Multi-Omic Temporal Landscape of Plasma and Synovial Fluid-Derived Extracellular Vesicles Using an Experimental Model of Equine Osteoarthritis.

Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active ß1-integrin in adult tendon cells.

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal-regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active ß1-integrin-containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp-mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5ß1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.

Proteomic profiling of murine biliary-derived hepatic organoids and their capacity for drug disposition, bioactivation and detoxification.

Hepatic organoids are a recent innovation in in vitro modeling. Initial studies suggest that organoids better recapitulate the liver phenotype in vitro compared to pre-existing proliferative cell models. However, their potential for drug metabolism and detoxification remains poorly characterized, and their global proteome has yet to be compared to their tissue of origin. This analysis is urgently needed to determine what gain-of-function this new model may represent for modeling the physiological and toxicological response of the liver to xenobiotics. Global proteomic profiling of undifferentiated and differentiated hepatic murine organoids and donor-matched livers was, therefore, performed to assess both their similarity to liver tissue, and the expression of drug-metabolizing enzymes and transporters. This analysis quantified 4405 proteins across all sample types. Data are available via ProteomeXchange (PXD017986). Differentiation of organoids significantly increased the expression of multiple cytochrome P450, phase II enzymes, liver biomarkers and hepatic transporters. While the final phenotype of differentiated organoids is distinct from liver tissue, the organoids contain multiple drug metabolizing and transporter proteins necessary for liver function and drug metabolism, such as cytochrome P450 3A, glutathione-S-transferase alpha and multidrug resistance protein 1A. Indeed, the differentiated organoids were shown to exhibit increased sensitivity to midazolam (10-1000 µM) and irinotecan (1-100 µM), when compared to the undifferentiated organoids. The predicted reduced activity of HNF4A and a resulting dysregulation of RNA polymerase II may explain the partial differentiation of the organoids. Although further experimentation, optimization and characterization is needed relative to pre-existing models to fully contextualize their use as an in vitro model of drug-induced liver injury, hepatic organoids represent an attractive novel model of the response of the liver to xenobiotics. The current study also highlights the utility of global proteomic analyses for rapid and accurate evaluation of organoid-based test systems.

The application of cytokeratin-18 as a biomarker for drug-induced liver injury.

Drug-induced liver injury (DILI) is a frequent and dangerous adverse effect faced during preclinical and clinical drug therapy. DILI is a leading cause of candidate drug attrition, withdrawal and in clinic, is the primary cause of acute liver failure. Traditional diagnostic markers for DILI include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Yet, these routinely used diagnostic markers have several noteworthy limitations, restricting their sensitivity, specificity and accuracy in diagnosing DILI. Consequently, new biomarkers for DILI need to be identified.A potential biomarker for DILI is cytokeratin-18 (CK18), an intermediate filament protein highly abundant in hepatocytes and cholangiocytes. Extensively researched in a variety of clinical settings, both full length and cleaved forms of CK18 can diagnose early-stage DILI and provide insight into the mechanism of hepatocellular injury compared to traditionally used diagnostic markers. However, relatively little research has been conducted on CK18 in preclinical models of DILI. In particular, CK18 and its relationship with DILI is yet to be characterised in an in vivo rat model. Such characterization of CK18 and ccCK18 responses may enable their use as translational biomarkers for hepatotoxicity and facilitate management of clinical DILI risk in drug development. The aim of this review is to discuss the application of CK18 as a biomarker for DILI. Specifically, this review will highlight the properties of CK18, summarise clinical research that utilised CK18 to diagnose DILI and examine the current challenges preventing the characterisation of CK18 in an in vivo rat model of DILI.

Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis.

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV-visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

Exosomal Transport of Hepatocyte-Derived Drug-Modified Proteins to the Immune System.

