Pingchuanning Decotion Alleviates Bronchial Asthma Airway Inflammation Through ROS/HMGB1/Beclin-1 Mediated Cell Autophagy.

Objective: Bronchial asthma is a prevalent respiratory disorder characterized by airway inflammation. This study aimed to investigate the protective effect of Pingchuanning decoction (PCN) on airway inflammation in bronchial asthma, focusing on the role of autophagy and its underlying molecular mechanism. Methods: Using an in vitro lipopolysaccharide (LPS)-induced inflammatory damage model of human airway epithelial cells (16HBE), we assessed the effect of PCN. Various experiments were performed to evaluate the expression of autophagy-related genes, autophagosome and vesicle counts, and reactive oxygen species (ROS) levels. Results: First, PCN reduced LPS-induced cellular inflammation. Second, PCN decreased the number of autophagosomes and autophagic vesicles. And third, PCN significantly reduced reactive oxygen species (ROS) levels. Most importantly, PCN also down-regulated LPS-induced expression of HMGB1, Beclin-1, and autophagy-related gene 5 (ATG5) while enhancing the expression of B-cell lymphoma 2 (Bcl-2), which further reduced the LC3II/I ratio. Conclusion: PCN reduces the 16HBE inflammatory response by inhibiting the overexpression of ROS/HMGB1/Beclin-1 mediated cell autophagy. Therefore, it may serve as a potential drug for treating bronchial asthma.

Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade.

OBJECTIVE: Investigating the effect of ferroptosis in the tumour microenvironment to identify combinatory therapy for liver cancer treatment. DESIGN: Glutathione peroxidase 4 (GPx4), which is considered the master regulator of ferroptosis, was genetically altered in murine models for hepatocellular carcinoma (HCC) and colorectal cancer (CRC) to analyse the effect of ferroptosis on tumour cells and the immune tumour microenvironment. The findings served as foundation for the identification of additional targets for combine therapy with ferroptotic inducer in the treatment of HCC and liver metastasis. RESULTS: Surprisingly, hepatocyte-restricted GPx4 loss does not suppress hepatocellular tumourigenesis. Instead, GPx4-associated ferroptotic hepatocyte death causes a tumour suppressive immune response characterised by a CXCL10-dependent infiltration of cytotoxic CD8+ T cells that is counterbalanced by PD-L1 upregulation on tumour cells as well as by a marked HMGB1-mediated myeloid derived suppressor cell (MDSC) infiltration. Blocking PD-1 or HMGB1 unleashes T cell activation and prolongs survival of mice with Gpx4-deficient liver tumours. A triple combination of the ferroptosis inducing natural compound withaferin A, the CXCR2 inhibitor SB225002 and α-PD-1 greatly improves survival of wild-type mice with liver tumours. In contrast, the same combination does not affect tumour growth of subcutaneously grown CRC organoids, while it decreases their metastatic growth in liver. CONCLUSION: Our data highlight a context-specific ferroptosis-induced immune response that could be therapeutically exploited for the treatment of primary liver tumours and liver metastases.

Variation of Plasma Damage-Associated Molecular Patterns in Patients with Advanced Solid Tumors after Standard of Care Systemic Treatment.

