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The Editors-in-Chief would like to alert readers that this article [1] is part of an investigation being conducted by the journal following the conclusions of an institutional enquiry at the University of Liverpool with respect to the quantitative mass spectrometry-generated results regarding acetylated and redox-modified HMGB1.
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The Editors-in-Chief would like to alert readers that this article (Sitapara et al. 2014) is part of an investigation being conducted by the journal following the conclusions of an institutional enquiry at the University of Liverpool with respect to the quantitative mass spectrometry-generated results regarding acetylated and redox-modified HMGB1.
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High-mobility group box-1 (HMGB1) is a damage-associated molecular pattern (DAMP) increased in response to liver injury. Because HMGB1 is a ligand for the receptor for advanced glycation endproducts (RAGE), we hypothesized that induction of HMGB1 could participate in the pathogenesis of liver fibrosis though RAGE cell-specific signaling mechanisms. Liver HMGB1 protein expression correlated with fibrosis stage in patients with chronic hepatitis C virus (HCV) infection, primary biliary cirrhosis (PBC), or alcoholic steatohepatitis (ASH). Hepatic HMGB1 protein expression and secretion increased in five mouse models of liver fibrosis attributed to drug-induced liver injury (DILI), cholestasis, ASH, or nonalcoholic steatohepatitis (NASH). HMGB1 was up-regulated and secreted mostly by hepatocytes and Kupffer cells (KCs) following CCl4 treatment. Neutralization of HMGB1 protected, whereas injection of recombinant HMGB1 promoted liver fibrosis. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep ) or in myeloid cells (Hmgb1ΔMye ) partially protected, whereas ablation in both (Hmgb1ΔHepΔMye ) prevented liver fibrosis in vivo. Coculture with hepatocytes or KCs from CCl4 -injected wild-type (WT) mice up-regulated Collagen type I production by hepatic stellate cells (HSCs); yet, coculture with hepatocytes from CCl4 -injected Hmgb1ΔHep or with KCs from CCl4 -injected Hmgb1ΔMye mice partially blunted this effect. Rage ablation in HSCs (RageΔHSC ) and RAGE neutralization prevented liver fibrosis. Last, we identified that HMGB1 stimulated HSC migration and signaled through RAGE to up-regulate Collagen type I expression by activating the phosphorylated mitogen-activated protein kinase kinase (pMEK)1/2, phosphorylated extracellular signal-regulated kinase (pERK)1/2 and pcJun signaling pathway. Conclusion: Hepatocyte and KC-derived HMGB1 participates in the pathogenesis of liver fibrosis by signaling through RAGE in HSCs to activate the pMEK1/2, pERK1/2 and pcJun pathway and increase Collagen type I deposition.
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Colágeno Tipo I/metabolismo , Proteína HMGB1/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/etiologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Intoxicação por Tetracloreto de Carbono/metabolismo , Estudos de Casos e Controles , Hepatócitos/metabolismo , Humanos , Células de Kupffer/metabolismo , Cirrose Hepática/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Células Mieloides/metabolismoRESUMO
OBJECTIVES: Inflammatory periosteal reaction (IPR) on the visceral surfaces of the ribs has been used in bioarchaeology as an indicator of lower respiratory tract disease. This article presents a detailed method for recording IPR on the ribs, even those in severely fragmented states, with the objectives of increasing the consistency of recording and producing true prevalence rates for skeletons so as to improve data comparability between future bioarchaeological studies of lower respiratory tract disease. MATERIALS AND METHODS: The presence and prevalence of respiratory-related IPR were recorded from three different Sudanese cemetery sites using a detailed method for identifying and recording IPR. Sites with variable preservation were chosen to test the applicability of the method. A flowchart to aid in identification of bony changes is presented. The method requires the recording of IPR on three separate sections of the rib (neck, angle, and shaft) and the allocation of ribs into rib cage regions of upper, upper-middle, middle, lower-middle, and lower. RESULTS: Results demonstrate differences in the distribution of IPR between sites and verify the method's applicability to archeological sites with various levels of skeletal preservation. DISCUSSION: While crude prevalence rates can indicate the number of individuals experiencing lower respiratory tract disease within a site, this method can provide information about the distribution of IPR within the rib cage. This should lead to new ways of distinguishing respiratory diseases within archeological populations. This method also allows for comparability between well-preserved and lesser-preserved sites by accommodating for rib fragmentation.
