High Mobility Group Box-1 Protein Research Articles

The Potential Application and Promising Role of Targeted Therapy in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a rare yet serious progressive disorder that is currently incurable. This female-predominant disease unfolds as a pan-vasculopathy that affects all layers of the vessel wall. Five classes of pharmacological agents currently exist to target the three major cellular signaling pathways identified in PAH but are incapable of effectively reversing the disease progression. While several targets have been identified for therapy, none of the current PAH specific therapies are curative and cost-effective as they fail to reverse vascular remodeling and do not address the cancer-like features of PAH. Our purpose is to review the current literature on the therapeutic management of PAH, as well as the molecular targets under consideration for therapy so as to shed light on the potential role and future promise of novel strategies in treating this high-mortality disease. This review study summarizes and discusses the potential therapeutic targets to be employed against PAH. In addition to the three major conventional pathways already used in PAH therapy, targeting PDGF/PDGFR signaling, regulators in glycolytic metabolism, PI3K/AKT pathways, mitochondrial heat shock protein 90 (HSP90), high-mobility group box-1 (HMGB1), and bromodomain and extra-terminal (BET) proteins by using their specific inhibitors, or a pharmacological induction of the p53 expression, could be attractive strategies for treating PAH.

Biomarkers of heatstroke-induced organ injury and repair.

What is the topic of this review? The status and potential role of novel biological markers (biomarkers) that can help identify the patients at risk of organ injury or long-term complications following heatstroke. What advances does it highlight? Numerous biomarkers were identified related to many aspects of generalized heatstroke-induced cellular injury and tissue damage, and heatstroke-provoked cardiovascular, renal, cerebral, intestinal and skeletal muscle injury. No novel biomarkers were identified for liver or lung injury. Classic and exertional heatstroke cause acute injury and damage across numerous organ systems. Moreover, heatstroke survivors may sustain long-term neurological, cardiovascular and renal complications with a persistent risk of death. In this context, biomarkers, defined as biological samples obtained from heatstroke patients, are needed to detect early organ injury, and predict outcomes to develop novel organ preservation therapeutic strategies. This narrative review provides preliminary insights that will guide the development and future utilization of these biomarkers. To this end, we have identified numerous biomarkers of widespread heatstroke-associated cellular injury, tissue damage and repair (extracellular heat shock proteins 72 and 60, high mobility group box protein 1, histone H3, and interleukin-1α), and other organ-specific biomarkers including those related to the cardiovascular system (cardiac troponin I, endothelium-derived factors, circulation endothelial cells, adhesion molecules, thrombomodulin and von Willebrand factor antigen), the kidneys (plasma and urinary neutrophil gelatinase-associated lipocalin), the intestines (intestinal fatty acid-binding protein 2), the brain (serum S100β and neuron-specific enolase) and skeletal muscle (creatine kinase, myoglobin). No specific biomarkers have been identified so far for liver or lung injury in heatstroke. Before translating the identified biomarkers into clinical practice, additional preclinical and clinical prospective studies are required to further understand their clinical utility, particularly for the biomarkers related to long-term post-heatstroke health outcomes.

Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) “histone code” for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx.

Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect

BackgroundSepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol (Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis.MethodsWe evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide (LPS). We screened for potential protein targets of Cel using activity-based protein profiling (ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays (CETSA) and surface plasmon resonance (SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown.ResultsCel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS (P < 0.05). Cel bound to Cys424 of pyruvate kinase M2 (PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis (Warburg effect). Cel also bound to Cys106 in high mobility group box 1 (HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin (IL)-1β. Cel bound to the Cys residues in lactate dehydrogenase A (LDHA).ConclusionCel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