BACKGROUND: Immunogenic cell death (ICD) is known for releasing damage-associated molecular patterns (DAMPs) from tumor cells. We aimed to find ICD signals by assessing the variation of plasmatic DAMPs (HMGB1, S100A8) before-after standard of care (SoC) systemic treatment in patients with advanced solid tumors. METHODS: Patients scheduled to start a new line of systemic treatment were included. Plasmatic concentrations of HMGB1 and S100A8 were measured (ng/mL) before and after three months of treatment. RESULTS: Fifty-two patients were included. Forty-four patients (85%) had metastases, and 8 (15%) were treated for stage III tumors. The most frequent tumor sites were colorectal (35%) and lung (25%). Forty-two patients (81%) received this treatment in the first-line setting. Thirty-six patients (69%) were treated chemotherapy (CT) alone, ten (19%) CT plus targeted therapy, two (3.8%) carboplatin-pemetrexed-pembrolizumab, three (5.8%) pembrolizumab alone and one (1.9%) cetuximab alone. Median plasmatic concentration of S100A8 was significantly higher before than after treatment in the whole population (3.78 vs. 2.91 ng/mL; p = 0.011) and more markedly in the subgroups of patients who experienced RECIST-assessed tumor response (5.70 vs. 2.63 ng/mL; p = 0.002). Median plasmatic concentration of HMGB1was not significantly different before and after treatment (10.23 vs. 11.85 ng/mL; p = 0.382) and did not differ depending on tumor response. Median PFS was not significantly different between patients whose plasma HMBG1 concentration decreased or increased (8.0 vs. 10.6 months; p = 0.29) after treatment. Median PFS was significantly longer in those patients in whom the plasma concentration of S100A8 decreased after treatment (12 vs. 4.7 months; p < 0.001). Median OS was not significantly different between patients whose plasma HMBG1 concentration decreased or increased (13.1 vs. 14.7 months; p = 0.46) after treatment. Median OS was significantly longer in those patients in whom the plasma concentration of S100A8 decreased after treatment (16.7 vs. 9.0 months; p < 0.001). CONCLUSIONS: Signals of ICD were not observed. S100A8 behaves as an inflammatory marker with decreased concentration after treatment, mostly in RECIST-responders. PFS and OS were significantly prolonged in those patients who experienced a decrease of S100A8 compared with those patients who experienced increase of plasma S100A8 at three months.

Etanercept ameliorates psoriasis progression through regulating high mobility group box 1 pathway.

BACKGROUND: As a common skin disease, psoriasis is related to inflammation and immune response. Due to the frequent recurrence of psoriasis, the treatment of psoriasis remains a clinical challenge. As an effective tumor necrosis factor-alpha (TNF-α) inhibitor, etanercept has been used for the treatment of psoriasis. However, some patients with psoriasis have no response to etanercept or discontinue treatment. To improve the therapeutic effect of etanercept, searching the potential biomarkers and investigating the related mechanisms of etanercept in the treatment of psoriasis are vital. MATERIALS AND METHODS: We stimulated HaCaT cells with lipopolysaccharide (LPS) to generate cellular psoriatic changes and established an imiquimod (IMQ)-induced psoriasis-like mouse model, and then used an etanercept to treat cell and mouse model. RESULTS: Etanercept alleviated IMQ-induced pathological changes and inflammation, and it also decreased the protein expression of high mobility group box 1 (HMGB1), receptor for advanced glycation end-products, and toll-like receptor 4. Moreover, the results of in vitro experiments showed that etanercept inhibited proliferation and inflammation, and promoted cell cycle arrest and apoptosis in LPS-treated HaCaT cells. Knockdown of HMGB1 further enhanced the inhibitory effects of etanercept on LPS-treated HaCaT cell viability and inflammation, while overexpression of HMGB1 notably reversed the inhibitory effects of etanercept on LPS-induced HaCaT cell viability and inflammation. CONCLUSION: Etanercept inhibited proliferation and inflammation and promoted cell cycle arrest and apoptosis in LPS-induced HaCaT cells, and etanercept ameliorated inflammation in a psoriasis-like mouse model.

Decreased levels of histidine-rich glycoprotein and increased levels of high-mobility group box 1 are risk factors for refractory Kawasaki disease.

OBJECTIVES: Histidine-rich glycoprotein (HRG) and high-mobility group box 1 (HMGB1) regulate the activation of neutrophils and vascular endothelium. The aim of this study was to quantify HRG and HMGB1 levels in patients with Kawasaki disease (KD) and evaluate their use in the clinical management of KD. METHODS: This study was prospectively performed. Patients were divided into two groups and analysed depending on whether KD symptoms improved by Day 10 of illness. HRG, HMGB1, and other laboratory variables were measured before the first treatment in all cases and, in most cases, afterwards for assessing trends. RESULTS: In this prospective study, we enrolled 60 patients with KD and 48 healthy controls. The HRG level in the KD group was significantly lower than that in the healthy control group; HMGB1 levels showed no obvious differences. In the KD group, HRG levels were negatively correlated with white blood cell and neutrophil counts. In the poor responders and responders groups, a tendency for a decrease in HRG and HMGB1 levels, respectively, was observed from pretreatment to post-treatment. CONCLUSIONS: HRG and HMGB1 are related to the pathogenesis of KD; low HRG and high HMGB1 levels cause resistance against KD treatment.

