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Refereed/Peer-reviewed Background and purpose: The aim was to evaluate urinary neopterin, a marker of pro-inflammatory state, as a potential biomarker of disease prognosis and progression in amyotrophic lateral sclerosis (ALS); and to compare its utility to urinary neurotrophin receptor p75 extracellular domain (p75ECD). Methods: This was an observational study including 21 healthy controls and 46 people with ALS, 29 of whom were sampled longitudinally. Neopterin and p75ECD were measured using enzyme-linked immunoassays. Baseline and longitudinal changes in clinical measures, neopterin and urinary p75ECD were examined, and prognostic utility was explored by survival analysis. ResultsAt baseline, urinary neopterin was higher in ALS compared to controls (181.7 ± 78.9 μmol/mol creatinine vs. 120.4 ± 60.8 μmol/mol creatinine, p = 0.002, Welch's t test) and correlated with the Revised ALS Functional Rating Scale (r = −0.36, p = 0.01). Combining previously published urinary p75ECD results from 22 ALS patients with a further 24 ALS patients, baseline urinary p75ECD was also higher compared to healthy controls (6.0 ± 2.7 vs. 3.2 ± 1.0 ng/mg creatinine, p < 0.0001) and correlated with the Revised ALS Functional Rating Scale (r = −0.36, p = 0.01). Urinary neopterin and p75ECD correlated with each other at baseline (r = 0.38, p = 0.009). In longitudinal analysis, urinary neopterin increased on average (±SE) by 6.8 ± 1.1 µmol/mol creatinine per month (p < 0.0001) and p75ECD by 0.19 ± 0.02 ng/mg creatinine per month (p < 0.0001) from diagnosis in 29 ALS patients. Conclusion: Urinary neopterin holds promise as marker of disease progression in ALS and is worthy of future evaluation for its potential to predict response to anti-inflammatory therapies.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare thromboembolic complication of adenoviral-vectored SARS-CoV2 vaccines, mediated by antibodies directed against platelet factor 4 (PF4). Given their causal role in VITT, identification of the molecular composition of anti-PF4 antibodies is crucial for developing better diagnostics and treatments. Here, we utilised a novel proteomic workflow to analyse the immunoglobulin variable (IgV) region composition of anti-PF4 antibodies at the level of the secreted proteome. Serum anti-PF4 IgG antibodies from five patients with VITT triggered by ChAdOx1 nCoV-19 vaccination were affinity purified by PF4-coupled magnetic beads and sequenced by mass spectrometry. We revealed a single IgG heavy (H)-chain species paired with a single lambda light (L)-chain species in all five unrelated patients. Remarkably, all L-chains were encoded by the identical IGLV3-21*02 gene subfamily with identical L-chain third complementarity determining region (LCDR3) lengths. Moreover, striking stereotypic features were also identified in heavy-chains anti-PF4 antibodies characterised by identical HCDR3 length and homologous sequences. In summary, we unravelled the molecular signature of highly stereotyped clonotypic anti-PF4 antibodies, indicating shared pathways of antibody production in VITT patients. These discoveries are critical to understand the molecular basis of this serious condition and develop novel therapies aimed at removing pathogenic clones.KEY POINTSAnti-PF4 antibodies in VITT comprise highly stereotyped clonotypeA single IGLV3-21*02 encoded light chain is found in unrelated patients

Clostridioides difficile infection is the leading cause of nosocomial diarrhoea globally. Immune responses to toxins produced by C. difficile are important in disease progression and outcome. Here, we analysed the anti-toxin A and anti-toxin B serum antibody proteomes following natural infection or vaccination with a C. difficile toxoid A/toxoid B vaccine using a modified miniaturised proteomic approach based on de novo mass spectrometric sequencing. Analysis of immunoglobulin variable region (IgV) subfamily expression in immunoprecipitated toxin A and toxin B antibodies from four and seven participants of a vaccine trial, respectively, revealed a polyclonal proteome with restricted IGHV, IGKV and IGLV subfamily usage. No dominant IGHV subfamily was observed in the toxin A response, however the dominant anti-toxin B heavy (H)-chain was encoded by IGHV3-23. Light (L)-chain usage was convergent for both anti-toxin A and anti-toxin B proteomes with IGKV3-11, 3-15, 3-20 and 4-1 shared among all subjects in both cohorts. Peptide mapping of common IgV families showed extensive public and private amino acid substitutions. The cohort responses to toxin A and toxin B showed limited similarity in shared IGHV subfamilies. L-chain subfamily usage was more similar in the anti-toxin A and anti-toxin B responses, however the mutational signatures for each subfamily were toxin-dependent. Samples taken both post vaccination (n = 5) or at baseline, indicating previous exposure (n = 2), showed similar anti-toxin B IgV subfamily usage and mutational profiles. In summary, this study provides the first sequence-based proteomic analysis of the antibody response to the major disease-mediating toxins of C. difficile, toxin A and toxin B, and demonstrates that despite the potential for extreme diversity, the immunoglobulin repertoire can raise convergent responses to specific pathogens whether through natural infection or following vaccination.