Pentachlorophenol mediated regulation of DAMPs and inflammation: In vitro study

Pentachlorophenol (PCP) was once a widely employed organochlorine pesticide and wood preservative in United States. Due to its toxicity, the U.S. Environmental Protection Agency has classified it as a restricted-use pesticide and established as a liver carcinogen. Earlier reports have indicated increased production of inflammatory mediators like IL-1β and TNF-α by immune cells, including NK cells, lymphocytes, or monocytes -on PCP exposure. Yet, there is only scant information available regarding the detailed molecular mechanisms affected by acute or chronic exposure of humans to PCP. Considering this, we examined PCP-induced inflammation and downstream signaling events in-(a) human lung adenocarcinoma cells (A549) with type II alveolar epithelial characteristics; and (b) human liver carcinoma cells (HepG2). Treatment of these cells with 1 μM and 10 μM concentration of PCP for 24 h duration resulted in a significant induction of cytokines/chemokines including IL-1β, IL-6, TNF-α, IL-8, CCL2, and CCL5. Assessment of mRNA expression showed upregulated levels of danger-associated molecular patterns (DAMPs)-high mobility group box-1 (HMGB1) and heat shock protein 70 (Hsp70) as well as TLR-4 receptor in PCP-challenged cells. Increased expression of transcription factors-NF-κB and STAT3 provide further insight into the molecular mechanisms underlying PCP-induced toxicity/pathology. Interestingly, antibody-mediated neutralization of DAMPs abrogates PCP-mediated transcriptional induction of cytokines, chemokines and transcription factors in HepG2 and A549 cells. Overall, our findings demonstrate the important role of DAMPs in PCP-induced inflammatory responses.

A Porcine Sepsis Model With Numerical Scoring for Early Prediction of Severity.

IntroductionSepsis can lead to organ dysfunctions with disturbed oxygen dynamics and life-threatening consequences. Since the results of organ-protective treatments cannot always be transferred from laboratory models into human therapies, increasing the translational potential of preclinical settings is an important goal. Our aim was to develop a standardized research protocol, where the progression of sepsis-related events can be characterized reproducibly in model experiments within clinically-relevant time frames.MethodsPeritonitis was induced in anesthetized minipigs injected intraperitoneally with autofeces inoculum (n = 27) or with saline (sham operation; n = 9). The microbial colony-forming units (CFUs) in the inoculum were retrospectively determined. After awakening, clinically relevant supportive therapies were conducted. Nineteen inoculated animals developed sepsis without a fulminant reaction. Sixteen hours later, these animals were re-anesthetized for invasive monitoring. Blood samples were taken to detect plasma TNF-α, IL-10, big endothelin (bET), high mobility group box protein1 (HMGB1) levels and blood gases, and sublingual microcirculatory measurements were conducted. Hemodynamic, respiratory, coagulation, liver and kidney dysfunctions were detected to characterize the septic status with a pig-specific Sequential Organ Failure Assessment (pSOFA) score and its simplified version (respiratory, cardiovascular and renal failure) between 16 and 24 h of the experiments.ResultsDespite the standardized sepsis induction, the animals could be clustered into two distinct levels of severity: a sepsis (n = 10; median pSOFA score = 2) and a septic shock (n = 9; median pSOFA score = 8) subgroup at 18 h of the experiments, when the decreased systemic vascular resistance, increased DO2 and VO2, and markedly increased ExO2 demonstrated a compensated hyperdynamic state. Septic animals showed severity-dependent scores for organ failure with reduced microcirculation despite the adequate oxygen dynamics. Sepsis severity characterized later with pSOFA scores was in correlation with the germ count in the induction inoculum (r = 0.664) and CFUs in hemocultures (r = 0.876). Early changes in plasma levels of TNF-α, bET and HMGB1 were all related to the late-onset organ dysfunctions characterized by pSOFA scores.ConclusionsThis microbiologically-monitored, large animal model of intraabdominal sepsis is suitable for clinically-relevant investigations. The methodology combines the advantages of conscious and anesthetized studies, and mimics human sepsis and septic shock closely with the possibility of numerical quantification of host responses.

Investigation of the relationship between community-acquired respiratory distress syndrome toxin and the high-mobility group box protein 1-toll-like receptors-myeloid differentiation factor 88 signaling pathway in Mycoplasma pneumoniae pneumonia

