulating the YAP-dependent HIF1α pathway and shed light on the development of metabolism-targeting therapeutics for HCC chemoprevention. (Hepatology 2018;67:1823-1841).

BACKGROUND

High mobility group protein B1 (HMGB1) is an important late inflammatory mediator in sepsis. Understanding the mechanisms that regulate HMGB1 release from cells and their downstream signal transduction pathways may lead to the ability to develop anti-HMGB1 therapies to treat inflammation.

METHODS

We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS+ ethylpyruvate (EP) and examined the resulting HMGB1 expression and release. We also studied the expression of related signal transduction factors (NF-κB, p38 MAPK, and CBP).

CONCLUSIONS

Gene expression of HMGB1 mRNA in RAW264.7 cell showed no significant change at 0-18 h after stimulation with LPS, but increased significantly at 24, 36, and 48 h. HMGB1 mRNA expression in the LPS+EP group was significantly lower than in LPS alone. HMGB1 was distributed mainly in the nucleus; the cytoplasmic level was low before LPS stimulation. After stimulation with LPS, cytoplasmic HMGB1 increased gradually and plateaued at a high level at 12-48 h. Nuclear HMGB1 decreased gradually at 12-24 h, then increased, maintaining a comparatively high level at 36-48 h. EP prevented this pattern significantly. LPS induced p38 MAPK activation and NF-κB signal pathways first, followed by CBP activation. Activated CBP acetylated HMGB1 was stored in a crino-lysosome and secreted activated NF-κB resulted in increased transcription and synthesis of HMGB1, but the expression of up-regulated HMGB1 mRNA was delayed. Extracellular HMGB1 originated from early synthetic reserves present in the nucleus. New HMGB1 protein was synthesized in the nucleus and transferred into the cytoplasm, causing an increase in HMGB1 in the nucleus and cytoplasm. EP inhibits HMGB1 mRNA up-regulation and release from LPS- stimulated macrophages. The molecular function of EP is to attenuate the activation p38 MAPK, NF-κB, and CBP signaling pathways.

BACKGROUND

High-mobility group protein box 1 (HMGB1) is essential in the response to injury during sepsis. We hypothesized that resveratrol (RESV) administration would inhibit nuclear-cytoplasmic HMGB1 translocation in hepatocytes, which is associated with sirtuin 1 (SIRT1) upregulation. We investigated the regulatory role of SIRT1 in HMGB1 nucleocytoplasmic translocation and its effect on sepsis-induced liver injury.

METHODS

Rats were randomly assigned to pretreatment with RESV (60 mg/kg per day), nicotinamide (60 mg/kg per day), or vehicle (olive oil), which was administered by gavage for 3 days directly before cecal ligation and puncture was performed to induce sepsis. Parallel control groups were established. Rats were killed 24 h after surgery, and cytokine production, histology, apoptosis, SIRT1, serum HMGB1, nuclear and cytoplasmic HMGB1/ac-HMGB1, and the interaction between SIRT1 and HMGB1 were evaluated. In vitro evaluations were performed in human liver L02 cells subjected to lipopolysaccharide-induced injury, and siRNA-mediated SIRT1 knockdown experiments were performed.

RESULTS

Sepsis-induced serum aminotransferase activities and proinflammatory chemokine levels were reduced by RESV pretreatment, which also improved liver histological parameters in association with SIRT1 upregulation. Resveratrol inhibited HMGB1 cytoplasmic translocation. Nicotinamide, an SIRT1 inhibitor, reduced the SIRT1-mediated suppression of HMGB1 translocation and aggravated cecal ligation and puncture-induced liver damage. Sirtuin 1 knockdown in vitro confirmed that RESV increased the SIRT1-mediated repression of HMGB1 translocation. In vivo, SIRT1 and HMGB1 physically interacted in the nucleus, and SIRT1 regulated HMGB1 acetylation in response to septic liver injury.

CONCLUSIONS

Resveratrol protects against sepsis-induced liver injury through the SIRT1-mediated HMGB1 nucleocytoplasmic translocation pathway, a new potential therapeutic target in sepsis-induced liver injury.

Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. In our previous study, we found that KLF4 mRNA was up-regulated more than 10-fold in adult mice lung tissues after endotoxin stimuli, and that KLF4 can regulate the expression of IL -10, an early inflammatory mediator. To determine whether KLF4 influences the expression and release of high-mobility group box 1 (HMGB1), an important late inflammatory mediator, which contains two potential KLF4-binding elements in its promoter, pcDNA3.1-KLF4 expression plasmid or KLF4 antisense oligonucleotide was transfected into RAW264.7 macrophages, the expression and release of HMGB1 were examined by reverse-transcriptase-polymerase chain reaction and Western blot, respectively. Electrophoretic mobility shift assay was performed to detect the binding activity of KLF4 to the HMGB1 promoter. The results showed that KLF4 overexpression led to an increased expression of HMGB1 in both cytoplasm and nucleus, whereas KLF4 deficiency led to a decrease in HMGB1. Moreover, compared with the control group, the release of HMGB1 was increased after KLF4 overexpression after LPS treatment, whereas the release of HMGB1 was decreased after KLF4 deficiency in response to LPS. Electrophoretic mobility shift assay results showed the binding of KLF4 to the oligonucleotides designed according to the HMGB1 promoter, and the binding activity was increased in response to LPS stimulation. These results indicate that KLF4 plays an important role in regulating the expression of HMGB1 in normal condition, as well as the translocation and release of HMGB1 in response to LPS.

OBJECTIVE

Receptor for advanced glycation end products (RAGE) plays a role in inflammatory reactions. Soluble RAGE (sRAGE) level is elevated in patients with acute respiratory distress syndrome (ARDS). However, which clinical parameters and inflammatory biomarkers including sRAGE are associated with death in ARDS patients remain unknown.

METHODS

We examined whether sRAGE level was independently associated with death in 20 ARDS patients with severe infection.

RESULTS

Compared with age- and sex-matched control subjects, blood pressure levels were lower and KL-6, high mobility group box 1 (HMGB1), interleukin-6 and sRAGE levels were higher in ARDS patients. In multivariate analysis, sRAGE was associated with death in ARDS patients, but severity of illness was not. HMGB1 was a sole independent correlate of sRAGE.

CONCLUSIONS

This study demonstrated that sRAGE was independently associated with death in ARDS patients. Our present results suggest active involvement of HMGB1-RAGE axis in poor prognosis of ARDS.

Barriers to successful lung xenotransplantation appear to be even greater than for other organs. This difficulty may be related to several macro anatomic factors, such as the uniquely fragile lung parenchyma and associated blood supply that results in heightened vulnerability of graft function to segmental or lobar airway flooding caused by loss of vascular integrity (also applicable to allotransplants). There are also micro-anatomic considerations, such as the presence of large numbers of resident inflammatory cells, such as pulmonary intravascular macrophages and natural killer (NK) T cells, and the high levels of von Willebrand factor (vWF) associated with the microvasculature. We have considered what developments would be necessary to allow successful clinical lung xenotransplantation. We suggest this will only be achieved by multiple genetic modifications of the organ-source pig, in particular to render the vasculature resistant to thrombosis. The major problems that require to be overcome are multiple and include (i) the innate immune response (antibody, complement, donor pulmonary and recipient macrophages, monocytes, neutrophils, and NK cells), (ii) the adaptive immune response (T and B cells), (iii) coagulation dysregulation, and (iv) an inflammatory response (e.g., TNF-α, IL-6, HMGB1, C-reactive protein). We propose that the genetic manipulation required to provide normal thromboregulation alone may include the introduction of genes for human thrombomodulin/endothelial protein C-receptor, and/or tissue factor pathway inhibitor, and/or CD39/CD73; the problem of pig vWF may also need to be addressed. It would appear that exploration of every available therapeutic path will be required if lung xenotransplantation is to be successful. To initiate a clinical trial of lung xenotransplantation, even as a bridge to allotransplantation (with a realistic possibility of survival long enough for a human lung allograft to be obtained), significant advances and much experimental work will be required. Nevertheless, with the steadily increasing developments in techniques of genetic engineering of pigs, we are optimistic that the goal of successful clinical lung xenotransplantation can be achieved within the foreseeable future. The optimistic view would be that if experimental pig lung xenotransplantation could be successfully managed, it is likely that clinical application of this and all other forms of xenotransplantation would become more feasible.