Idiosyncratic drug-induced liver injury (DILI) is a rare, often difficult-to-predict adverse reaction with complex pathomechanisms. However, it is now evident that certain forms of DILI are immune-mediated and may involve the activation of drug-specific T cells. Exosomes are cell-derived vesicles that carry RNA, lipids, and protein cargo from their cell of origin to distant cells, and they may play a role in immune activation. Herein, primary human hepatocytes were treated with drugs associated with a high incidence of DILI (flucloxacillin, amoxicillin, isoniazid, and nitroso-sulfamethoxazole) to characterize the proteins packaged within exosomes that are subsequently transported to dendritic cells for processing. Exosomes measured between 50 and 100 nm and expressed enriched CD63. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) identified 2,109 proteins, with 608 proteins being quantified across all exosome samples. Data are available through ProteomeXchange with identifier PXD010760. Analysis of gene ontologies revealed that exosomes mirrored whole human liver tissue in terms of the families of proteins present, regardless of drug treatment. However, exosomes from nitroso-sulfamethoxazole-treated hepatocytes selectively packaged a specific subset of proteins. LC/MS-MS also revealed the presence of hepatocyte-derived exosomal proteins covalently modified with amoxicillin, flucloxacillin, and nitroso-sulfamethoxazole. Uptake of exosomes by monocyte-derived dendritic cells occurred silently, mainly through phagocytosis, and was inhibited by latrunculin A. An amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SRY (sex determining region Y)-box 30 activated naïve T cells from human leukocyte antigen A*02:01-positive human donors. Conclusion: This study shows that exosomes have the potential to transmit drug-specific hepatocyte-derived signals to the immune system and provide a pathway for the induction of drug hapten-specific T-cell responses.

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) modifies energy metabolism via 5' AMP-activated protein kinase signalling in malignant cells.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an adverse biomarker across many malignancies. Using K562 cells engineered to have high or low CIP2A expression, we show that high CIP2A levels significantly bias cellular energy production towards oxidative phosphorylation (OXPHOS) rather than glycolysis. Mass spectrometric analysis of CIP2A interactors and isobaric tagging for relative and absolute protein quantitation (ITRAQ) experiments identified many associated proteins, several of which co-vary with CIP2A level. Many of these CIP2A associating and co-varying proteins are involved in energy metabolism including OXPHOS, or in 5' AMP-activated protein kinase (AMPK) signalling, and manipulating AMPK activity mimics the effects of low/high CIP2A on OXPHOS. These effects are dependent on the availability of nutrients, driven by metabolic changes caused by CIP2A. CIP2A level did not affect starvation-induced AMPK phosphorylation of Unc-51 autophagy activating kinase 1 (ULK-1) at Ser555, but autophagy activity correlated with an increase in AMPK activity, to suggest that some AMPK processes are uncoupled by CIP2A, likely via its inhibition of protein phosphatase 2A (PP2A). The data demonstrate that AMPK mediates this novel CIP2A effect on energy generation in malignant cells.

Definition of the Nature and Hapten Threshold of the ß-Lactam Antigen Required for T Cell Activation In Vitro and in Patients.

Covalent modification of protein by drugs may disrupt self-tolerance, leading to lymphocyte activation. Until now, determination of the threshold required for this process has not been possible. Therefore, we performed quantitative mass spectrometric analyses to define the epitopes formed in tolerant and hypersensitive patients taking the ß-lactam antibiotic piperacillin and the threshold required for T cell activation. A hydrolyzed piperacillin hapten was detected on four lysine residues of human serum albumin (HSA) isolated from tolerant patients. The level of modified Lys541 ranged from 2.6 to 4.8%. Analysis of plasma from hypersensitive patients revealed the same pattern and levels of modification 1-10 d after the commencement of therapy. Piperacillin-responsive skin-homing CD4+ clones expressing an array of Vß receptors were activated in a dose-, time-, and processing-dependent manner; analysis of incubation medium revealed that 2.6% of Lys541 in HSA was modified when T cells were activated. Piperacillin-HSA conjugates that had levels and epitopes identical to those detected in patients were shown to selectively stimulate additional CD4+ clones, which expressed a more restricted Vß repertoire. To conclude, the levels of piperacillin-HSA modification that activated T cells are equivalent to the ones formed in hypersensitive and tolerant patients, which indicates that threshold levels of drug Ag are formed in all patients. Thus, the propensity to develop hypersensitivity is dependent on other factors, such as the presence of T cells within an individual's repertoire that can be activated with the ß-lactam hapten and/or an imbalance in immune regulation.

Assessment of Antipiperacillin IgG Binding to Structurally Related Drug Protein Adducts.

The risk of developing hypersensitivity to alternative antibiotics is a concern for penicillin hypersensitive patients and healthcare providers. Herein we use piperacillin hypersensitivity as a model to explore the reactivity of drug-specific IgG against alternative ß-lactam protein adducts. Mass spectrometry was used to show the drugs (amoxicillin, flucloxacillin, benzyl penicillin, aztreonam, and piperacillin) bind to similar lysine residues on the protein carrier bovine serum albumin. However, the hapten-specific IgG antibodies found in piperacillin hypersensitive patient plasma did not bind to other ß-lactam protein conjugates. These data outline the fine specificity of piperacillin-specific IgG antibodies that circulate in patients with hypersensitivity.

Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from Epoxide Metabolites of Carbamazepine in Patients.

Carbamazepine (CBZ) is an effective antiepileptic drug that has been associated with hypersensitivity reactions. The pathogenesis of those reactions is incompletely understood but is postulated to involve a complex interplay between the drug's metabolism, genetic variation in human leukocyte antigens, and adverse activation of the immune system. Multiple T-cell activation mechanisms have been hypothesized including activation by drug-peptide conjugates derived from proteins haptenated by reactive metabolites. However, definitive evidence of the drug-protein adducts in patients has been lacking. In this study, mass spectrometry was used to characterize protein modifications by microsomally-generated metabolites of CBZ and in patients taking CBZ therapy. CBZ 10,11-epoxide (CBZE), a major electrophilic plasma metabolite of CBZ, formed adducts with glutathione-S-transferase pi (GSTP; Cys47) and human serum albumin (HSA; His146 and His338, but not Cys34) in vitro via notably divergent side-chain selectivity. Both proteins were adducted at the same residues by undefined monoxygenated metabolites ([O]CBZ) when they were incubated with human liver microsomes, NADPH and CBZ. There was also evidence for formation of a CBZ adduct at His146 and His338 of HSA derived via dehydration from an intermediate arene oxide adduct. Glutathione trapping of reactive metabolites confirmed microsomal production of CBZE and indicated simultaneous production of arene oxides. In 15 patients prescribed CBZ therapy, [O]CBZ-modified HSA (His146) was detected in all subjects. The relative amount of adduct was moderately positively correlated with plasma concentrations of CBZ (r2 = 0.44, p = 0.002) and CBZE (r2 = 0.35, p = 0.006). Our results have provided the first chemical evidence for microsomal production of [O]CBZ species that are able to escape the microsomal domain to react covalently with soluble proteins. This study has also demonstrated the presence of circulating [O]CBZ-modified HSA in patients without hypersensitivity reactions who were receiving standard CBZ therapy. The implications of those circulating adducts for susceptibility to CBZ hypersensitivity merit further immunological investigation in hypersensitive patients.

Plasma biomarkers to study mechanisms of liver injury in patients with hypoxic hepatitis.

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.

Total proteome analysis identifies migration defects as a major pathogenetic factor in immunoglobulin heavy chain variable region (IGHV)-unmutated chronic lymphocytic leukemia.

The mutational status of the immunoglobulin heavy chain variable region defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and unmutated (UM-CLL). To elucidate the molecular mechanisms underlying the adverse clinical outcome associated with UM-CLL, total proteomes from nine UM-CLL and nine M-CLL samples were analyzed by isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry. Based on the expression of 3521 identified proteins, principal component analysis separated CLL samples into two groups corresponding to immunoglobulin heavy chain variable region mutational status. Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells underexpressed proteins associated with cytoskeletal remodeling and overexpressed proteins associated with transcriptional and translational activity. Taken together, our findings indicate that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes, where they are exposed to proliferative stimuli. In keeping with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed a strong and specific association between UM-CLL and lymphadenopathy. Our study illustrates the potential of total proteome analysis to elucidate pathogenetic mechanisms in cancer.

Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients.

Amoxicillin-clavulanate (AC) is one of the most common causes of drug induced liver injury (DILI). The association between AC-DILI and HLA alleles and the detection of drug-specific T cells in patients with AC-DILI indicate that the adaptive immune system is involved in the disease pathogenesis. In this study, mass spectrometric methods were employed to characterize the antigen formed by AC in exposed patients and the antigenic determinants that stimulate T cells. Amoxicillin formed penicilloyl adducts with lysine residues on human serum albumin (HSA) in vitro, with K190 and K199 being the most reactive sites. Amoxicillin-modified K190 and K199 have also been detected in all patients, and more extensive modification was observed in patients exposed to higher doses of amoxicillin. In contrast, the binding of clavulanic acid to HSA was more complicated. Multiple adducts were identified at high concentrations in vitro, including those formed by direct binding of clavulanic acid to lysine residues, novel pyrazine adducts derived from binding to the degradation products of clavulanic acid, and a cross-linking adduct. Stable adducts derived from formylacetic acid were detected in all patients exposed to the drug. Importantly, analysis of hapten-protein adducts formed in the cell culture medium revealed that the highly drug-specific T-cell responses were likely driven by the markedly different haptenic structures formed by these two drugs. In this study, the unique haptenic structures on albumin in patients formed by amoxicillin and clavulanic acid have been characterized and shown to function as chemically distinct antigens which can stimulate separate, specific T-cell clones.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney.

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 h, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared with those of wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared with those of vehicle control. In the light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis, and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.

Extracorporeal liver assist device to exchange albumin and remove endotoxin in acute liver failure: Results of a pivotal pre-clinical study.

BACKGROUND & AIMS: In acute liver failure, severity of liver injury and clinical progression of disease are in part consequent upon activation of the innate immune system. Endotoxaemia contributes to innate immune system activation and the detoxifying function of albumin, critical to recovery from liver injury, is irreversibly destroyed in acute liver failure. University College London-Liver Dialysis Device is a novel artificial extracorporeal liver assist device, which is used with albumin infusion, to achieve removal and replacement of dysfunctional albumin and reduction in endotoxaemia. We aimed to test the effect of this device on survival in a pig model of acetaminophen-induced acute liver failure. METHODS: Pigs were randomised to three groups: Acetaminophen plus University College London-Liver Dialysis Device (n=9); Acetaminophen plus Control Device (n=7); and Control plus Control Device (n=4). Device treatment was initiated two h after onset of irreversible acute liver failure. RESULTS: The Liver Dialysis Device resulted in 67% reduced risk of death in acetaminophen-induced acute liver failure compared to Control Device (hazard ratio=0.33, p=0.0439). This was associated with 27% decrease in circulating irreversibly oxidised human non-mercaptalbumin-2 throughout treatment (p=0.046); 54% reduction in overall severity of endotoxaemia (p=0.024); delay in development of vasoplegia and acute lung injury; and delay in systemic activation of the TLR4 signalling pathway. Liver Dialysis Device-associated adverse clinical effects were not seen. CONCLUSIONS: The survival benefit and lack of adverse effects would support clinical trials of University College London-Liver Dialysis Device in acute liver failure patients.

Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis.

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models.

Characterization of Peroxidases Expressed in Human Antigen Presenting Cells and Analysis of the Covalent Binding of Nitroso Sulfamethoxazole to Myeloperoxidase.

Drug hypersensitivity remains a major concern, as it causes high morbidity and mortality. Understanding the mechanistic basis of drug hypersensitivity is complicated by the multiple risk factors implicated. This study utilized sulfamethoxazole (SMX) as a model drug to (1) relate SMX metabolism in antigen presenting cells (APCs) to the activation of T-cells and (2) characterize covalent adducts of SMX and myeloperoxidase, which might represent antigenic determinants for T-cells. The SMX metabolite nitroso-SMX (SMX-NO) was found to bind irreversibly to APCs. Time- and concentration-dependent drug-protein adducts were also detected when APCs were cultured with SMX. Metabolic activation of SMX was significantly reduced by the oxygenase/peroxidase inhibitor methimazole. Similarly, SMX-NO-specific T-cells were activated by APCs pulsed with SMX, and the response was inhibited by pretreatment with methimazole or glutaraldehyde, which blocks antigen processing. Western blotting, real-time polymerase chain reaction (RT-PCR), and mass spectrometry analyses suggested the presence of low concentrations of myeloperoxidase in APCs. RT-PCR revealed mRNA expression for flavin-containing monooxygenases (FMO1-5), thyroid peroxidase, and lactoperoxidase, but the corresponding proteins were not detected. Mass spectrometric characterization of SMX-NO-modified myeloperoxidase revealed the formation of N-hydroxysulfinamide adducts on Cys309 and Cys398. These data show that SMX's metabolism in APCs generates antigenic determinants for T-cells. Peptides derived from SMX-NO-modified myeloperoxidase may represent one form of functional antigen.