BackgroundIn recent years, reports of refractory Mycoplasma pneumoniae pneumonia (RMPP) have gradually increased, including reports on how these conditions threaten the lives of children. However, the specific mechanism of Mycoplasma pneumoniae pneumonia (MPP) remains unclear. This study aimed to investigate the relationship between community-acquired respiratory distress syndrome toxin (CARDS TX) and High-mobility group box protein 1-Toll-like receptors-Myeloid differentiation factor 88 (HMGB1-TLRs-MyD88) in MPP and to examine the immune pathogenesis of Mycoplasma pneumoniae infection.MethodsChildren who were diagnosed with MPP and examined by bronchoscopy were included in the MPP group. Additionally, children who underwent bronchoscopy because of bronchial foreign bodies in the same period were included in the control group. Gene expression of CARDS TX, HMGB1, Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), MyD88, and cluster of differentiation 14 (CD14) in bronchoalveolar lavage fluid (BALF) were detected using real-time reverse transcription-polymerase chain reaction. Correlations between CARDS TX and HMGB1-TLRs-MyD88 were analyzed.ResultsCARDS TX, HMGB1, TLR2, MyD88, and CD14 mRNA expression in BALF in the MPP group was significantly higher than that in the control group (all P < 0.05). CARDS TX mRNA expression was positively correlated with HMGB1, TLR2, MyD88, and CD14 mRNA expression (all P < 0.05). Furthermore, HMGB1 mRNA expression was positively correlated with TLR2, MyD88, and CD14 mRNA expression (all P < 0.05).ConclusionsCARDS TX may participate in the immune pathogenesis of MPP through the HMGB1-TLRs/CD14-MyD88 pathway.

Cysteine 106 Controls HMGB1 Nucleocytoplasmic Distribution and Function: Role of Sex and Implication to Pulmonary Hypertension

IntroductionHigh mobility group box protein 1 (HMGB1) is a nuclear protein that gets released during cell damage and stimulates inflammatory and proliferative pathways in the survived neighboring cells. Our previous research discovered a significant sex difference in HMGB1 release and downstream signaling and confirmed its critical importance in the pathobiology of pulmonary hypertension (PH).HypothesisHMGB1 contains three cysteine residues (23/45/106) involved in the extracellular HMGB1 oligomerization and receptor binding, with Cys106 being especially critical for HMGB1 signaling. However, the role of cysteines in the intracellular HMGB1 activity is less established. We hypothesize that Cys106 regulates intracellular HMGB1 homeostasis and function and contributes to the pathogenic mechanisms relevant to PH.MethodsTo study the activities of HMGB1 cysteines, we designed peptides that bind to the specific regions of HMGB1 and, by shielding either Cys106 or Cys23/45, prevent their interaction with the downstream targets. The peptides with cell‐penetrating TAT sequences were used to study the intracellular role of HMGB1 cysteines, while peptides without TAT served as extracellular controls. Fractionating of pulmonary artery smooth muscle cells (PASMC) allowed the evaluation of HMGB1 cytosol/nuclear distribution. Calf thymus double‐stranded DNA cellulose was used to study the binding of recombinant fully reduced HMGB1 in the presence or absence of shielding peptide. The protective role of peptide in vivo was estimated using the rat Sugen/Hypoxia model of PH. Given the discovered sex difference in the HMGB1‐mediated signaling, the in vitro and in vivo studies were replicated in male and female animals and cells.ResultsThe cytosol and nuclear levels of HMGB1 were affected by cell‐penetrating peptide designed to shield Cys106 (TAT‐α‐HMGB1Cys106, or α‐Cys106) and not any other peptide. Further investigation revealed that α‐Cys106 peptide produced ~ 5.9 fold decrease in HMGB1 binding to DNA, enhanced the exit of nuclear HMGB1 to the cytosol, which was especially evident in male cells, and induced its proteasomal degradation (1±0.097 vs. 0.428±0.045 vs. 0.885±0.049 in untreated, α‐Cys106 and α‐Cys106/proteasome inhibitor (MG132, 10μM, 1hr) treated PASMC, p&lt;0.001 and p&lt;0.003, N=6). The peptide‐mediated decrease in HMGB1/DNA binding negatively affected the expression of TLR4 and p53 and reduced cell apoptosis. In females, treatment with α‐Cys106 (2.5 mg/kg/day, i.p during the wks 1‐2 or wks 3‐5) produced an anti‐apoptotic effect and reduced plasma HMGB1 levels, which corresponded to a decreased right ventricle (RV) systolic pressure at the early stage of PH and protected the RV at the later stage. In contrast, male PH rats showed no apoptosis activation in the lungs, and α‐Cys106 treatment produced no changes in plasma HMGB1 or hemodynamic parameters.ConclusionHMGB1Cys106 is the critical regulator of intracellular HMGB1 function, including HMGB1/DNA binding, regulation of p53 expression and apoptosis activation, the intracellular distribution of HMGB1, and its extracellular release. This study also confirms the significant sex difference in the levels of pulmonary apoptosis, HMGB1 release, and its contribution to the pathobiology of PH.