Identification of tumor biomarkers for pathological complete response to neoadjuvant treatment in locally advanced breast cancer.

BACKGROUND: Therapeutic response predictors like age, nodal status, and tumor grade and markers, like ER/PR, HER2, and Ki67, are not reliable in predicting the response to a specific form of chemotherapy. The current study aims to identify and validate reliable markers that can predict pathological complete response (pCR) in fluorouracil, epirubicin, and cyclophosphamide (FEC)-based neoadjuvant therapy with (NACT/RT) and without concurrent radiation (NACT). MATERIALS AND METHODS: Tandem mass tag (TMT) quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins from core needle breast biopsy between pCR (n = 4) and no-pCR (n = 4). Immunoblotting of shortlisted proteins with the tissue lysates confirmed the differential expression of the markers. Further, immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded sections of treatment-naive core needle biopsies. In the NACT, 29 pCR and 130 no-pCR and in NACT/RT, 32 pCR and 71 no-pCR were used. RESULTS: 733 and 807 proteins were identified in NACT and NACT/RT groups, respectively. Ten proteins were shortlisted for validation as potential pCR-predictive markers. THBS1, TNC, and DCN were significantly overexpressed in no-pCR in both the groups. In NACT, CPA3 was significantly upregulated in the no-pCR. In NACT/RT, HnRNPAB was significantly upregulated and HMGB1 significantly downregulated in the no-pCR. HMGB1 was the only marker to show prognostic significance. CONCLUSION: Quantitative proteomics followed by IHC identified and validated potential biomarkers for predicting patient response to therapy. These markers can be used, following larger-scale validation, in combination with routine histological analysis providing vital indications of treatment response.

High-mobility group box 1 in Parkinson's disease: from pathogenesis to therapeutic approaches.

Parkinson's disease (PD) presents the second most common neurodegenerative disorder with largely unknown pathogenesis and inefficient therapeutic management. Accumulating data indicate that neuroinflammation, autophagy impairment, α-synuclein aggregation, and mitochondrial dysfunction may contribute to PD onset; however, the molecular mechanisms underlying these pathophysiological processes are still under elucidation. Interestingly, recent evidence has indicated that High-mobility group box 1 (HMGB1), a DNA-binding protein that can be actively secreted by inflammatory cells and passively released by necrotic cells may play a key role in PD pathogenesis. HMGB1 has been shown to participate in neuroinflammation, modulate autophagy and apoptosis as well as regulate gene transcription. Furthermore, therapeutic targeting of HMGB1 with either anti-HMGB1 antibodies or selective inhibitors, such as glycyrrhizin, has been shown to inhibit neurodegeneration in animal models. This review provides an update of recent data on the emerging role of HMGB1 in PD pathogenesis and discusses potential therapeutic approaches.

HMGB1 a-Box Reverses Brain Edema and Deterioration of Neurological Function in a Traumatic Brain Injury Mouse Model.