While peanut oral immunotherapy (POIT) represents a promising treatment for peanut allergies in children, safety concerns remain a common barrier to widespread adoption. We aimed to systematically assess available evidence to determine the risk and frequency of adverse events occurring during POIT, and examine study-level characteristics associated with their occurrence and severity. A systematic search of MEDLINE, EMBASE, and Web of Science was conducted through April 2019. Controlled and non-controlled studies evaluating POIT were eligible. Twenty-seven studies, involving 1488 subjects, were included. Adverse events to POIT were common and led to treatment discontinuation in 6.6% of children (95% CI 4.4–9.0; 27 studies, I2 = 48.7%). Adverse events requiring treatment with epinephrine occurred among 7.6% (4.5–11.4; 26 studies, I2 = 75.5%) of participants, at a rate of 2.0 per 10,000 doses (0.8–3.7; 15 studies, I2 = 64.4). Use of a rush treatment phase and targeting a higher maintenance dose were associated with a higher risk and frequency of epinephrine use, while using co-treatments in addition to POIT was associated with a lower risk of treatment discontinuation due to adverse events. While adverse events to POIT are common, this study provides promising explorative evidence that certain modifications to existing treatment protocols could significantly improve treatment outcomes.

ObjectiveTo comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren’s syndrome (SjS) using an integrated omics workflow.MethodsPeptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS.ResultsAffected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification.ConclusionThe ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab.

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn2+, Co2+, and Fe2+ increased the alginate degrading activity, but it was inhibited in the presence of Zn2+ and Cu2+. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.

Proteinaceous α-synuclein-containing inclusions are found in affected brain regions in patients with Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These appear in neurons as Lewy bodies in both PD and DLB and as glial cytoplasmic inclusions (GCIs) in oligodendrocytes in MSA. The role they play in the pathology of the diseases is unknown, and relatively little is still known about their composition. By purifying the inclusions from the surrounding tissue and comprehensively analysing their protein composition, vital clues to the formation mechanism and role in the disease process may be found. In this study, Lewy bodies were purified from postmortem brain tissue from DLB cases (n = 2) and GCIs were purified from MSA cases (n = 5) using a recently improved purification method, and the purified inclusions were analysed by mass spectrometry. Twenty-one percent of the proteins found consistently in the GCIs and LBs were synaptic-vesicle related. Identified proteins included those associated with exosomes (CD9), clathrin-mediated endocytosis (clathrin, AP-2 complex, dynamin), retrograde transport (dynein, dynactin, spectrin) and synaptic vesicle fusion (synaptosomal-associated protein 25, vesicle-associated membrane protein 2, syntaxin-1). This suggests that the misfolded or excess α-synuclein may be targeted to inclusions via vesicle-mediated transport, which also explains the presence of the neuronal protein α-synuclein within GCIs.

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. The Advax-SM adjuvanted vaccine induced high titers of binding antibody against spike protein that were able to neutralise the original wildtype virus on which the vaccine was based as well as the variant B.1.1.7 lineage virus. The Covax-19 vaccine also induced potent spike-specific CD4+ and CD8+ memory T-cells with a dominant Th1 phenotype, and this was shown to be associated with cytotoxic T lymphocyte killing of spike labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus 2 weeks after the second immunisation. Notably, ferrets that received two 25 or 50μg doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting the possibility that Covax-19 vaccine may in addition to protection against lung infection also have the potential to block virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

Glucagon is secreted by pancreatic α cells in response to hypoglycemia and increases hepatic glucose output through hepatic glucagon receptors (GCGRs). There is evidence supporting the notion of extrapancreatic glucagon but its source and physiological functions remain elusive. Intestinal tissue samples were obtained from patients undergoing surgical resection of cancer. Mass spectrometry analysis was used to detect glucagon from mucosal lysate. Static incubations of mucosal tissue were performed to assess glucagon secretory response. Glucagon concentration was quantitated using a highly specific sandwich enzyme-linked immunosorbent assay. A cholesterol uptake assay and an isolated murine colonic motility assay were used to assess the physiological functions of intestinal GCGRs. Fully processed glucagon was detected by mass spectrometry in human intestinal mucosal lysate. High glucose evoked significant glucagon secretion from human ileal tissue independent of sodium glucose cotransporter and KATP channels, contrasting glucose-induced glucagon-like peptide 1 (GLP-1) secretion. The GLP-1 receptor agonist Exendin-4 attenuated glucose-induced glucagon secretion from the human ileum. GCGR blockade significantly increased cholesterol uptake in human ileal crypt culture and markedly slowed ex vivo colonic motility. Our findings describe the human gut as a potential source of extrapancreatic glucagon and demonstrate a novel enteric glucagon/GCGR circuit with important physiological functions beyond glycemic regulation.