Böbreküstü Bezinde HMGB1 ile Obezite İlişkisinin Araştırılması

Aim: The interaction between obesity and increased production of pro-inflammatory cytokines results the existence of inflammation HMGB1 secreted from the adrenal gland can play a role in inflammation pathways. The aim of this study is to explain the link between HMGB1 and obesity in the adrenal gland. Material and Methods: In this study; eighteen female Wistar Albino rats were divided into two groups: untreated control group (n=8) and obese group (n=10). The rats in obese group were fed with high fat diet for ten weeks. Morphometric parameters of adrenal gland were assessed by using stereological techniques. The expression of high mobility group box protein 1 (HMGB1) in adrenal gland was evaluated. Results: At the end of the analyses; mean volumes of zona fasciculate, zona reticularis, and medulla were significantly increased in obese group. Also, the number of HMGB1 stained cells was significantly increased in the obese group in comparison to control group.Conclusion: The results suggest that obesity may be one of the reasons of inflammation and hypertrophy in the adrenal gland. HMGB1 may provide a novel perspective into the anti-inflammatory therapeutic strategies in obese patients.

Abstract 552: Sustained Inhibition Of High Mobility Box 1 By Antisense Oligonucleotide

Background: High-mobility group box 1 (HMGB1), a highly conserved nonhistone DNA-binding nuclear protein, may contribute to vascular diseases including atherosclerosis, pulmonary hypertension, and abdominal aortic aneurysm. Since whole-body genetic deletion of HMGB1 is embryonic lethal, pharmacological approaches, such as neutralizing antibodies and functional inhibitors, have been used to manipulate HMGB1 in mice. However, it remains desirable to genetically manipulate HMGB1 for further understanding its role. In the current study, we assessed the efficacy of a novel antisense oligonucleotides (ASOs) approach to deplete HMGB1 in mice. Methods and Results: First, abundance of HMGB1 mRNA was assessed by qPCR in major organs and tissues of male C57BL/6J mice (N=6). Hmgb1 mRNA was expressed ubiquitously, but it was most highly abundant in the lung, while least abundant in the testes. Next, we examined the efficacy of ASOs to reduce HMGB1 protein abundance at selected intervals. Either ASOs (25 mg/kg/day) or phosphate-buffered saline (PBS) were injected intraperitoneally into male C57BL/6J mice (8-10-week-old) at day 0 and 3 in the initial week and then once a week during the remainder of the study. Mice were terminated at either 2, 6, or 12 weeks after the initial injection of ASOs (N=5/group). Subsequently, mRNA and protein abundance of HMGB1 were determined in the lungs. Since previous studies have shown that systemic administration of ASOs mainly targets kidney and liver, HMGB1 mRNA or protein abundance was also examined in these organs. We also assessed HMGB1 protein abundance in the heart. In lungs, kidney, and liver, ASO administration resulted in a consistent &gt;90% decrease of Hmgb1 mRNA abundance at 2 weeks of ASO injection (P&gt;0.001). The decreased Hmbg1 mRNA abundance was also consistent at both weeks 6 and 12 (P&gt;0.001). ASOs decreased HMGB1 protein abundance significantly at 6- and 12-week time intervals. Heart tissue also indicated a consistent Hmgb1 mRNA decrease in response to ASO administration. Conclusion: ASO approach decreased HMGB1 at mRNA and protein abundance for 12 weeks in the liver, kidney, lungs, and heart. This mode may provide more clear insights into understanding the biological functions of HMGB1.

High Mobility Group Box 1 (HMGB1) is Central to the Cytomix‐Induced Pro‐Inflammatory Response in Human Pulmonary Microvascular Endothelial Cells

