Non-histone Nuclear Protein Research Articles

AI-powered precision: breast carcinoma diagnosis through digital proliferation index (Ki-67) assessment in pathological anatomy

PurposeThe primary objective of the study is to enhance the accuracy and efficiency of assessing the proliferation index in cancer cells, specifically focusing on the role of Ki-67. The purpose is to address the limitations of traditional visual assessments conducted by pathologists by integrating AI technologies, particularly deep learning. By accurately computing the percentage of Ki-67-labeled cells, the research aims to streamline the diagnostic process, reduce subjectivity and contribute to the advancement of diagnostic precision in pathological anatomy.Design/methodology/approachThe research employs a methodological approach that integrates Ki-67, a non-histone nuclear protein, as a vital biomarker for assessing the proliferative status of cancer cells. Given the challenges associated with traditional visual assessments by pathologists, including inter- and intra-observer variability and time-consuming efforts, the study adopts a novel methodology leveraging artificial intelligence (AI) solutions. Deep learning is applied to precisely calculate the percentage of Ki-67-labeled cells. The process involves pathologists delineating the tumor area at x40 magnification, enabling the segmentation of various cell types (positive, negative and tumor-infiltrating lymphocytes). The subsequent percentage calculation enhances efficiency and minimizes subjectivity in the diagnostic process.FindingsDespite inherent errors, the research findings indicate that the model surpasses existing benchmarks, showcasing superior accuracy in terms of average error measurement. The comparison with diverse datasets and benchmarking against pathologists’ diagnoses contributes empirical evidence to support the effectiveness of the AI-based model in accurately computing the percentage of Ki-67-labeled cells. These findings signify a noteworthy advancement in diagnostic methodologies and reinforce the potential of AI technologies in improving the precision of cancer diagnostics within the realm of pathological anatomy.Originality/valueThe research contributes to the field by introducing an innovative approach that combines Ki-67 as a biomarker and AI technologies for improved diagnostic precision. The originality lies in the utilization of deep learning to calculate the percentage of labeled cells, mitigating the challenges associated with manual assessments. The validation of the model against diverse datasets and benchmarking against pathologists’ diagnoses demonstrates its superior accuracy, highlighting the value of integrating AI in pathological anatomy for enhanced diagnostic outcomes. The study represents a significant stride in original research, offering novel insights and methodologies in the pursuit of more precise cancer diagnostics.

Crystal structure of the HMGA AT-hook 1 domain bound to the minor groove of AT-rich DNA and inhibition by antikinetoplastid drugs

High mobility group (HMG) proteins are intrinsically disordered nuclear non-histone chromosomal proteins that play an essential role in many biological processes by regulating the expression of numerous genes in eukaryote cells. HMGA proteins contain three DNA binding motifs, the “AT-hooks”, that bind preferentially to AT-rich sequences in the minor groove of B-form DNA. Understanding the interactions of AT-hook domains with DNA is very relevant from a medical point of view because HMGA proteins are involved in different conditions including cancer and parasitic diseases. We present here the first crystal structure (1.40 Å resolution) of the HMGA AT-hook 1 domain, bound to the minor groove of AT-rich DNA. In contrast to AT-hook 3 which bends DNA and shows a larger minor groove widening, AT-hook 1 binds neighbouring DNA molecules and displays moderate widening of DNA upon binding. The binding affinity and thermodynamics of binding were studied in solution with surface plasmon resonance (SPR)-biosensor and isothermal titration calorimetry (ITC) experiments. AT-hook 1 forms an entropy-driven 2:1 complex with (TTAA)2-containing DNA with relatively slow kinetics of association/dissociation. We show that N-phenylbenzamide-derived antikinetoplastid compounds (1–3) bind strongly and specifically to the minor groove of AT-DNA and compete with AT-hook 1 for binding. The central core of the molecule is the basis for the observed sequence selectivity of these compounds. These findings provide clues regarding a possible mode of action of DNA minor groove binding compounds that are relevant to major neglected tropical diseases such as leishmaniasis and trypanosomiasis.

Plasmodium berghei HMGB1 controls the host immune responses and splenic clearance by regulating the expression of pir genes

High mobility group box (HMGB) proteins belong to high mobility group (HMG) superfamily of non-histone nuclear proteins that are involved in chromatin remodeling, regulation of gene expression and DNA repair. When extracellular, HMGBs serve as alarmins inducing inflammation and this is attributed to the proinflammatory activity of box B. Here, we show that Plasmodium HMGB1 has key amino acid changes in box B resulting in the loss of TNF-α stimulatory activity. Site-directed mutagenesis of the critical amino acids in box B with respect to mouse HMGB1 renders recombinant Plasmodium berghei (Pb) HMGB1 capable of inducing TNF-α release. Targeted deletion of PbHMGB1 and a detailed in vivo phenotyping show that PbHMGB1 knockout (KO) parasites can undergo asexual stage development. Interestingly, Balb/c mice-infected with PbHMGB1KO parasites display a protective phenotype with subsequent clearance of blood parasitemia, and develop long-lasting protective immunity against the challenges performed with Pb wildtype parasites. The characterization of splenic responses show prominent germinal centres leading to effective humoral responses and enhanced T follicular helper cells. There is also a complete protection from experimental cerebral malaria in CBA/CaJ mice susceptible for cerebral pathogenesis with subsequent parasite clearance. Transcriptomic studies suggest the involvement of PbHMGB1 in pir expression. Our findings highlight the gene regulatory function of parasite HMGB1 and its in vivo significance in modulating the host immune responses. Further, clearance of asexual stages in PbHMGB1KO-infected mice underscores the important role of parasite HMGB1 in host immune evasion. These findings have implications in developing attenuated blood-stage vaccine for malaria.

Ki-67 AS A PREDICTING FACTOR OF NASOPHARYNGEAL, BREAST, AND CERVICAL CANCER: A LITERATURE REVIEW

Background: Although the exact role remains unknown, Ki-67 is vital in cell division. Ki-67 is a nuclear nonhistone protein present in all active phases of the cell cycle. It has the potential to predict the prognosis of responsiveness to radiotherapy, chemotherapy, or endocrine therapy, estimate residual risk, and predict recurrence in many types of cancer, including nasopharyngeal cancer (NPC), breast cancer (BC), and cervical cancer (CX), and be a marker for treatment efficacy. We want to review the efficacy of Ki-67 as a predicting factor for three types of cancer, including NPC, breast, and cervical cancer, from several pieces of literature. Materials and Methods: We are conducting a literature review to describe the role of Ki-67 as a predicting factor in NPC, BC, and CX. Results: Pretreatment Ki-67 is used as an independent prognostic marker in NPC, and elevated values are associated with poor prognosis. High Ki-67 expression may indicate poorer recurrence-free survival and overall survival. A high Ki-67 index significantly correlated with adverse prognostic factors. Conclusion: A high expression of Ki-67 in NPC, breast cancer, and cervical cancer is associated with poor prognosis, poorer recurrence, and overall survival rates. Ki-67 can be considered as a valuable biomarker of many cancers. Keywords: Radiotherapy, Ki-67, nasopharyngeal cancer, breast cancer, cervical cancer

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7144.

High Mobility Group Box Protein (HMGB1): A Potential Therapeutic Target for Diabetic Encephalopathy.

HMGB (high mobility group B) is one of the ubiquitous non-histone nuclear protein superfamilies that make up the HMG (high mobility group) protein group. HMGB1 is involved in a variety of physiological and pathological processes in the human body, including a structural role in the cell nucleus as well as replication, repair, DNA transcription, and assembly of nuclear proteins. It functions as a signaling regulator in the cytoplasm and a pro-inflammatory cytokine in the extracellular environment. Among several studies, HMGB1 protein is also emerging as a crucial factor involved in the development and progression of diabetic encephalopathy (DE) along with other factors such as hyperglycaemia-induced oxidative and nitrosative stress. Diabetes' chronic side effect is DE, which manifests as cognitive and psychoneurological dysfunction. The HMGB1 is released outside to the extracellular medium in diabetes condition through active or passive routes, where it functions as a damage-associated molecular pattern (DAMP) molecule to activate several signaling pathways by interacting with receptors for advanced glycosylation end-products (RAGE)/toll like receptors (TLR). HMGB1 reportedly activates inflammatory pathways, disrupts the blood-brain barrier, causes glutamate toxicity and oxidative stress, and promotes neuroinflammation, contributing to the development of cognitive impairment and neuronal damage which is suggestive of the involvement of HMGB1 in the enhancement of the diabetes-induced encephalopathic condition. Additionally, HMGB1 is reported to induce insulin resistance, further exacerbating the metabolic dysfunction associated with diabetes mellitus (DM). Thus, the present review explores the possible pathways associated with DM-induced hyperactivation of HMGB1 ultimately leading to DE.

Expression of HMGB1 and NF-κB in Fetal Membranes of Premature Rupture of Membranes

Background: Inflammation, either sterile or infection-related, may lead to premature rupture of membranes (PROM). The non-histone nuclear proteins, high mobility group box-1 (HMGB1), and nuclear factor kappa-B (NF-κB) transcription factors have been extensively investigated in many disorders involving inflammatory reactions. This study aimed to determine the expression of HMGB1 and NF-κB in fetal membranes of PROM compared with non-PROM.Methods: This study was an analytical observational study with a case-control design, performed from November 2021 to January 2022, including 40 fetal membrane samples (20 PROM and 20 non-PROM), which were obtained from pregnant women treated in the emergency unit of a hospital in Surabaya from August to November 2019 using the non-probability sampling method. The HMGB1 and NF-κB expressions were examined using the immunohistochemical method and further viewed under a light microscope (400x magnification), then assessed by Image-J software. The values were then compared between PROM and non-PROM, and analyzed using the Mann-Whitney U test.Results: There was a significant difference (p<0.001) in the expression of HMGB1 and NF-κB in PROM and non-PROM for HMGB1 45.86± 14.21% vs. 8.50± 5.66% expression/mm2; and NF-κB 33.47±5.45% vs. 7.29±4.90% expression/mm2, respectively.Conclusions: PROM groups have significantly higher expression of HMGB1 and NF-κB s, indicating their higher activity and contribution to PROM.

High-mobility group box 1 emerges as a therapeutic target for asthma.

High-mobility group box 1 (HMGB1) is a highly conserved nonhistone nuclear protein found in the calf thymus and participates in a variety of intracellular processes such as DNA transcription, replication and repair. In the cytoplasm, HMGB1 promotes mitochondrial autophagy and is involved in in cellular stress response. Once released into the extracellular, HMGB1 becomes an inflammatory factor that triggers inflammatory responses and a variety of immune responses. In addition, HMGB1 binding with the corresponding receptor can activate the downstream substrate to carry out several biological effects. Meanwhile, HMGB1 is involved in various signaling pathways, such as the HMGB1/RAGE pathway, HMGB1/NF-κB pathway, and HMGB1/JAK/STAT pathway, which ultimately promote inflammation. Moreover, HMGB1 may be involved in the pathogenesis of asthma by regulating downstream signaling pathways through corresponding receptors and mediates a number of signaling pathways in asthma, such as HMGB1/TLR4/NF-κB, HMGB1/RAGE, HMGB1/TGF-β, and so forth. Accordingly, HMGB1 emerges as a therapeutic target for asthma.

Npro of classical swine fever virus enhances HMGB1 acetylation and its degradation by lysosomes to evade from HMGB1-mediated antiviral immunity

Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral Npro. High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or Npro-expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV Npro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent Npro-mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote Npro-mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its Npro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation.

Contributing Role of High Mobility Group Box 1 Signaling in Oral Cancer Development and Therapy.

Oral squamous cell carcinoma (OSCC) is the most frequent type of oral cancer of multifactorial origin, characterized by histological and clinical manifestations. To date, there are no specific biomarkers or treatment modalities available to efficiently manage this neoplasia, demanding further research on the molecular background of OSCC pathology. Elucidation of signal transduction pathways and associated molecules with differential expression and function in OSCC are expected to enhance the future development of molecular targeted therapies. Among signaling proteins with a potential functional role in OSCC, the High Mobility Group Box 1 (HMGB1) protein has stimulated scientific interest due to frequent upregulation, and implication in the progression of many types of head and neck cancer types. HMGB1 is a nuclear nonhistone protein and an extracellularly secreted cytokine that can interact with several signaling molecules implicated in the pathogenic pathways of OSCC. Binding of HMGB1 to specific receptors on OSCC cells such as the receptor of AGE (RAGE) and the toll-like receptor (TLR) has been shown to initiate several intercellular signaling cascades that can promote OSCC growth, invasion, and metastasis, indicating a potential target for patient prognosis and therapeutic approaches. The purpose of this review is to explore the functional role and associated signaling of HMGB1 in OSCC in order to reveal potential therapeutic targeting options.

Structure and Functions of HMGB2 Protein

High-Mobility Group (HMG) chromosomal proteins are the most numerous nuclear non-histone proteins. HMGB domain proteins are the most abundant and well-studied HMG proteins. They are involved in variety of biological processes. HMGB1 and HMGB2 were the first members of HMGB-family to be discovered and are found in all studied eukaryotes. Despite the high degree of homology, HMGB1 and HMGB2 proteins differ from each other both in structure and functions. In contrast to HMGB2, there is a large pool of works devoted to the HMGB1 protein whose structure-function properties have been described in detail in our previous review in 2020. In this review, we attempted to bring together diverse data about the structure and functions of the HMGB2 protein. The review also describes post-translational modifications of the HMGB2 protein and its role in the development of a number of diseases. Particular attention is paid to its interaction with various targets, including DNA and protein partners. The influence of the level of HMGB2 expression on various processes associated with cell differentiation and aging and its ability to mediate the differentiation of embryonic and adult stem cells are also discussed.

HMGB1 cleavage by complement C1s and its potent anti-inflammatory product.

Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation.

The Redox Protein High-Mobility Group Box 1 in Cell Death and Cancer.

Significance: As a redox-sensitive protein, high-mobility group box 1 (HMGB1) is implicated in regulating stress responses to oxidative damage and cell death, which are closely related to the pathology of inflammatory diseases, including cancer. Recent Advances: HMGB1 is a nonhistone nuclear protein that acts as a deoxyribonucleic acid chaperone to control chromosomal structure and function. HMGB1 can also be released into the extracellular space and function as a damage-associated molecular pattern protein during cell death, including during apoptosis, necrosis, necroptosis, pyroptosis, ferroptosis, alkaliptosis, and cuproptosis. Once released, HMGB1 binds to membrane receptors to shape immune and metabolic responses. In addition to subcellular localization, the function and activity of HMGB1 also depend on its redox state and protein posttranslational modifications. Abnormal HMGB1 plays a dual role in tumorigenesis and anticancer therapy (e.g., chemotherapy, radiation therapy, and immunotherapy) depending on the tumor types and stages. Critical Issues: A comprehensive understanding of the role of HMGB1 in cellular redox homeostasis is important for deciphering normal cellular functions and pathological manifestations. In this review, we discuss compartmental-defined roles of HMGB1 in regulating cell death and cancer. Understanding these advances may help us develop potential HMGB1-targeting drugs or approaches to treat oxidative stress-related diseases or pathological conditions. Future Directions: Further studies are required to dissect the mechanism by which HMGB1 maintains redox homeostasis under different stress conditions. A multidisciplinary effort is also required to evaluate the potential applications of precisely targeting the HMGB1 pathway in human health and disease. Antioxid. Redox Signal. 39, 569-590.

HMGB1 coordinates with Brahma-related gene 1 to promote epithelial-mesenchymal transition via the PI3K/Akt/mTOR pathway in BEAS-2B cells

High mobility group protein B1 (HMGB1), a highly conserved non-histone nuclear protein, is highly expressed in fibrotic diseases; however, the role of HMGB1 in pulmonary fibrosis has not been fully elucidated. In this study, an epithelial-mesenchymal transition (EMT) model was constructed using transforming growth factor-β1 (TGF-β1) to stimulate BEAS-2B cells in vitro, and HMGB1 was knocked down or overexpressed to observe its effects on cell proliferation, migration and EMT. Meanwhile, string system, immunoprecipitation and immunofluorescence analyses were applied to identify and examine the relationship between HMGB1 and its potential interacting protein Brahma-related gene 1 (BRG1), and to explore the mechanism of interaction between HMGB1 and BRG1 in EMT. The results indicate that exogenous increase in HMGB1 promotes cell proliferation and migration and facilitates EMT by enhancing the PI3K/Akt/mTOR signaling pathway, whereas silencing HMGB1 has the opposite effect. Mechanistically, HMGB1 exerts these functions by interacting with BRG1, which may enhance BRG1 function and activate the PI3K/Akt/mTOR signaling pathway, thereby promoting EMT. These results suggest that HMGB1 is important for EMT and is a potential therapeutic target for the treatment of pulmonary fibrosis.

Structural Characteristics of High-Mobility Group Proteins HMGB1 and HMGB2 and Their Interaction with DNA.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.

The redox protein HMGB1 in cell death and cancer.

As a redox-sensitive protein, high-mobility group box 1 (HMGB1) is implicated in regulating stress responses to oxidative damage and cell death, which are closely related to the pathology of inflammatory diseases, including cancer. HMGB1 is a non-histone nuclear protein that acts as a DNA chaperone to control chromosomal structure and function. HMGB1 can also be released into the extracellular space and function as a damage-associated molecular pattern protein during cell death, including during apoptosis, necrosis, necroptosis, pyroptosis, ferroptosis, alkaliptosis, and cuproptosis. Once released, HMGB1 binds to membrane receptors to shape immune and metabolic responses. In addition to subcellular localization, the function and activity of HMGB1 also depends on its redox state and protein posttranslational modifications. Abnormal HMGB1 plays a dual role in tumorigenesis and anticancer therapy (e.g., chemotherapy, radiation therapy, and immunotherapy) depending on tumor types and stages. A comprehensive understanding of the role of HMGB1 in cellular redox homeostasis is important for deciphering normal cellular functions and pathological manifestations. In this review, we discuss compartmental-defined roles of HMGB1 in regulating cell death and cancer. Understanding these advances may help us develop potential HMGB1-targeting drugs or approaches to treat oxidative stress-related diseases or pathological conditions. Further studies are required to dissect the mechanism by which HMGB1 maintains redox homeostasis under different stress conditions. A multidisciplinary effort is also required to evaluate the potential applications of precisely targeting the HMGB1 pathway in human health and disease.

The regulation and function of acetylated high-mobility group box 1 during implantation and decidualization.

High-mobility group box 1 (HMGB1) is a non-histone nuclear protein and can be extracellularly secreted to induce sterile inflammation. Although uterine deletion of HMGB1 causes implantation and decidualization defects, how secreted HMGB1 is involved in mouse early pregnancy is still unknown. Mouse models, mouse primary endometrial cells and human endometrial cell lines were used in this study. Both immunofluorescence and Western blot were performed to show the localization and relative level of HMGB1 and acetylated HMGB1, respectively. Relative mRNA levels were analyzed by real time RT-PCR. The secreted HMGB1 was detected in uterine lumen fluid in mouse periimplantation uterus. There is an obvious difference for secreted HMGB1 levels in uterine fluid between day 4 of pregnancy and day 4 of pseudopregnancy, suggesting the involvement of blastocysts during HMGB1 secretion. Trypsin is clearly detected in mouse blastocyst cavity and in the supernatant of cultured blastocysts. Trypsin significantly stimulates HB-EGF production through activating PAR2 and ADAM17. Uterine injection of PAR2 inhibitor into day 4 pregnant mice significantly reduces the number of implantation sites. HB-EGF released from luminal epithelium can induce mouse in vitro decidualization. The conditioned medium collected from trypsin-treated luminal epithelium is able to induce in vitro decidualization, which is suppressed by EGFR inhibitor. Intrauterine injection of glycyrrhizin (HMGB1 inhibitor) can significantly inhibit mouse embryo implantation. We also showed that exogenous HMGB1 released from human epithelial cells are able to induce human in vitro decidualization. Trypsin can induce decidualization of stromal cells via PAR2-HMGB1-ADAM17-HB-EGF from luminal epithelium.

Histamine induced high mobility group box-1 release from vascular endothelial cells through H1 receptor.

BackgroundSystemic allergic reaction is characterized by vasodilation and vascular leakage, which causes a rapid, precipitous and sustained decrease in arterial blood pressure with a concomitant decrease of cardiac output. Histamine is a major mediator released by mast cells in allergic inflammation and response. It causes a cascade of inflammation and strongly increases vascular permeability within minutes through its four G-protein-coupled receptors (GPCRs) on endothelial cells. High mobility group box-1 (HMGB1), a nonhistone chromatin-binding nuclear protein, can be actively secreted into the extracellular space by endothelial cells. HMGB1 has been reported to exert pro-inflammatory effects on endothelial cells and to increase vascular endothelial permeability. However, the relationship between histamine and HMGB1-mediated signaling in vascular endothelial cells and the role of HMGB1 in anaphylactic-induced hypotension have never been studied.Methods and resultsEA.hy 926 cells were treated with different concentrations of histamine for the indicated periods. The results showed that histamine induced HMGB1 translocation and release from the endothelial cells in a concentration- and time-dependent manner. These effects of histamine were concentration-dependently inhibited by d-chlorpheniramine, a specific H1 receptor antagonist, but not by H2 or H3/4 receptor antagonists. Moreover, an H1-specific agonist, 2-pyridylethylamine, mimicked the effects of histamine, whereas an H2-receptor agonist, 4-methylhistamine, did not. Adrenaline and noradrenaline, which are commonly used in the clinical treatment of anaphylactic shock, also inhibited the histamine-induced HMGB1 translocation in endothelial cells. We therefore established a rat model of allergic shock by i.v. injection of compound 48/80, a potent histamine-releasing agent. The plasma HMGB1 levels in compound 48/80-injected rats were higher than those in controls. Moreover, the treatment with anti-HMGB1 antibody successfully facilitated the recovery from compound 48/80-induced hypotension.ConclusionHistamine induces HMGB1 release from vascular endothelial cells solely through H1 receptor stimulation. Anti-HMGB1 therapy may provide a novel treatment for life-threatening systemic anaphylaxis.

Endothelial Dysfunction, HMGB1, and Dengue: An Enigma to Solve.

Dengue is a viral infection caused by dengue virus (DENV), which has a significant impact on public health worldwide. Although most infections are asymptomatic, a series of severe clinical manifestations such as hemorrhage and plasma leakage can occur during the severe presentation of the disease. This suggests that the virus or host immune response may affect the protective function of endothelial barriers, ultimately being considered the most relevant event in severe and fatal dengue pathogenesis. The mechanisms that induce these alterations are diverse. It has been suggested that the high mobility group box 1 protein (HMGB1) may be involved in endothelial dysfunction. This non-histone nuclear protein has different immunomodulatory activities and belongs to the alarmin group. High concentrations of HMGB1 have been detected in patients with several infectious diseases, including dengue, and it could be considered as a biomarker for the early diagnosis of dengue and a predictor of complications of the disease. This review summarizes the main features of dengue infection and describes the known causes associated with endothelial dysfunction, highlighting the involvement and possible relationship between HMGB1 and DENV.

Studies On the Composition of The Low Molecular Weight Thiols Present in Tumour Cells– Is There "An Elephant in The Room”? An Autobiographical Review

This is an account of the author’s research investigations on the identification of the low molecular weight thiols (LMWT) present in a variety of eukaryote cells, particularly cancer cells. The work was carried out periodically over some 60 years in a number of university and private laboratories as, and when, resources permitted: as a result, this research can be roughly divided into 7 phases. The first phase arose from initial research into ways to improve cancer treatment by radiation treatment. The second was involved with studies of the thiols present in isolated nuclei and nucleoli. The third consisted of attempts to develop new techniques to isolate and identify the various nuclear protein thiol containing moieties of a rat hepatoma tumour. The fourth phase involved the successful isolation of the nuclear nonhistone proteins which contain the bulk of the nuclear thiol material, leading to study nuclear thiol changes during rat liver carcinogenesis. The fifth phase consisted of the discovery and isolation of “hidden” low molecular weight thiols in isolated nuclei from rat tumours and other tissues. After a break of some 14 years, when bulk preparations of whole human cells became available, it became possible to continue work again on the low molecular weight thiols. Phase six consisted of studies on the thiols found in the ASF of various tumour cell lines using the new analytical technologies which were developing at that time. The seventh phase evolved into studies on human prostate cancer cell lines. Here a method was developed which clearly demonstrated that, among the low molecular weight thiols present in these cells, there was “an elephant in the room”. This new thiol moiety, constituting 56.4% of the total cell thiols, was found to be concealed within the protein matrices of the cell, held in place, not covalently but possibly by ionic and hydrophobic bonding; deemed to be a “Conthiol”. It does not contain any of the constituents of known cellular thiols and has proved difficult to analyse due to its reactive nature.