BACKGROUND/AIMS: Traumatic brain injury (TBI) is a complex neurological injury in young adults lacking effective treatment. Emerging evidences suggest that inflammation contributes to the secondary brain injury following TBI, including breakdown of the blood brain barrier (BBB), subsequent edema and neurological deterioration. High mobility group box-1 (HMGB1) has been identified as a key cytokine in the inflammation reaction following TBI. Here, we investigated the therapeutic efficacy of HMGB1 A-box fragment, an antagonist competing with full-length HMGB1 for receptor binding, against TBI. METHODS: TBI was induced by controlled cortical impact (CCI) in adult male mice. HMGB1 A-box fragment was given intravenously at 2 mg/kg/day for 3 days after CCI. HMGB1 A-box-treated CCI mice were compared with saline-treated CCI mice and sham mice in terms of BBB disruption evaluated by Evan's blue extravasation, brain edema by brain water content, cell death by propidium iodide staining, inflammation by Western blot and ELISA assay for cytokine productions, as well as neurological functions by the modified Neurological Severity Score, wire grip and beam walking tests. RESULTS: HMGB1 A-box reversed brain damages in the mice following TBI. It significantly reduced brain edema by protecting integrity of the BBB, ameliorated cell degeneration, and decreased expression of pro-inflammatory cytokines released in injured brain after TBI. These cellular and molecular effects were accompanied by improved behavioral performance in TBI mice. Notably, HMGB1 A-box blocked IL-1ß-induced HMGB1 release, and preferentially attenuated TLR4, Myd88 and P65 in astrocyte cultures. CONCLUSION: Our data suggest that HMGB1 is involved in CCI-induced TBI, which can be inhibited by HMGB1 A-box fragment. Therefore, HMGB1 A-box fragment may have therapeutic potential for the secondary brain damages in TBI.

A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and postinjury inflammation in mice.

Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP-induced acute liver injury (APAP-ALI) and justifies development of anti-inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N-acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP-ALI. The anti-HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti-HMGB1 polyclonal antibody treatment improves survival in a model of APAP-ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti-HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP-ALI. The mouse anti-HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody-Fc backbones. Effector function-deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP-ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP-induced serum elevations of alanine aminotransferase and microRNA-122 and completely abrogated markers of APAP-induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C-X-C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. CONCLUSION: This is the first report describing the generation of a partly humanized HMGB1-neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP-ALI. The therapeutic effect was mediated by HMGB1 neutralization and attenuation of postinjury inflammation. These results represent important progress toward clinical implementation of HMGB1-specific therapy as a means to treat APAP-ALI and other inflammatory conditions. (Hepatology 2016;64:1699-1710).

Preconditioning with recombinant high-mobility group box 1 induces ischemic tolerance in a rat model of focal cerebral ischemia-reperfusion.

Preconditioning with ligands of toll-like receptors (TLRs) is a powerful neuroprotective approach whereby a low dose of stimulus confers significant protection against subsequent substantial brain damage by reprogramming the ischemia-activated TLRs signaling. Herein, we aim to explore whether preconditioning with recombinant high-mobility group box 1 (rHMGB1), one of the TLRs ligands, decreases cerebral ischemia-reperfusion injury (IRI). Rats were intracerebroventricularly pretreated with rHMGB1, 1 or 3 days before induction of middle cerebral artery occlusion. Results showed that preconditioning with rHMGB1 1 day, but not 3 days, prior to ischemia dramatically reduced neurological deficits, infarct size, brain swelling, cell apoptosis, and blood-brain barrier permeability. Interleukin-1R-associated kinase-M (IRAK-M), a critical negative regulator of TLRs signaling, was robustly increased in response to brain IRI and was further elevated by rHMGB1 pretreatment, indicating its role associated with the rHMGB1 preconditioning-mediated ischemic tolerance. In vitro and in vivo assays indicated that the induced IRAK-M expression was localized in microglia. In addition, TLR4 specific inhibitor TAK-242 abolished the neuroprotective effects and the induction of IRAK-M offered by rHMGB1 preconditioning. Collectively, our study demonstrates that rHMGB1 preconditioning is neuroprotective during cerebral IRI, which is associated with activated TLR4/IRAK-M signaling in microglia. We found that high-mobility group box 1 (HMGB1) pretreatment conditioned the brain against subsequent ischemia-reperfusion injury. We propose the following mechanism for HMGB1 preconditioning-mediated ischemic tolerance: through toll-like receptor TLR4, HMGB1 preconditioning magnifies the up-regulation of interleukin-1R-associated kinase-M (IRAK-M) induced by ischemia-reperfusion in microglia, resulting in the decreased phosphorylation of IRAK-1. These findings are helpful in understanding the endogenous mechanisms that counteract ischemic insults.

Preconditioning with recombinant high-mobility group box 1 protein protects the kidney against ischemia-reperfusion injury in mice.