Endothelial cell death is an early event in acute lung injury (ALI) leading to acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). We have previously shown that cytomix (lipopolysaccharide (LPS), interleukin (IL)‐1β, interferon‐γ, and tumor necrosis factor‐α), but not LPS alone, results in robust cell death in human pulmonary microvascular endothelial cells (hPMVEC). We hypothesized that the inflammatory cascade associated with cytomix treatment in hPMVEC would result in the release of HMGB1, a damage‐associated molecular pattern (DAMP), which would enhance the pro‐inflammatory response. Treatment of hPMVEC with cytomix for 24 hours resulted in a robust (25‐fold) induction of HMGB1 protein levels in the media compared to controls. Cytomix treatment of hPMVEC resulted in nearly 5‐fold greater cleaved caspase‐1 protein levels and nearly 60‐fold greater cleaved IL‐1β protein levels than in control hPMVEC. Viable hPMVEC numbers were significantly reduced after cytomix treatment. To assess the role of TLR4 in the inflammatory response of hPMVEC following cytomix treatment, we utilized TAK‐242, a TLR4 antagonist. The cytomix‐induced increase in media HMGB1 levels was significantly attenuated by TAK‐242. TAK‐242 also significantly attenuated the cytomix‐induced increases in cleaved caspase‐1 and cleaved IL‐1β. To assess the role of caspase 1 in the inflammatory response of hPMVEC following cytomix treatment, we utilized Ac‐YVAD‐CMK, a caspase‐1 antagonist. Treatment with Ac‐YVAD‐CMK attenuated the cytomix‐induced increase in media HMGB1 and cleaved IL‐1β; and partially restored viable cell numbers following cytomix treatment. To further understand the role of HMGB1 in the pro‐inflammatory process we treated hPMVEC with an HMGB1 antibody; and found that the HMGB1 antibody significantly attenuated the increases in HMGB1 and cleaved IL‐1β release, as well as endothelial cell death caused by cytomix treatment. Our results demonstrate that treatment with cytomix results in release of HMGB1, which activates caspase 1 leading to greater HMGB1 production. Furthermore, HMGB1 activation of caspase‐1 leads to cleaved IL‐1β production as well as hPMVEC death. Preventing the interaction of HMGB1 and TLR4 attenuated HMGB1 and cleaved IL‐1β release, and attenuated cytomix‐induced reductions in viable cell numbers. We speculate that the release of damage‐associated molecular patterns (DAMPs), such as HMGB1, may be crucial in the innate immune response of hPMVECs. These findings may have implications for the pathogenesis of ALI in inflammatory lung diseases.

Human HMGB1 does not induce eryptosis &lt;i&gt;in vitro&lt;/i&gt;

Aim: To study the ability of human high mobility group box protein 1 (HMGB1) to induce eryptosis in vitro. Material and methods: Blood collected from six healthy volunteers was incubated with HMGB1 (0-0.2-1-5 ng per ml). Eryptosis of red blood cells was assessed by Annexin V staining and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining by flow cytometry. The forward scatter (FSC) fluorescence was used to evaluate the morphology of red blood cells. Results: Exposure of erythrocytes to HMGB1 did not affect the morphology of erythrocytes, evidenced by no changes in the percentage of cells with small volume, i.e. shrunken cells, and erythrocytes with large volume, i.e. enlarged cells. HMGB1 had no impact on phosphatidylserine externalization, which is confirmed by the absence of statistically significant changes in the amount of phosphatidylserine-displaying cells and the mean fluorescence intensity (MFI) values of Annexin V-FITC in cells exposed to different concentrations of HMGB1. Furthermore, H2DCFDA staining revealed that the HMGB1 did not induce oxidative stress. Conclusion: HMGB1 does not promote eryptosis of human erythrocytes at concentrations of up to 5 ng/ml. Key words: high mobility group box protein 1, erythrocytes, inflammation, cell death

Highly transmissible cytoplasmic incompatibility by the extracellular insect symbiont Spiroplasma

SummaryCytoplasmic incompatibility (CI) is a form of reproductive manipulation caused by maternally inherited endosymbionts infecting arthropods, like Wolbachia, whereby matings between infected males and uninfected females produce few or no offspring. We report the discovery of a new CI symbiont, a strain of Spiroplasma causing CI in the parasitoid wasp Lariophagus distinguendus. Its extracellular occurrence enabled us to establish CI in uninfected adult insects by transferring Spiroplasma-infected hemolymph. We sequenced the CI-Spiroplasma genome and did not find any homologues of any of the cif genes discovered to cause CI in Wolbachia, suggesting independent evolution of CI. Instead, the genome contains other potential CI-causing candidate genes, such as homologues of high-mobility group (HMG) box proteins that are crucial in eukaryotic development but rare in bacterial genomes. Spiroplasma’s extracellular nature and broad host range encompassing medically and agriculturally important arthropods make it a promising tool to study CI and its applications.