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Doenças Pleurais , Costelas/patologia , Adolescente , Adulto , Arqueologia , Cemitérios , História Antiga , História Medieval , Humanos , Paleopatologia , Doenças Pleurais/epidemiologia , Doenças Pleurais/história , Doenças Pleurais/patologia , Sudão , Adulto JovemRESUMO
The publisher regrets that this article has been temporarily removed. A replacement will appear as soon as possible in which the reason for the removal of the article will be specified, or the article will be reinstated. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.
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Sterile liver inflammation, such as liver ischemia-reperfusion, hemorrhagic shock after trauma, and drug-induced liver injury, is initiated and regulated by endogenous mediators including DNA and reactive oxygen species. Here, we identify a mechanism for redox-mediated regulation of absent in melanoma 2 (AIM2) inflammasome activation in hepatocytes after redox stress in mice, which occurs through interaction with cytosolic high mobility group box 1 (HMGB1). We show that in liver during hemorrhagic shock in mice and in hepatocytes after hypoxia with reoxygenation, cytosolic HMGB1 associates with AIM2 and is required for activation of caspase-1 in response to cytosolic DNA. Activation of caspase-1 through AIM2 leads to subsequent hepatoprotective responses such as autophagy. HMGB1 binds to AIM2 at a non-DNA-binding site on the hematopoietic interferon-inducible nuclear antigen domain of AIM2 to facilitate inflammasome and caspase-1 activation in hepatocytes. Furthermore, binding of HMGB1 to AIM2 is stronger with fully reduced all-thiol HMGB1 than with partially oxidized disulfide-HMGB1, and binding strength corresponds to caspase-1 activation. These data suggest that HMGB1 redox status regulates AIM2 inflammasome activation. CONCLUSION: These findings suggest a novel and important mechanism for regulation of AIM2 inflammasome activation in hepatocytes during redox stress and may suggest broader implications for how this and other inflammasomes are activated and how their activation is regulated during cell stress, as well as the mechanisms of inflammasome regulation in nonimmune cell types. (Hepatology 2017;65:253-268).
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Proteínas de Ligação a DNA/fisiologia , Hepatócitos/metabolismo , Inflamassomos/metabolismo , Hepatopatias/etiologia , Animais , Caspase 1/metabolismo , Proteína HMGB1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , OxirreduçãoRESUMO
Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).
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Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Testes de Toxicidade , Células Cultivadas/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes/metabolismo , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1-/- chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.
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Plaquetas/metabolismo , Proteína HMGB1/fisiologia , Trombose Venosa/genética , Animais , Plaquetas/patologia , Dissulfetos/química , Dissulfetos/metabolismo , Proteína HMGB1/química , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Trombose Venosa/metabolismo , Trombose Venosa/patologiaRESUMO
Approximately 30% of epilepsy patients experience seizures that are not controlled by the available drugs. Moreover, these drugs provide mainly a symptomatic treatment since they do not interfere with the disease's mechanisms. A mechanistic approach to the discovery of key pathogenic brain modifications causing seizure onset, recurrence and progression is instrumental for designing novel and rationale therapeutic interventions that could modify the disease course or prevent its development. In this regard, increasing evidence shows that neuroinflammation is a pathogenic factor in drug-resistant epilepsies. The High Mobility Group Box 1 (HMGB1)/Toll-like receptor 4 axis is a key initiator of neuroinflammation following brain injuries leading to epilepsy, and its activation contributes to seizure mechanisms in animal models. Recent findings have shown dynamic changes in HMGB1 and its isoforms in the brain and blood of animals exposed to acute brain injuries and undergoing epileptogenesis, and in surgically resected epileptic foci in humans. HMGB1 isoforms reflect different pathophysiological processes, and the disulfide isoform, which is generated in the brain during oxidative stress, is implicated in seizures, cell loss and cognitive dysfunctions. Interfering with disulfide HMGB1-activated cell signaling mediates significant therapeutic effects in epilepsy models. Moreover, both clinical and experimental data suggest that HMGB1 isoforms may serve as mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy. These novel findings suggest that the HMGB1 system could be targeted to prevent seizure generation and may provide clinically useful prognostic biomarkers which may also predict the patient's response to therapy.
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Epilepsia/patologia , Proteína HMGB1/metabolismo , Alarminas/metabolismo , Alarminas/fisiologia , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Disfunção Cognitiva/complicações , Modelos Animais de Doenças , Epilepsia/metabolismo , Proteína HMGB1/fisiologia , Humanos , Convulsões/etiologia , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismoRESUMO
Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of disulfide high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented disulfide HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.
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Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Epilepsia/tratamento farmacológico , Domínios HMG-Box/efeitos dos fármacos , Proteína HMGB1/sangue , Proteína HMGB1/metabolismo , Isotiocianatos/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Disfunção Cognitiva/complicações , Disfunção Cognitiva/tratamento farmacológico , Modelos Animais de Doenças , Quimioterapia Combinada , Epilepsia/metabolismo , Proteína HMGB1/biossíntese , Hipocampo/metabolismo , Isotiocianatos/farmacologia , Masculino , Degeneração Neural/dietoterapia , Neurônios/metabolismo , Ratos , SulfóxidosRESUMO
PURPOSE: Paracetamol is one of the world's most commonly used drugs. In overdose, it is well established to be hepatotoxic. The aim of this review was to identify factors that have been, or actually are, associated with the development of liver injury after paracetamol exposure in humans. METHOD: Google Scholar and PubMed were searched on various dates between December 2016 and March 2017. Papers identified had their references analysed for further studies that might be relevant. RESULTS: At the time of writing, there was little good quality clinical evidence-from studies of paracetamol overdose or therapeutic use-to suggest that any groups of people are relatively protected from, or are at greater risk of, liver injury. The factors that were historically used to indicate higher risk in the UK have no good quality clinical evidence to support their re-introduction into clinical practice. The safe (and still effective) oral dose of paracetamol in patients weighing less than 50 kg has not been established. CONCLUSION: There is no patient group that is unequivocally at elevated risk of paracetamol-induced liver toxicity. We propose two clinical scenarios that warrant further research. Firstly, there is a need to establish whether the dose of paracetamol should be reduced in patients with low body weight. Secondly, if or when genomic information regarding individual patients becomes readily available to inform prescribing, we propose the contribution of the genome to paracetamol toxicity should be re-investigated with robustly designed studies. Such studies could enhance the safe use of one of the most frequently taken drugs.
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Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Analgésicos não Narcóticos/efeitos adversos , Humanos , Fatores de RiscoRESUMO
The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1ß, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1ß, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.
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Proteína HMGB1/metabolismo , Inflamassomos/metabolismo , eIF-2 Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Antígenos de Bactérias/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/farmacologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Cristalinas/metabolismo , Escherichia coli/imunologia , Escherichia coli/fisiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Feminino , Proteína HMGB1/sangue , Humanos , Inflamassomos/agonistas , Interleucina-18/sangue , Interleucina-1beta/sangue , Interleucina-6/análise , Interleucina-6/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas NLR , Peritonite/metabolismo , Fosforilação , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/farmacologia , Rotenona/farmacologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Salmonella typhimurium/fisiologia , Transfecção , Ácido Úrico/farmacologia , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/deficiência , eIF-2 Quinase/genéticaRESUMO
OBJECTIVE: Liver fibrosis is associated with significant collagen-I deposition largely produced by activated hepatic stellate cells (HSCs); yet, the link between hepatocyte damage and the HSC profibrogenic response remains unclear. Here we show significant induction of osteopontin (OPN) and high-mobility group box-1 (HMGB1) in liver fibrosis. Since OPN was identified as upstream of HMGB1, we hypothesised that OPN could participate in the pathogenesis of liver fibrosis by increasing HMGB1 to upregulate collagen-I expression. DESIGN AND RESULTS: Patients with long-term hepatitis C virus (HCV) progressing in disease stage displayed enhanced hepatic OPN and HMGB1 immunostaining, which correlated with fibrosis stage, whereas it remained similar in non-progressors. Hepatocyte cytoplasmic OPN and HMGB1 expression was significant while loss of nuclear HMGB1 occurred in patients with HCV-induced fibrosis compared with healthy explants. Well-established liver fibrosis along with marked induction of HMGB1 occurred in CCl4-injected OpnHep transgenic yet it was less in wild type and almost absent in Opn-/- mice. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep) protected mice from CCl4-induced liver fibrosis. Coculture with hepatocytes that secrete OPN plus HMGB1 and challenge with recombinant OPN (rOPN) or HMGB1 (rHMGB1) enhanced collagen-I expression in HSCs, which was blunted by neutralising antibodies (Abs) and by Opn or Hmgb1 ablation. rOPN induced acetylation of HMGB1 in HSCs due to increased NADPH oxidase activity and the associated decrease in histone deacetylases 1/2 leading to upregulation of collagen-I. Last, rHMGB1 signalled via receptor for advanced glycation end-products and activated the PI3K-pAkt1/2/3 pathway to upregulate collagen-I. CONCLUSIONS: During liver fibrosis, the increase in OPN induces HMGB1, which acts as a downstream alarmin driving collagen-I synthesis in HSCs.