OBJECTIVE

Ischemic acute kidney injury may be exacerbated by an inflammatory response. How injury elicits inflammation remains a major question in understanding acute kidney injury. The present review examines the hypothesis that molecules released by injured cells elicit inflammation.

RESULTS

After necrotic death, intracellular molecules find their way into the extracellular space. These molecules include heat shock proteins and HMGB1. Receptors for these proteins include TLR4, TLR2, CD91 and RAGE. These proinflammatory mechanisms may be so useful that nature has evolved mechanisms for programming necrotic death via poly(ADP-ribose) polymerase and cyclophilin D. In addition, apoptosis may also elicit inflammation.

CONCLUSIONS

The concepts discussed in this review are important for clinical medicine. Drugs and genetic manipulation may ameliorate ischemic kidney injury by regulating the inflammatory response to cell injury.

Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEDeltacyto) (TE671/RAGEDeltacyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH(2) terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEDeltacyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential.

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrate species are inactive due to the accumulation of mutations. A representative of a subfamily of fish elements estimated to be last active>> 10 million years ago has been reconstructed, and named Sleeping Beauty(SB). This element opened up new avenues for studies on DNA transposition in vertebrates, and for the development of transposon tools for genetic manipulation in important model species and in humans. Multiple transposase binding sites within the terminal inverted repeats, a transpositional enhancer sequence, unequal affinity of the transposase to the binding sites and the activity of the cellular HMGB1 protein all contribute to a highly regulated assembly of SB synaptic complexes, which is likely a requirement for the subsequent catalytic steps. Host proteins involved in double-strand DNA break repair are limiting factors of SB transposition in mammalian cells, underscoring evolutionary, structural and functional links between DNA transposition, retroviral integration and V(D)J recombination. SB catalyzes efficient cut-and-paste transposition in a wide range of vertebrate cells in tissue culture, and in somatic tissues as well as the germline of the mouse and zebrafish in vivo, indicating its usefulness as a vector for transgenesis and insertional mutagenesis.

OBJECTIVE

To investigate plasma levels of high mobility group box-1 protein (HMGB1), a marker of tissue necrosis and immune activation, as well as lipopolysaccharide (LPS), a marker of bacterial translocation, in HIV-1-infected patients.

METHODS

We studied 32 HIV-1-positive patients who had responded to antiretroviral therapy with undetectable viremia after 2 years, 10 nonresponders and 19 healthy controls.

METHODS

HMGB1 was analyzed by ELISA, and LPS by Lamilus colometric assay. Nonparametric statistics were applied.

RESULTS

In naive HIV-1 patients, HMGB1 and LPS were elevated as compared with controls (P < 0.001). LPS levels were higher in African and Oriental patients compared with whites (P = 0.007). Notably, viral load was two-fold higher in patients with LPS, and HMGB1 was above median as compared with other patients (P = 0.005). This association was largely driven by African patients, who had a five-fold increased viral load in the presence of elevated LPS and HMGB1. After 2 years of effective antiretroviral therapy, LPS was reduced to the same median level as in the control group (P < 0.001), and HMGB1 was also reduced (P = 0.001), whereas no reductions were seen in nonresponders.

CONCLUSIONS

The new findings are the association of elevated plasma levels of LPS and HMGB1 with high viral load, as well as the normalized levels of LPS, and the reduction of HMGB1 after 2 years of effective antiretroviral therapy. As LPS and HMGB1 tend to form immunologically active complexes in vitro, we propose that such complexes may be involved in the immune activation and pathogenesis of HIV-1 infection.

Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized (15)N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear (1)H-(15)N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ~17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules.