Sample Preparation Strategies for Antibody-Free Quantitative Analysis of High Mobility Group Box 1 Protein.
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ABSTRACT:
SUBMITTER: Kvivik I
PROVIDER: S-EPMC8230321 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We evaluated the efficacy of a neutralizing anti-high mobility group box 1 (HMGB1) monoclonal antibody in MRL/lpr lupus-prone mice. The anti-HMGB1 monoclonal antibody (5 mg/kg weight) or class-matched control immunoglobulin G2a (IgG2a) was administered intravenously twice a week for 4-15 weeks. Urine albumin was monitored, and histological evaluation of the kidneys was conducted at 16 weeks. Lymphadenopathies were evaluated by 1-(2'-deoxy-2'-[18F]fluoro-?-D-arabinofuranosyl)cytosine ([18F]FAC) positron emission tomography/computed tomography (PET/CT) at 12 weeks. Following 4-week treatment, [18F]FAC-PET/CT showed similar accumulation in cervical and axillary lymph nodes at 12 weeks of age. However, anti-HMGB1 monoclonal antibody sufficiently inhibited the increase in albuminuria compared to an isotype control following 15-week treatment. Complement deposition was also improved; however, there were no significant differences in IgG deposition and renal pathological scores between the two groups. Anti-double-stranded DNA (dsDNA) antibody titers and cytokine and chemokine levels were also unaltered. Although there were no significant differences in glomerular macrophage infiltration, neutrophil infiltration was significantly decreased by the anti-HMGB1 monoclonal antibody. Antagonizing HMGB1 treatment suppressed HMGB1 translocation from nuclei in the kidney and suppressed neutrophil extracellular traps. The anti-HMGB1 monoclonal antibody demonstrated therapeutic potential against albuminuria in lupus nephritis by inhibiting neutrophil recruitment and neutrophil extracellular traps.
| S-EPMC5472134 | biostudies-literature
Project description:High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein in mammals. When released into the extracellular space, it acts as a damage-associated molecular pattern. This study investigates whether increased HMGB1 levels are found in the intestinal mucosa of ulcerative colitis (UC) patients, and whether an anti-HMGB1 neutralizing-antibody (HnAb) can inhibit the intestinal inflammation elicited by dextran sulfate sodium (DSS) in mice. Because toll-like receptor 4 (TLR4) is implicated in HMGB1-mediated immune cell activation, DSS colitis was also elicited in TLR4-deficient mice in the presence and absence of HnAb. The expression of HMGB1 in UC patients was examined. HnAb was administered via intraperitoneal injection to TLR4 deficient mice and their wild-type littermates, both being induced to colitis with DSS. Finally, the protective effect of HnAb and TLR4 deficiency were evaluated. In UC patients, HMGB1 was up-regulated in the inflamed colon. When administered during DSS application, HnAb alleviated the severity of colitis with a lower disease activity index, limited histological damages, and reduced production of proinflammatory cytokines. This antibody also limited colonic barrier loss, decreased colonic lamina propria macrophages and partially reversed the DSS treatment-associated dysbiosis. The protective effect of this antibody was enhanced in TLR4-deficient mice in some aspects, indicating that both additional HMGB1-mediated as well as TLR4-mediated inflammatory signaling pathways were involved in the induction of colitis by DSS. HnAb ameliorated colitis via macrophages inhibition and colonic barrier protection. It may therefore be a novel treatment option in colitis.
| S-EPMC7661783 | biostudies-literature
Project description:High mobility group box 1 (HMGB1) is an extremely versatile protein that is located predominantly in the nucleus of quiescent eukaryotic cells, where it is critically involved in maintaining genomic structure and function. During cellular stress, however, this multifaceted, cytokine-like protein undergoes posttranslational modifications that promote its translocation to the cytosol, from where it is released extracellularly, either actively or passively, according to cell type and stressor. In the extracellular milieu, HMGB1 triggers innate inflammatory responses that may be beneficial or harmful, depending on the magnitude and duration of release of this pro-inflammatory protein at sites of tissue injury. Heightened awareness of the potentially harmful activities of HMGB1, together with a considerable body of innovative, recent research, have revealed that excessive production of HMGB1, resulting from misdirected, chronic inflammatory responses, appears to contribute to all the stages of tumorigenesis. In the setting of established cancers, the production of HMGB1 by tumor cells per se may also exacerbate inflammation-related immunosuppression. These pro-inflammatory mechanisms of HMGB1-orchestrated tumorigenesis, as well as the prognostic potential of detection of elevated expression of this protein in the tumor microenvironment, represent the major thrusts of this review.
| S-EPMC7407638 | biostudies-literature
Project description:Purpose: Determine the mechanism of high mobility group box 1 (HMGB1)-induced signaling in melanocytes. Method: Primary human epidermal melanocytes were treated with HMGB1 (1 μg/ml) and incubated for 24 h. Total RNA (500 ng) from melanocytes were extracted and subjected to library synthesis. Results: HMGB1-treated melanocytes exhibited upregulation of cell death and type 1 interferon-related genes. Conclusion: HMGB1-induced melanocyte signaling was well evaluated using RNA sequencing.