A preconditioning effect occurs when exposure to a nonharmful quantity of a mediator of injury provides protection against injury upon subsequent reexposure. High-mobility group box 1 (HMGB1) protein, an endogenous ligand for Toll-like receptor (TLR) 4, is a TLR4-dependent mediator of kidney ischemia-reperfusion injury. Here we determined whether preconditioning with recombinant HMGB1 can block kidney ischemia-reperfusion injury, whether this effect is TLR4 dependent and, if so, how preconditioning downregulates TLR signaling. Wild-type mice pretreated with rHMGB1 before ischemia were protected against kidney ischemia-reperfusion injury, indicated by lower serum creatinine, less tubular damage, less tubulointerstitial neutrophil and macrophage infiltration, and less tubular epithelial cell apoptosis versus control mice. Gene expression of TLR-downstream cytokines and chemokines in ischemia-reperfusion injury kidney were also significantly reduced. While TLR4 and TLR2 knockout mice were protected against kidney ischemia-reperfusion injury, HMGB1 preconditioning provided additional protection to TLR2 but not TLR4 knockout mice. The protective effect of rHMGB1 preconditioning involved Siglec-G upregulation, a negative regulator of HMGB1-mediated TLR4 pathway activation. Thus, preconditioning with rHMGB1 affords significant protection from TLR4-dependent kidney ischemia-reperfusion injury, indicating therapeutic potential.

Andrographolide anti-proliferation and metastasis of hepatocellular carcinoma through LncRNA MIR22HG regulation.

Hepatocellular carcinoma (HCC) treatment is a major challenge. Although andrographolide (Andro) has an anti-proliferation effect on HCC, its underlying mechanism is not yet elucidated, and whether Andro can inhibit HCC metastasis has not been reported. The present study aimed to clarify whether Andro inhibits SK-Hep-1 cell proliferation and HCC metastasis, and the mechanisms. The results showed that Andro significantly reduced the survival of HCC cells and tumor weight and volume in tumor-bearing nude mice. Andro also triggered apoptosis of HCC cells and upregulated MIR22HG, Cleaved Caspase 9/7/3 expression levels, and downregulated BCL-2 mRNA, BCL-2 expression levels. Knockdown of MIR22HG or overexpression of HuR attenuated the effects of Andro on the signal transduction of mitochondrial apoptotic pathway and proliferation ability in HCC cells. Moreover, Andro significantly reduced the invasive ability of the cells and the level of HCC cell lung metastasis, upregulated miR-22-3p expression level and downregulated HMGB1 and MMP-9 expression levels. MIR22HG or miR-22-3p knockdown attenuated the effects of Andro on the signaling of HMGB1/MMP-9 pathway and invasive ability in HCC cells, while the overexpression of HMGB1 attenuated the inhibitory effects of Andro on the MMP-9 expression level and invasive ability in HCC cells. Thus, the regulation of MIR22HG-HuR/BCL-2 and MIR22HG/HMGB1 signaling pathways is involved in the anti-HCC proliferation and metastasis effects of Andro. This study provided a new pharmacological basis for Andro in HCC treatment and, for the first time, identified a natural product molecule capable of positively regulating MIR22HG, which has a robust biological function.

Exogenous high-mobility group box 1 protein prevents postinfarction adverse myocardial remodeling through TGF-ß/Smad signaling pathway.