Background Progressive advances in proteomic technology has improved our understanding of the chronic rhinosinusitis (CRS) pathogenesis and endotypes. This scoping review aims to present a comprehensive and descriptive analysis of nasal mucosa and mucus proteome of CRS patients. Methodology Studies investigating the proteome of nasal mucosa and mucus from healthy and CRS patients via mass spectrometry were included. Critical appraisal of methodological quality was conducted with extraction of protein lists. Gene set enrichment analysis (GSEA) was performed on studies including CRS patients. Results 2962 proteins were identified in the 21 studies included in this review. Eleven studies investigated the nasal mucus proteome and ten studies investigated the nasal mucosa proteome. Studies demonstrated heterogeneity in patients, sampling and mass spectrometry methodology. Samples from CRS patients suggested a trend in enrichment of immune system and programmed cell death pathways. Increased expression of proteins involved in cellular components including the cytoskeleton and adherens junctions was also present in CRS. Conclusions Alterations in the healthy sinonasal proteome may lead to the increased immunological, metabolic and tissue remodeling processes observed in CRS. However, it is difficult to draw significant conclusions from the GSEA due to the heterogeneity present in the limited literature available. These findings allow us to direct further research to better understand CRS pathogenesis and its endotypes.

Chronic rhinosinusitis (CRS) has a complex and multifactorial pathogenesis with a heterogeneous inflammatory profile. Proteomic analysis of nasal mucus may enable further understanding of protein abundances and biologic processes present in CRS and its endotypes compared with in healthy patients.Our aim was to determine differences in the nasal mucus proteome of healthy patients and patients with CRS.Nasal mucus was obtained from healthy patients, patients with CRS without nasal polyps (CRSsNP), and patients with CRS with nasal polyps (CRSwNP) before surgery. Gel electrophoresis was performed to fractionate the complex protein extracts before mass spectrometry analysis. Gene set enrichment analysis was performed on differentially expressed proteins.A total of 33 patients were included in this study (12 healthy, 10 with CRSsNP, and 11 with CRSwNP). In all, 1142 proteins were identified in mucus samples from healthy patients, 761 in mucus samples from patients with CRSsNP, and 998 in mucus samples from patients with CRSwNP. Dysfunction in immunologic pathways, reduced cellular signaling, and increased cellular metabolism with associated tissue remodeling pathways were present in patients with CRS compared with in healthy patients.Significant downregulation of mucosal immunity and antioxidant pathways with increased tissue modeling processes may account for the clinical manifestations of CRS. Ultimately, the differing proteome and biologic processes provide further insight into CRS pathogenesis and its endotypes.

Bone marrow transplantation may transfer the production of food-specific IgE to the recipient, but this may not be sufficient to cause allergy; we undertook detailed immunological workup in a strongly sensitized but nonallergic hematopoietic stem cell transplant recipient to elucidate the potential mechanisms of tolerance. Refereed/Peer-reviewed

Ca2+ entry through store-operated Ca2+ channels (SOCs) in the plasma membrane (PM) of hepatocytes plays a central role in the hormonal regulation of liver metabolism. SOCs are composed of Orai1, the channel pore protein, and STIM1, the activator protein, and are regulated by hormones and reactive oxygen species (ROS). In addition to Orai1, STIM1 also interacts with several other intracellular proteins. Most previous studies of the cellular functions of Orai1 and STIM1 have employed immortalised cells in culture expressing ectopic proteins tagged with a fluorescent polypeptide such as GFP. Little is known about the intracellular distributions of endogenous Orai1 and STIM1. The aims are to determine the intracellular distribution of endogenous Orai1 and STIM1 in hepatocytes and to identify novel STIM1 binding proteins. Subcellular fractions of rat liver were prepared by homogenisation and differential centrifugation. Orai1 and STIM1 were identified and quantified by western blot. Orai1 was found in the PM (0.03%), heavy (44%) and light (27%) microsomal fractions, and STIM1 in the PM (0.09%), and heavy (85%) and light (13%) microsomal fractions. Immunoprecipitation of STIM1 followed by LC/MS or western blot identified peroxiredoxin-4 (Prx-4) as a potential STIM1 binding protein. Prx-4 was found principally in the heavy microsomal fraction. Knockdown of Prx-4 using siRNA, or inhibition of Prx-4 using conoidin A, did not affect Ca2+ entry through SOCs but rendered SOCs susceptible to inhibition by H2O2. It is concluded that, in hepatocytes, a considerable proportion of endogenous Orai1 and STIM1 is located in the rough ER. In the rough ER, STIM1 interacts with Prx-4, and this interaction may contribute to the regulation by ROS of STIM1 and SOCs.