Silibinin induces immunogenic cell death in cancer cells and enhances the induced immunogenicity by chemotherapy.

Introduction: Silibinin is a natural flavonoid compound known to induce apoptosis in cancer cells. Despite silibinin's safety and efficacy as an anticancer drug, its effects on inducing immunogenic cell death (ICD) are largely unknown. Herein, we have evaluated the stimulating effects of silibinin on ICD in cancer cells treated with silibinin alone or in combination with chemotherapy. Methods: The anticancer effect of silibinin, alone or in combination with doxorubicin or oxaliplatin (OXP), was assessed using the MTT assay. Compusyn software was used to analyze the combination therapy data. Western blotting was conducted to examine the level of STAT3 activity. Flow cytometry was used to analyze calreticulin (CRT) and apoptosis. The heat shock protein (HSP70), high mobility group box protein1 (HMGB1), and IL-12 levels were assessed by ELISA. Results: Compared to the negative control groups, silibinin induced ICD in CT26 and B16F10 cells and significantly enhanced the induction of this type of cell death by doxorubicin, and these changes were allied with substantial increases in the level of damage-associated molecular patterns (DAMPs) including CRT, HSP70, and HMGB1. Furthermore, conditioned media from cancer cells exposed to silibinin and doxorubicin was found to stimulate IL-12 secretion in dendritic cells (DCs), suggesting the link of this treatment with the induction of Th1 response. Silibinin did not augment the ICD response induced by OXP. Conclusion: Our findings showed that silibinin can induce ICD and it potentiates the induction of this type of cell death induced by chemotherapy in cancer cells.

Cell type-specific response of colon cancer tumor cell lines to oncolytic HSV-1 virotherapy in hypoxia

BackgroundNovel strategies are required since the hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. High mobility group box 1 (HMGB1) protein can block aerobic respiration in cancer cells. We hypothesized that HMGB1could also kill the colorectal cancer cells during hypoxia.MethodsIn this study, we developed oncolytic herpes simplex virus type 1 expressing HMGB1 protein (HSV-HMGB1) and investigated the cytotoxic effect of HSV-HMGB1 and its parental virus (HSV-ble) on three colorectal cancer cells (HCT116, SW480, and HT29) under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. We further identified potential autophagy- related genes in HT29 cells by retrieving mRNA expression microarray datasets from the Gene Expression Omnibus database. These genes were then detected in HT29 cells infected with HSV-HMGB1 and HSV-ble during normoxia and hypoxia by Real-Time quantitative PCR (qRT-PCR).ResultsThe cytotoxic effect of HSV-HMGB1 was significantly higher than that of HSV-ble during normoxia; however, during hypoxia, HSV-HMGB1 enhanced the viability of HT29 cells at MOI 0.1. Analyzing the cell death pathway revealed that HSV-HMGB1 induced autophagy in HT29 cells under hypoxic conditions.ConclusionIn conclusion, it appears that oncolytic virotherapy is cell context-dependent. Therefore, understanding the cancer cells’ characteristics, microenvironment, and cell signaling are essential to improve the therapeutic strategies.

HMGB1 in Pediatric COVID-19 Infection and MIS-C: A Pilot Study.

ObjectiveSince the beginning of the coronavirus disease 2019 (COVID-19) pandemic, a novel syndrome known as a multisystem inflammatory syndrome in children (MIS-C) was reported in previously healthy children. A possible pro-inflammatory molecule, high-mobility group box 1 (HMGB1), may be assumed to play an important role in the pathogenesis and clinical presentation of MIS-C. We described the clinical picture of patients with MIS-C and we also aimed to test and compare HMGB1 serum levels of MIS-C patients with those of patients with previous SARS-CoV2 infection and healthy children.Study designWe determined HMGB1 levels by Western blot in 46 patients and divided them into three groups, namely, five patients with MIS-C (median age: 8.36 years), 20 children with a history of SARS-CoV-2 infection (median age: 10.45 years), and 21 healthy children (controls) (median age: 4.84 years), without evidence of respiratory infection in the last 3 months.ResultsThe median level of HMGB1 in the serum of five patients with MIS-C was found to be significantly higher compared with both patients with a recent history of COVID-19 (1,151.38 vs. 545.90 densitometric units (DU), p = 0.001) and control (1,151.38 vs. 320.33 DU, p = 0.001) groups. The HMGB1 level in MIS-C patients with coronary involvement had a slightly higher value with respect to patients without coronary dilatation (1,225.36 vs. 1,030.49 DU, p = 0.248). In two of the five children with MIS-C that performed a follow-up, the HMGB1 value decreased to levels that were superimposable to the ones of the control group.ConclusionThe significantly high level of HMGB1 protein found in the serum of COVID-19 and patients with MIS-C supports its involvement in inflammatory manifestations, suggesting HMGB1 as a potential biomarker and therapeutic target in patients with severe illness.

Serum Level of High-Mobility Group Box Protein 1 as a Potential Treatment Target in Egyptian Sickle Cell Disease Patients

Background:&#x0D; During tissue injury, high mobility group box 1 (HMGB1) is passively released from necrotic cells and actively secreted by inflammatory cells. Extracellular HMGB1 acts as an amplifier of Toll-Like Receptor (TLR)-dependent inflammation rather than a primary trigger of inflammation. We studied HMGB1 quantitative trait locus reference sequence 2249825 (rs2249825) and its serum level in both sickle cell disease (SCD) patients and healthy subjects to explore its possible role in the pathogenesis of vaso-occlusive crises (VOCs).&#x0D; Methods:&#x0D; HMGB1 rs2249825 was assayed in peripheral blood samples using real-time polymerase chain reaction (RT-PCR). While the serum level was assayed using a two-site enzyme-linked immunosorbent technique (ELISA).&#x0D; Results:&#x0D; Both the SCD patients and the control group had comparable HMGB1 rs2249825 genotype frequencies (P-value &gt;0.05). SCD patients at their steady-state showed statistically significantly higher serum HMGB1 levels than the healthy controls, a median of 0.6 ng/ml with a range of 0.1- 85 ng/ml versus a median of 0.3 ng/ml and a range of 0.1-3 ng/ml (P-value &lt;0.001), respectively. Statistically significant skewed high serum HMGB1 in the VOC samples in contrast to the steady-state samples was observed in the SCD patients with a median of 3.2 ng/ml and a range of 0.3-76.4 ng/ml versus a median of 0.2 ng/ml and a range 0.2-7.4 ng/ml (P-value &lt;0.0001), respectively.&#x0D; &#x0D; &#x0D; Conclusion:&#x0D; HMGB1 could have a role in the VOC pathogenesis, hence it is suggested as a potential additive therapeutic target in SCD in general and in vaso-occlusions in specific.&#x0D; Keywords:&#x0D; Sickle cell disease, HMGB1, Hemoglobin S

Effects of ivacaftor on systemic inflammation and the plasma proteome in people with CF and G551D

BackgroundIvacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator for people with CF and the G551D mutation. We aimed to investigate the biology of CFTR modulation and systemic effects of CFTR restoration by examining changes in circulating measurements of inflammation and growth and novel proteins with ivacaftor treatment. MethodsBlood samples from 64 CF subjects with G551D-CFTR were analyzed for inflammatory and growth-related proteins at baseline, 1 and 6 months after ivacaftor initiation. In 30 subjects, plasma was assayed for 1,322 proteins using the SomaScan proteomic platform at baseline and 6 months post-ivacaftor. Correlations with clinical outcomes were assessed. Measurements and Main ResultsSignificant reductions in high mobility group box-1 protein (HMGB-1), calprotectin, serum amyloid A, and granulocyte colony-stimulating factor (G-CSF), and an increase in insulin-like growth factor (IGF-1) occurred 1 month after ivacaftor. This treatment effect was sustained at 6 months for HMGB-1 and calprotectin. Correcting for multiple comparisons in the proteomic analysis, 9 proteins (albumin, afamin, leptin, trypsin, pancreatic stone protein [PSP], pituitary adenylate cyclase-activating polypeptide-38, repulsive guidance molecule A [RGMA], calreticulin, GTPase KRas) changed significantly with ivacaftor. Proteins changing with treatment are involved in lipid digestion and transport and extracellular matrix organization biological processes. Reductions in calprotectin and G-CSF and increases in calreticulin, and RGMA correlated with improved lung function, while increasing IGF-1, leptin and afamin and decreasing PSP correlated with increased weight. ConclusionsIvacaftor led to changes in inflammatory, lipid digestion, and extracellular matrix proteins, lending insights into the extrapulmonary effects of CFTR modulation.