2023-07-28 | GSE216423 | GEO
Project description:In eukaryotes, the high-mobility-group (HMG) nuclear factors are highly conserved throughout evolution and are divided into three families, including HGMB, characterized by an HMG box domain. Some HMGB factors are DNA structure specific and preferentially interact with distorted DNA sequences, trigger DNA bending, and hence facilitate the binding of nucleoprotein complexes that in turn activate or repress transcription. In Plasmodium falciparum, two HMGB factors were predicted: PfHMGB1 and PfHMGB2. They are small proteins, under 100 amino acids long, encompassing a characteristic HMG box domain closely related to box B of metazoan factors, which comprises two HMG box domains, A and B, in tandem. Computational analyses supported the conclusion that the Plasmodium proteins were genuine architectural HMGB factors, and in vitro analyses performed with both recombinant proteins established that they were able to interact with distorted DNA structures and bend linear DNA with different affinities. These proteins were detected in both asexual- and gametocyte-stage cells in Western blotting experiments and mainly in the parasite nuclei. PfHMGB1 is preferentially expressed in asexual erythrocytic stages and PfHMGB2 in gametocytes, in good correlation with transcript levels of expression. Finally, immunofluorescence studies revealed differential subcellular localizations: both factors were observed in the nucleus of asexual- and sexual-stage cells, and PfHMGB2 was also detected in the cytoplasm of gametocytes. In conclusion, in light of differences in their levels of expression, subcellular localizations, and capacities for binding and bending DNA, these factors are likely to play nonredundant roles in transcriptional regulation of Plasmodium development in erythrocytes.
| S-EPMC1459676 | biostudies-literature
Project description:PurposeTo determine high-mobility group box 1 (HMGB1) expression during experimental dry eye (EDE) and dry eye-like culture conditions and elucidate its role in corneal dry eye-related inflammation.MethodsEDE was induced in 8- to 12-week-old C57BL/6 mice. Corneal tissue sections and lysates from EDE and untreated mice were evaluated for HMGB1 expression by immunostaining and quantitative real-time PCR (qPCR). For in vitro studies, human corneal epithelial cells (HCEC) were treated with hyperosmolar media, toll-like receptor (TLR) agonists, or proinflammatory cytokines to determine HMGB1 expression. HCEC were also treated with human recombinant HMGB1 (hrHMGB1) alone or in combination with inflammatory stimuli, and TNFα, IL-6, and IL-8 expression evaluated by qPCR and ELISA. Nuclear factor-κB (NF-κB) p65 nuclear translocation was determined by immunostaining.ResultsEDE mice had higher corneal HMGB1 RNA and protein expression compared to untreated animals. In HCEC, hyperosmolar stress and TNFα treatment stimulated HMGB1 production and secretion into culture supernatants. However, in vitro stimulation with hrHMGB1 did not induce secretion of TNFα, IL-6, or IL-8 or NF-κB p65 nuclear translocation. In addition, the inflammatory response elicited by TLR agonists fibroblast-stimulating lipopeptide-1 and lipopolysaccharide was not enhanced by hrHMGB1 treatment.ConclusionsHMGB1 expression was enhanced by dry eye conditions in vivo as well as in vitro, during hyperosmolar stress and cytokine exposure, suggesting an important role for HMGB1 in dry eye disease. However, no direct inflammatory effect was observed with HMGB1 treatment. Therefore, under these conditions, HMGB1 does not contribute directly to dry eye-induced inflammation and its function at the ocular surface needs to be explored further.