High-mobility group box 1 (HMGB1) has been reported to attenuate ventricular remodeling, but its mechanism remains mostly unresolved. Transforming growth factor-beta (TGF-ß) is a crucial mediator in the pathogenesis of post-infarction remodeling. Our study focused on the effects of HMGB1 on ventricular remodeling, and explored whether or not these effects were depended upon the TGF-ß signaling pathway. Rats underwent coronary artery ligation. An intramyocardium injection of phosphate buffered saline (PBS) with or without HMGB1 was administered 3 weeks after myocardial infarction (MI). At 4 weeks after the treatment, HMGB1 significantly increased the left ventricular ejection fraction (LVEF) (P < 0.05), decreased the left ventricular end diastolic dimension (LVEDD; P < 0.05), left ventricular end systolic dimension (LVESD) (P < 0.05) and the infarct size (P < 0.05) compared with control group. The expressions of collagen I, collagen III, and tissue inhibitor of metalloproteinase 2 (TIMP2) were also decreased, while the matrix metalloproteinases 2 (MMP2) and MMP9 expressions were upregulated by HMGB1 injection (P < 0.05) compared with control group. No effect on TIMP3 was observed. Furthermore, TGF-ß1 and phosphor-Smad2 (p-Smad2) were significantly suppressed and Smad7 was increased in HMGB1-treated group (P < 0.05) compared with control group, no effects on p-Smad3 and p-p38 were observed. HMGB1 also upregulated Smad 7 expression and decreased the level of collagen I on cardiac fibroblasts (P < 0.05). Silencing of Smad7 gene by small interfering RNA abolished the fibrogenic effects of HMGB1 on cardiac fibroblasts (P < 0.05). These finding suggested that HMGB1 injection modulated ventricular remodeling may function through the possible inhibition of TGF-ß/Smad signaling pathway.

Epithelial alarmins: a new target to treat chronic respiratory diseases.

INTRODUCTION: In response to injury, epithelial cells release alarmins including thymic stromal lymphopoietin (TSLP), high mobility group-box-1 (HMGB1), interleukin (IL)-33 and -25 that can initiate innate immune responses. These alarmins are recognized as activators of T2-immune responses characteristic for asthma, but recent evidence highlighted their role in non-T2 inflammation, airway remodeling, and pulmonary fibrosis making them an attractive therapeutic target for chronic respiratory diseases (CRD). AREAS COVERED: In this review, firstly we discuss the role of TSLP, IL-33, IL-25, and HMGB1 in the pathogenesis of asthma, COPD, idiopathic pulmonary fibrosis, and cystic fibrosis according to the published data. In the second part, we summarize the current evidence concerning the efficacy of the antialarmin therapies in CRD. Recent clinical trials showed that anti-TSLP and IL-33/R antibodies can improve severe asthma outcomes. Blocking the IL-33-mediated pathway decreased the exacerbation rate in COPD patients with more important benefit for former-smokers. EXPERT OPINION: Despite progress in the understanding of the alarmins' role in the pathogenesis of CRD, all their mechanisms of action are not yet identified. Blocking IL-33 and TSLP pathways offers an interesting option to treat severe asthma and COPD, but future investigations are needed to establish their place in the treatment strategies.

Therapeutic Effect of Nicotinamide Mononucleotide for Hypoxic-Ischemic Brain Injury in Neonatal Mice.

SUMMARY STATEMENT: Neonatal hypoxia-ischemia reduces nicotinamide adenine dinucleotide (NAD+) and SIRT6 levels in the injured hippocampus.Hippocampal high mobility group box-1 (HMGB1) release is significantly increased after neonatal hypoxia-ischemia.Nicotinamide mononucleotide (NMN) treatment normalizes hippocampal NAD+ and SIRT6 levels, with significant decrease in caspase-3 activity and HMGB1 release.NMN improves early developmental behavior, as well as motor and memory function.

FLOT and CROSS chemotherapy regimens alter the frequency of CD27+ and CD69+ T cells in oesophagogastric adenocarcinomas: implications for combination with immunotherapy.

Combining immunostimulatory chemotherapies with immunotherapy is an attractive strategy to enhance treatment responses in oesophagogastric junctional adenocarcinoma (OGJ). This study investigates the immunostimulatory properties of FLOT, CROSS and MAGIC chemotherapy regimens in the context of OGJ using in vitro and ex vivo models of the treatment-naïve and post-chemotherapy treated tumour microenvironment. FLOT and CROSS chemotherapy regimens increased surrogate markers of immunogenic cell death (HMGB1 and HLA-DR), whereas the MAGIC treatment regimen decreased HMGB1 and HLA-DR on OGJ cells (markedly for epirubicin). Tumour-infiltrating and circulating T cells had significantly lower CD27 expression and significantly higher CD69 expression post-FLOT and post-CROSS treatment. Similarly, the supernatant from FLOT- and CROSS-treated OGJ cell lines and from FLOT- and CROSS-treated OGJ biopsies cultured ex vivo also decreased CD27 and increased CD69 expression on T cells. Following 48 h treatment with post-FLOT and post-CROSS tumour conditioned media the frequency of CD69+ T cells in culture negatively correlated with the levels of soluble immunosuppressive pro-angiogenic factors in the conditioned media from ex vivo explants. Supernatant from FLOT- and CROSS-treated OGJ cell lines also increased the cytotoxic potential of healthy donor T cells ex vivo and enhanced OGJ patient-derived lymphocyte mediated-killing of OE33 cells ex vivo. Collectively, this data demonstrate that FLOT and CROSS chemotherapy regimens possess immunostimulatory properties, identifying these chemotherapy regimens as rational synergistic partners to test in combination with immunotherapy and determine if this combinatorial approach could boost anti-tumour immunity in OGJ patients and improve clinical outcomes.

Radiotherapy combined with docetaxel alters the immune phenotype of HNSCC cells and results in increased surface expression of CD137 and release of HMGB1 of specifically HPV-positive tumor cells.

PURPOSE: Human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) tumors respond significantly better to anticancer treatments. It is assumed to be due to a better response to radiotherapy (RT), and presumably to an increased immunogenicity. However, little is known how the immune phenotype of HNSCC tumor cells is modulated by standard treatment, namely by radiochemotherapy (RCT). METHODS: Therefore, we aimed to examine the impact of the HPV status on the immune phenotype of HNSCC cell lines following RCT with 5 × 3Gy or 1 × 19.3Gy and/or docetaxel, by analyzing cell death, release of damage-associated molecular patterns (DAMPs), surface expression of immune checkpoint molecules (ICMs) and the impact on activation of human monocyte-derived dendritic cells (hmDCs). RESULTS: Cell death induction and Hsp70 release following RCT was independent of the HPV status, and RCT significantly increased the expression of the immune suppressive ICMs PD-L1, PD-L2 and HVEM. An immune stimulatory ICM, CD137, was significantly increased following RCT only on HPV-positive cell lines, as well as the release of HMGB1. Although the treatment increased cell death and modulated ICM expression in HNSCC, the hmDCs were not activated after co-incubation with treated tumor cells. CONCLUSION: Our data with the HPV-dependent release of HMGB1 and increased expression of CD137 following RCT provide a hint for increased immunogenicity underlining the better prognosis for HPV positive tumors following RCT.

Syringic acid suppresses ferroptosis of skeletal muscle cells to alleviate lower limb ischemia/reperfusion injury in mice via the HMGB1 pathway.

Ischemia/reperfusion (I/R) of skeletal muscle in the lower limbs is an important factor affecting the outcome of lower limbs ischemia patients, with no effective preventive or therapeutic approaches available. The study was to investigate the effect of syringic acid (SA) on I/R skeletal muscle in the lower limbs injury. Mice femoral artery I/R models and C2C12 cell hypoxia/reoxygenation (H/R) models was establish, tissue damage, inflammatory status, and high mobility group box 1 (HMGB1) pathway were evaluated using histological analysis, enzyme-linked immunosorbent assay, and western blotting. Further, the study detected the effect of SA on cell apoptosis, lipid peroxidation, Fe2+ level, and ferroptosis-related proteins expression. Finally, the effect of HMGB1 expression on SA in H/R stimulation was studied. SA alleviated pathological damage and reduced levels of IL-1ß, IL-6, and TNF-α in muscle tissues from femoral artery I/R mouse models. SA upregulated Bcl-2 and SOD as well as downregulated Bax, MDA, TBARS content, and Fe2+ level in H/R-induced cells. SA inhibited HMGB1 expression and promoted Nrf2, HO-1, GPX4, and SLC7A11 expressions in the injured tissues and cells. Such effects of SA on H/R-induced cells were rescued by HMGB1 overexpression. SA suppressed ferroptosis of skeletal muscle cells to alleviate lower limb I/R injury in mice by blocking the HMGB1 pathway, providing new insights for the treatment of lower limb ischemia-reperfusion injury.