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Colágeno Tipo I/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Cirrose Hepática/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Acetilação/efeitos dos fármacos , Animais , Anticorpos Neutralizantes , Tetracloreto de Carbono , Estudos de Casos e Controles , Núcleo Celular/química , Células Cultivadas , Citoplasma/química , Progressão da Doença , Expressão Gênica , Proteína HMGB1/análise , Células Estreladas do Fígado/metabolismo , Hepatite C Crônica/complicações , Hepatócitos/química , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , NADPH Oxidases/metabolismo , Osteopontina/análise , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
Infection of macrophages by bacterial pathogens can trigger Toll-like receptor (TLR) activation as well as Nod-like receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase-1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming-free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damage-associated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re-introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4-agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system.
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Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 1/metabolismo , Proteína HMGB1/metabolismo , Macrófagos/imunologia , Proteína Inibidora de Apoptose Neuronal/metabolismo , Receptores Toll-Like/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Apoptose , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao Cálcio/agonistas , Proteínas de Ligação ao Cálcio/imunologia , Morte Celular , Linhagem Celular , Expressão Gênica , Proteína HMGB1/análise , Interações Hospedeiro-Patógeno , Inflamassomos/imunologia , Inflamassomos/metabolismo , Ativação de Macrófagos/fisiologia , Macrófagos/microbiologia , Macrófagos/fisiologia , Camundongos , Dados de Sequência Molecular , Proteína Inibidora de Apoptose Neuronal/agonistas , Proteína Inibidora de Apoptose Neuronal/imunologia , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Receptores Toll-Like/imunologiaRESUMO
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).
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Anticorpos Neutralizantes/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Proteína HMGB1/uso terapêutico , Inflamação/tratamento farmacológico , Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Animais , Antipiréticos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The prediction and understanding of acetaminophen (APAP)-induced liver injury (APAP-ILI) and the response to therapeutic interventions is complex. This is due in part to sensitivity and specificity limitations of currently used assessment techniques. Here we sought to determine the utility of integrating translational non-invasive photoacoustic imaging of liver function with mechanistic circulating biomarkers of hepatotoxicity with histological assessment to facilitate the more accurate and precise characterization of APAP-ILI and the efficacy of therapeutic intervention. Perturbation of liver function and cellular viability was assessed in C57BL/6J male mice by Indocyanine green (ICG) clearance (Multispectral Optoacoustic Tomography (MSOT)) and by measurement of mechanistic (miR-122, HMGB1) and established (ALT, bilirubin) circulating biomarkers in response to the acetaminophen and its treatment with acetylcysteine (NAC) in vivo. We utilised a 60% partial hepatectomy model as a situation of defined hepatic functional mass loss to compared acetaminophen-induced changes to. Integration of these mechanistic markers correlated with histological features of APAP hepatotoxicity in a time-dependent manner. They accurately reflected the onset and recovery from hepatotoxicity compared to traditional biomarkers and also reported the efficacy of NAC with high sensitivity. ICG clearance kinetics correlated with histological scores for acute liver damage for APAP (i.e. 3h timepoint; r=0.90, P<0.0001) and elevations in both of the mechanistic biomarkers, miR-122 (e.g. 6h timepoint; r=0.70, P=0.005) and HMGB1 (e.g. 6h timepoint; r=0.56, P=0.04). For the first time we report the utility of this non-invasive longitudinal imaging approach to provide direct visualisation of the liver function coupled with mechanistic biomarkers, in the same animal, allowing the investigation of the toxicological and pharmacological aspects of APAP-ILI and hepatic regeneration.
Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico por imagem , Fígado/efeitos dos fármacos , Técnicas Fotoacústicas , Acetilcisteína/administração & dosagem , Alanina Transaminase/sangue , Animais , Bilirrubina/sangue , Biomarcadores/sangue , Sobrevivência Celular/efeitos dos fármacos , Glutationa/sangue , Proteína HMGB1/sangue , Fígado/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/sangueRESUMO
BACKGROUND & AIMS: Hypoxic hepatitis is a clinical condition precipitated by prolonged periods of oxygen deprivation to the liver. It can have several underlying causes. Despite its prevalence in critically ill patients, which can reach upwards of 10%, very little is known about the mechanisms of injury. Thus, we set out to measure previously identified circulating biomarkers in an attempt to describe mechanisms of injury following hypoxic hepatitis. METHODS: Plasma from patients diagnosed with hypoxic hepatitis was collected for this study. Biomarkers of hepatocellular injury, mitochondrial damage and cell death were measured. These results were compared against results obtained from well-characterized acetaminophen overdose patients. RESULTS: At peak injury, ALT measured 4082±606 U/L and gradually decreased over 5 days, corresponding to the clinically observed pattern of hypoxic hepatitis. Levels of GDH showed a similar pattern, but neither ALT nor GDH were significantly higher in these patients than in acetaminophen patients. Plasma levels of DNA fragments mimicked hepatocellular injury as measured by ALT and miRNA-122. Interestingly, we found a significant increase in caspase-cleaved cytokeratin-18; however, the full-length form greatly exceeded the cleaved form at the time of maximum injury (45837±12085 vs 2528±1074 U/L). We also found an increase in acHMGB1 at later time points indicating a possible role of inflammation, but cytokine levels at these times were actually decreased relative to early time points. CONCLUSIONS: The mechanism of injury following hypoxic hepatitis involves mitochondrial damage and DNA fragmentation. Importantly, necrosis, rather than apoptosis, is the main mode of cell death.
Assuntos
Hepatite/sangue , Hipóxia/sangue , Isquemia/sangue , Fígado/fisiopatologia , Acetaminofen/toxicidade , Adolescente , Adulto , Alanina Transaminase/sangue , Apoptose , Biomarcadores/sangue , Fragmentação do DNA , DNA Mitocondrial/sangue , Feminino , Proteína HMGB1/sangue , Humanos , Isquemia/etiologia , Queratina-18/sangue , Modelos Lineares , Fígado/irrigação sanguínea , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Mitocôndrias/patologia , Necrose/etiologia , Estados Unidos , Adulto JovemRESUMO
CONTEXT: There is an ongoing search for specific and translational biomarkers of drug-induced liver injury (DILI). MicroRNA-122 (miR-122) has previously shown potential as a sensitive, specific, and translational biomarker of DILI in both rodent, and human studies. OBJECTIVE: To build on previous work within the field, we examined biomarker kinetics in a rat model of acetaminophen (APAP)-induced liver injury to confirm the sensitivity, and specificity of miR-122 and glutamate dehydrogenase (GLDH). MATERIALS AND METHODS: qRT-PCR and a standard enzymatic assay were used for biomarker analysis. RESULTS: Both miR-122 and GLDH were demonstrated to be more readily-detectable biomarkers of APAP-DILI than alanine aminotransferase (ALT). Peak levels for all biomarkers were detected at 2 days after APAP. At day 3, miR-122 had returned to baseline; however, other biomarkers remained elevated between 3 and 4 days. We were also able to demonstrate that, although miR-122 is present in greater quantities in exosome-free form, both exosome-bound and non-vesicle bound miR-122 are released in a similar profile throughout the course of DILI. DISCUSSION AND CONCLUSIONS: Together, this study demonstrates that both GLDH and miR-122 could be used during preclinical drug-development as complementary biomarkers to ALT to increase the chance of early detection of hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Acetaminofen , Alanina Transaminase , Animais , Biomarcadores/sangue , Diagnóstico Precoce , Glutamato Desidrogenase/sangue , MicroRNAs/sangue , Farmacocinética , Ratos , Sensibilidade e EspecificidadeRESUMO
AIMS: To determine whether a correlation exists between paired cerebrospinal fluid (CSF) and serum levels of a novel inflammatory biomarker, high-mobility group box 1 (HMGB1), in different neurological conditions. METHODS: HMGB1 was measured in the serum and CSF of 46 neurological patients (18 idiopathic intracranial hypertension [IIH], 18 neurological infection/inflammation [NII] and 10 Rasmussen's encephalitis [RE]). RESULTS: Mean serum (± SD) HMGB1 levels were 1.43 ± 0.54, 25.28 ± 27.9 and 1.89 ± 1.49 ng/ml for the patients with IIH, NII and RE, respectively. Corresponding mean (± SD) CSF levels were 0.35 ± 0.22, 4.48 ± 6.56 and 2.24 ± 2.35 ng/ml. Both CSF and serum HMGB1 was elevated in NII. Elevated CSF HMGB1 was demonstrated in RE. There was no direct correlation between CSF and serum levels of HMGB1. CONCLUSION: Serum HMGB1 cannot be used as a surrogate measure for CSF levels. CSF HMGB1 was elevated in NII and RE, its role as a prognostic/stratification biomarker needs further study.