| S-EPMC5886030 | biostudies-literature
Project description:The unicellular eukaryote Entamoeba histolytica is a human parasite that causes amebic dysentery and liver abscess. A genome-wide analysis of gene expression modulated by intestinal colonization and invasion identified an up-regulated transcript that encoded a putative high-mobility-group box (HMGB) protein, EhHMGB1. We tested if EhHMGB1 encoded a functional HMGB protein, and determined its role in control of parasite gene expression. Recombinant EhHMGB1 was able to bend DNA in vitro, a characteristic of HMGB proteins. Core conserved residues required for DNA bending activity in other HMGB proteins were demonstrated by mutational analysis to be essential for EhHMGB1 activity. EhHMGB1 was also able to enhance the binding of human p53 to its cognate DNA sequence in vitro, which is expected for an HMGB1 protein. Confocal microscopy, using antibodies against the recombinant protein, confirmed its nuclear localization. Overexpression of EhHMGB1 in HM1: IMSS trophozoites led to modulation of 33 transcripts involved in a variety of cellular functions. Of these, twenty were also modulated at either day one or day 29 in the mouse model of intestinal amebiasis. Notably, four transcripts with known roles in virulence, including two encoding Gal/GalNAc lectin light chains, were modulated in response to EhHMGB1 overexpression. We concluded that EhHMGB1 was a bona fide HMGB protein with the capacity to recapitulate part of the modulation of parasite gene expression seen during adaptation to the host intestine. 6 samples for two groups: 3M and Gir. Each groups contain 3 samples
2008-11-19 | E-GEOD-12204 | biostudies-arrayexpress
Project description:The intestinal microbe-derived metabolite trimethylamine N-oxide (TMAO) is implicated in the pathogenesis of cardiovascular diseases (CVDs). The molecular mechanisms of how TMAO induces atherosclerosis and CVDs' progression are still unclear. In this regard, high-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to disrupt cell-cell junctions, resulting in vascular endothelial hyper permeability leading to endothelial dysfunction. The present study tested whether TMAO associated endothelial dysfunction results via HMGB1 activation. Biochemical and RT-PCR analysis showed that TMAO increased the HMGB1 expression in a dose-dependent manner in endothelial cells. However, prior treatment with glycyrrhizin, an HMGB1 binder, abolished the TMAO-induced HMGB1 production in endothelial cells. Furthermore, Western blot and immunofluorescent analysis showed significant decrease in the expression of cell-cell junction proteins ZO-2, Occludin, and VE-cadherin in TMAO treated endothelial cells compared with control cells. However, prior treatment with glycyrrhizin attenuated the TMAO-induced cell-cell junction proteins' disruption. TMAO increased toll-like receptor 4 (TLR4) expression in endothelial cells. Inhibition of TLR4 expression by TLR4 siRNA protected the endothelial cells from TMAO associated tight junction protein disruption via HMGB1. In conclusion, our results demonstrate that HMGB1 is one of the important mediators of TMAO-induced endothelial dysfunction.
| S-EPMC6678463 | biostudies-literature
Project description:Thymocyte selection-associated high-mobility group box (TOX) is a DNA-binding factor that is able to regulate transcription by modifying local chromatin structure and modulating the formation of multi-protein complexes. TOX has multiple roles in the development of the adaptive immune system including development of CD4 T cells, NK cells and lymph node organogenesis. However very few antibodies recognizing this molecule have been reported and no extensive study of the expression of TOX in reactive and neoplastic lymphoid tissue has been performed to date. In the present study, we have investigated TOX expression in normal and neoplastic lymphoid tissues using a novel rat monoclonal antibody that recognizes its target molecule in paraffin-embedded tissue sections. A large series of normal tissues and B- and T-cell lymphomas was studied, using whole sections and tissue microarrays. We found that the majority of precursor B/T lymphoblastic, follicular and diffuse large B-cell lymphomas, nodular lymphocyte-predominant Hodgkin lymphomas and angioimmunoblastic T-cell lymphomas strongly expressed the TOX protein. Burkitt and mantle cell lymphomas showed TOX expression in a small percentage of cases. TOX was not found in the majority of chronic lymphocytic leukemia, myelomas, marginal zone lymphomas and classical Hodgkin lymphomas. In conclusion, we describe for the first time the expression of TOX in normal and neoplastic lymphoid tissues. The co-expression of TOX and PD-1 identified in normal and neoplastic T cells is consistent with recent studies identifying TOX as a critical regulator of T-cell exhaustion and a potential immunotherapy target. Its differential expression may be of diagnostic relevance in the differential diagnosis of follicular lymphoma, the identification of the phenotype of diffuse large B-cell lymphoma and the recognition of peripheral T-cell lymphoma with a follicular helper T phenotype.
| S-EPMC7046285 | biostudies-literature
Project description:UnlabelledHigh-mobility group box 1 (HMGB1) is an abundant chromatin-associated nuclear protein and released into the extracellular milieu during liver ischemia-reperfusion (I/R), signaling activation of proinflammatory cascades. Because the intracellular function of HMGB1 during sterile inflammation of I/R is currently unknown, we sought to determine the role of intracellular HMGB1 in hepatocytes after liver I/R. When hepatocyte-specific HMGB1 knockout (HMGB1-HC-KO) and control mice were subjected to a nonlethal warm liver I/R, it was found that HMGB1-HC-KO mice had significantly greater hepatocellular injury after I/R, compared to control mice. Additionally, there was significantly greater DNA damage and decreased chromatin accessibility to repair with lack of HMGB1. Furthermore, lack of hepatocyte HMGB1 led to excessive poly(ADP-ribose)polymerase 1 activation, exhausting nicotinamide adenine dinucleotide and adenosine triphosphate stores, exacerbating mitochondrial instability and damage, and, consequently, leading to increased cell death. We found that this was also associated with significantly more oxidative stress (OS) in HMGB1-HC-KO mice, compared to control. Increased nuclear instability led to a resultant increase in the release of histones with subsequently more inflammatory cytokine production and organ damage through activation of Toll-like receptor 9.ConclusionThe lack of HMGB1 within hepatocytes leads to increased susceptibility to cellular death after OS conditions.
| S-EPMC3999251 | biostudies-literature