A Proline-Rich Structural Protein of the Surface Sheath of Larval Brugia Filarial Nematode Parasites

Both cDNA and genomic DNA sequences have been isolated which encode a proline-rich precursor protein of the sheath from microfilariae, the first stage larvae of the filarial nematode parasites Brugia pahangi andBrugia malayi. This 22-kDa protein is soluble only under reducing conditions and is extensively cross- linked by both disulfide and nonreducible bonds. Immunogold electron microscopy shows that the protein is localized exclusively in the sheath, a vestigial remnant of the eggshell, which is retained by and encloses the mature microfilaria. Analysis by Western blotting confirms that the protein is expressed only in microfilariae and adult female worms, although transcripts are detectable only in adult females. The deduced amino acid sequence contains a short N-terminal hydrophobic putative leader sequence, a central repetitive domain that contains 14 copies of a degenerate 5-amino acid repeat with the consensus sequence Met- Pro-Pro-Gln-Gly, and a C-terminal prolinerich do- main flanked by clusters of cysteine residues. These clusters can be aligned with cysteine residues implicated in cross-linking of a family of cuticular collagens originally identified in Caenorhabditiselegans but which extends to other nematodes

Absolute Quantitation of GTPase Protein Abundance.

Ras proteins and other small molecular weight GTPases are molecular switches controlling a wide range of cellular functions. High homology and functional redundancy between closely related family members are commonly observed. Antibody-based methods are commonly used to characterize their protein expression. However, these approaches are typically semi-quantitative, and the requirement to use different antibodies means that this strategy is not suited for comparative analysis of the relative expression of proteins expressed by different genes. We present a mass spectrometry-based method that precisely quantifies the protein copy number per cell of a protein of interest. We provide detailed protocols for the generation of isotopically labeled protein standards, cell/tissue processing, mass-spectrometry optimization, and subsequent utilization for the absolute quantitation of the abundance of a protein of interest. As examples, we provide instructions for the quantification of HRAS, KRAS4A, KRAS4B, NRAS, RALA, and RALB in cell line and tissue-derived samples

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

The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis

In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high–mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow–derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis

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

Background & AimsIn 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.MethodsPigs 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.ResultsThe 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.ConclusionsThe survival benefit and lack of adverse effects would support clinical trials of University College London-Liver Dialysis Device in acute liver failure patients

Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

Approximately 30% of epilepsy patients do not respond to antiepileptic drugs, representing an unmet medical need. There is evidence that neuroinflammation plays a pathogenic role in drug-resistant epilepsy. The high-mobility group box 1 (HMGB1)/TLR4 axis is a key initiator of neuroinflammation following epileptogenic injuries, and its activation contributes to seizure generation in animal models. However, further work is required to understand the role of HMGB1 and its isoforms in epileptogenesis and drug resistance. Using a combination of animal models and sera from clinically well-characterized patients, we have demonstrated that there are dynamic changes in HMGB1 isoforms in the brain and blood of animals undergoing epileptogenesis. The pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset and prospectively identified animals that developed the disease. Consistent with animal data, we observed early expression of disulfide HMGB1 in patients with newly diagnosed epilepsy, and its persistence was associated with subsequent seizures. In contrast with patients with well-controlled epilepsy, patients with chronic, drug-refractory epilepsy persistently expressed the acetylated, disulfide HMGB1 isoforms. Moreover, treatment of animals with antiinflammatory drugs during epileptogenesis prevented both disease progression and blood increase in HMGB1 isoforms. Our data suggest that HMGB1 isoforms are mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy in humans, necessitating evaluation in larger-scale prospective studies

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) <i>in vitro</i>, 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 <i>in vitro</i>, 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

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI

Assessing technical and biological variation in SWATH-MS-based proteomic analysis of chronic lymphocytic leukaemia cells.

Chronic lymphocytic leukaemia (CLL) exhibits variable clinical course and response to therapy, but the molecular basis of this variability remains incompletely understood. Data independent acquisition (DIA)-MS technologies, such as SWATH (Sequential Windowed Acquisition of all THeoretical fragments), provide an opportunity to study the pathophysiology of CLL at the proteome level. Here, a CLL-specific spectral library (7736 proteins) is described alongside an analysis of sample replication and data handling requirements for quantitative SWATH-MS analysis of clinical samples. The analysis was performed on 6 CLL samples, incorporating biological (IGHV mutational status), sample preparation and MS technical replicates. Quantitative information was obtained for 5169 proteins across 54 SWATH-MS acquisitions: the sources of variation and different computational approaches for batch correction were assessed. Functional enrichment analysis of proteins associated with IGHV mutational status showed significant overlap with previous studies based on gene expression profiling. Finally, an approach to perform statistical power analysis in proteomics studies was implemented. This study provides a valuable resource for researchers working on the proteomics of CLL. It also establishes a sound framework for the design of sufficiently powered clinical proteomics studies. Indeed, this study shows that it is possible to derive biologically plausible hypotheses from a relatively small dataset

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 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 50-100 nm and expressed enriched CD63. LC-MS/MS identified 2109 proteins, with 608 proteins being quantified across all exosome samples. Data are available via 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 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 via phagocytosis, and was inhibited by latrunculin A. An, amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SOX30 activated naïve T-cells from HLA-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 provides a pathway for the induction of drug hapten-specific T-cell responses. This article is protected by copyright. All rights reserved