Effect of resveratrol on high mobility group box-1 protein signaling pathway in cartilage endplate degeneration caused by inflammation

To investigate the effect of resveratrol (RES) on inflammation-induced cartilage endplate (CEP) degeneration, and its regulatory mechanism on high mobility group box-1 protein (HMGB1) signaling pathway. The intervertebral CEP cells of Sprague Dawley (SD) rats aged 3 weeks were extracted and identified by toluidine blue staining and immunofluorescence staining of rabbit anti-rat collagen type Ⅱ. The cell counting kit 8 (CCK-8) method was used to screen the optimal concentration of RES on intervertebral CEP cells. Gene chip analysis was used to determine the target of RES on intervertebral CEP cells. Interleukin 1β (IL-1β) was used to construct the intervertebral CEP cell degeneration model caused by inflammation and the 7-8-week-old SD rat intervertebral disc degeneration model, and pcDNA3.1-HMGB1 (pcDNA3.1) was used as the control of RES effect. Flow cytometry and TUNEL staining were used to detect the apoptotic rate of intervertebral CEP cells and rat intervertebral disc tissue cells, respectively. ELISA kit was used to detect the content of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the cell supernatant and rat serum. Western blot was used to detect the expressions of HMGB1, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), B cell lymphoma/leukemia 2 gene (Bcl-2), and Bcl-2-associated X protein (Bax). The extracted cells were identified as rat intervertebral CEP cells. CCK-8 method screened out the highest activity of intervertebral CEP cells treated with 30 μmol/L RES. The gene chip analysis confirmed that the HMGB1-ERK signal was the target of RES. Both cell experiments and animal experiments showed that RES treatment can significantly down-regulate the apoptosis rate of intervertebral CEP cells, inhibit the release of TNF-α, and increase the content of IL-10; and down-regulate the expressions of HMGB1, p-ERK, and Bax, and increase Bcl-2; and pcDNA3.1 could partially reverse these effects of RES, and the differences were all significant (P<0.05). RES can significantly inhibit the apoptosis of intervertebral CEP cells induced by inflammation, which is related to inhibiting the expression of HMGB1.

The Modulation of Interferon Regulatory Factor-1 via Caspase-1-Mediated Alveolar Macrophage Pyroptosis in Ventilator-Induced Lung Injury.

Background To examine the role of interferon regulatory factor-1 (IRF-1) and to explore the potential molecular mechanism in ventilator-induced lung injury. Methods Wild-type C57BL/6 mice and IRF-1 gene knockout mice/caspase-1 knockout mice were mechanically ventilated with a high tidal volume to establish a ventilator-related lung injury model. The supernatant of the alveolar lavage solution and the lung tissues of these mice were collected. The degree of lung injury was examined by hematoxylin and eosin staining. The protein and mRNA expression levels of IRF-1, caspase-1 (p10), and interleukin (IL)-1β (p17) in lung tissues were measured by western blot and quantitative real-time polymerase chain reaction, respectively. Pyroptosis of alveolar macrophages was detected by flow cytometry and western blotting for active caspase-1 and cleaved GSDMD. An enzyme-linked immunosorbent assay was used to measure the levels of IL-1β, IL-18, IL-6, TNF-α, and high mobility group box protein 1 (HMGB-1) in alveolar lavage fluid. Results IRF-1 expression and caspase-1-dependent pyroptosis in lung tissues of wild-type mice were significantly upregulated after mechanical ventilation with a high tidal volume. The degree of ventilator-related lung injury in IRF-1 gene knockout mice and caspase-1 knockout mice was significantly improved compared to that in wild-type mice, and the levels of GSDMD, IL-1β, IL-18, IL-6, and HMGB-1 in alveolar lavage solution were significantly reduced (P < 0.05). The expression levels of caspase-1 (p10), cleaved GSDMD, and IL-1β (p17) proteins in lung tissues of IRF-1 knockout mice with ventilator-related lung injury were significantly lower than those of wild-type mice, and the level of pyroptosis of macrophages in alveolar lavage solution was significantly reduced. Conclusions IRF-1 may aggravate ventilator-induced lung injury by regulating the activation of caspase-1 and the focal death of alveolar macrophages.