Longitudinal monitoring of response to chemotherapy in patients with malignant pleural mesothelioma by biomarkers.

BACKGROUND: The aim of the study was to longitudinally investigate the serum levels of mesothelin, sestrin1, hyaluronan synthase 2 (HAS2), midkine, and high mobility group box 1 (HMGB1) before and after chemotherapy and at the time of relapse in malignant pleural mesothelioma (MPM) patients treated with chemotherapy and to compare the changes in biomarker levels with radiological treatment outcome. METHODS: A total of 64 MPM patients treated with chemotherapy were enrolled in the study and longitudinally followed for changes in biomarker levels in response to treatment. Biomarkers levels were measured in serum using a human ELISA kit. Relative and absolute changes in biomarker levels were compared with the best radiological overall response at each time point. RESULTS: Median survival was 20.0 ± 2.4 (15.3-24.7) months in patients with partial and complete response, 17.0 ± 1.0 (15.0-19.0) months in patients with stable disease, and 9.0 ± 1.0 (7.0-11.0) months in patients with progressive disease. A significant decrease in serum levels of mesothelin, midkine, and HMGB1 was observed in patients with radiologically partial and complete responses to chemotherapy (p< 0.001, p= 0.016, and p= 0.039, respectively). In these patients, mesothelin levels decreased by 15%, midkine levels by 7%, and HMGB1 levels by 15%. In addition, HMGB1 serum levels were found to significantly increase by 15% in patients with radiologically progressive responses to chemotherapy compared to pretreatment serum levels (p= 0.035). In patients with partial and complete response to chemotherapy, mesothelin levels increased by 15%, midkine by 12%, and sestrin1 by 8% when the disease recurred (p= 0.004, p= 0.004 and p= 0.044, respectively). CONCLUSION: Biomarkers may be useful in the longitudinal monitoring of response to treatment in MPM. However, the results of our study should be validated in larger groups with sufficient case numbers from multicenter institutions.

Paeonol ameliorates diabetic erectile dysfunction by inhibiting HMGB1/RAGE/NF-kB pathway.

BACKGROUND: The management of diabetes mellitus-induced erectile dysfunction (DMED) is progressively becoming tricky due to the surge in the number of patients and the poor efficiency of phosphodiesterase type 5 inhibitors in DMED. Paeonol (Pae), as a traditional Chinese medicine, has been more and more widely used in the treatment of diabetic complications. However, whether Pae could be a potential therapeutic drug of DMED needs to be further evaluated. OBJECTIVES: To investigate the pharmacological effect and possible mechanism of Pae in the treatment of DMED. METHODS: Intraperitoneal streptozotocin injection and an apomorphine test were used to construct the model of DMED. Seventeen DMED rats were divided into two groups: DMED group (n = 8) and DMED+Pae group (Pae; 100 mg/kg/d; oral administration; n = 9). In addition, there were still 10 normal age-matched male rats as control group. Four weeks later, the cavernous nerve electric stimulation was carried out to measure the erectile response. Moreover, the corpus cavernosum smooth muscle cells (CCSMCs) were primarily isolated and exposed to high glucose (HG) stimulation, Pae treatment and glycyrrhizin (GL; the selective inhibitor of HMGB1). After an incubation for 1 week, the CCSMCs were harvested for detection. RESULTS: The impairment of erectile function was observed in DMED rats compared with control samples, accompanied by the upregulation of HMGB1/RAGE/NF-κB Pathway. The lower nitric oxide and cGMP level and the higher level of inflammation, fibrosis, and apoptosis were also observed in DMED rats. It showed contrast that Pae treatment could improve the erectile function, as well as histologic alteration and related molecular changes. In addition, Pae could downregulate the HMGB1/RAGE/NF-κB pathway to regulate the apoptosis and inflammation levels of CCSMCs in high-glucose conditions, which is similar to the results of GL treatment. CONCLUSION: Pae alleviated ED in DMED rats, likely by inhibiting HMGB1/RAGE/NF-κB Pathway, inflammatory, apoptosis, and fibrotic activity, and moderating endothelial dysfunction. Our study provide evidence for a potential new therapy for DMED.