Objective Rheumatoid factors (RFs) are associated with systemic disease in primary Sjogren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. Methods Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. Results Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. Conclusion Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.

Oral immunotherapy (OIT) using roasted peanut flour can effectively desensitize peanut-allergic children [1] but is considered not to be ready for clinical practice [2] due to high rates (≥45%) of adverse events (AEs) [3] [4]. This necessitates medically supervised up-dosing in hospital and limits the number of patients that can be treated. In 2001 Beyer et. al proposed that the prevalence of peanut allergy in China was lower than that of the Western world because peanuts consumed in China were boiled, not roasted [5]. They demonstrated that boiling peanuts for 20 minutes reduced IgE binding in vitro when compared to roasted peanut. We have subsequently shown that extended boiling progressively reduced peanut IgE binding to 12.5% at 2 hours and to 5.3% at 12 hours compared to raw peanut while still retaining T cell reactivity [6]. Further, Inhibition ELISAs demonstrated that boiled peanuts have restricted ability (2-h ~70%, 12-h ~50%) to block the binding of patient IgE to raw peanut [6] suggesting boiled peanuts possess an incomplete repertoire of epitopes. This indicates that boiled peanuts alone are unlikely to expose a patient to the full spectrum of peanut epitopes and will therefore require a roasted peanut phase following the initial boiled peanut therapy. We hypothesize that AEs can be reduced by commencing OIT with hypoallergenic boiled peanut. Here we describe a pilot study that aims to characterize the incidence of AEs and successful desensitization in mild/moderate peanut allergic children using hypoallergenic 2-hour boiled peanut prior to roasted peanut OIT. Due to the home-based up-dosing procedure, a cautious approach was adopted which excluded severely allergic children. This article is protected by copyright. All rights reserved.

Background: Fuchs endothelial corneal dystrophy (FECD) is a progressive and potentially a sight threatening disease, and a common indication for corneal grafting in the elderly. Aberrant thickening of Descemet's membrane, formation of microscopic excrescences (guttae) and gradual loss of corneal endothelial cells are the hallmarks of the disease. The aim of this study was to identify differentially abundant proteins between FECD-affected and unaffected Descemet's membrane. Methods: Label-free quantitative proteomics using nanoscale ultra-performance liquid chromatography-mass spectrometry (nUPLC-MSE ) was employed on affected and unaffected Descemet's membrane extracts, and interesting findings were further investigated using quantitative reverse transcription-polymerase chain reaction and immunohistochemical techniques. Results: Quantitative proteomics revealed significantly lower abundance of apolipoprotein E (APOE) and immunoglobulin heavy constant gamma 1 protein (IGHG1) in affected Descemet's membrane. The difference in the distribution of APOE between affected and unaffected Descemet's membrane and of IGHG1 detected by immunohistochemistry support their down-regulation in the disease. Comparative gene expression analysis showed significantly lower APOE mRNA levels in FECD-affected than unaffected corneal endothelium. IGHG1 gene is expressed at extremely low levels in the corneal endothelium, precluding relative expression analysis. Conclusions: This is the first study to report comparative proteomics of Descemet's membrane tissue, and implicates dysregulation of APOE and IGHG1 proteins in the pathogenesis of Fuchs endothelial corneal dystrophy.

Comparison with existing methods. Background Neurodegenerative disorders affect a large proportion of the elderly population. A group of disorders, known as the α-synucleinopathies, are characterised by the presence of α-synuclein-containing protein inclusions, such as Lewy Bodies (LBs) found in neurons from Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB), and Glial Cytoplasmic Inclusions (GCIs) found in oligodendrocytes from Multiple System Atrophy (MSA). The analysis of the protein composition of inclusions has been hindered by limitations of methods for isolating the inclusions from the surrounding tissue. Method Four modifications were made to the published method for GCI purification by Gai et al. (1999) which were: collecting the entire inclusion-containing part of the Percoll gradient; lysis of nuclei prior to DNAse digestion; limited tryptic digestion to release inclusions from the cytoskeletal meshwork; and increased antibody and magnetic bead concentrations/volumes to capture the larger amounts of inclusions. Results The optimised method gave a 28-fold increase in yield compared to the published method of Gai et al. (1999) . A 2D-DIGE comparison revealed a 3.8-fold increase in α-synuclein enrichment and a corresponding 5.2-fold reduction in tubulin contamination. This method was also successfully adapted to the purification of LBs from DLB tissue. A 2D-DIGE comparison of purified GCIs ( n = 2) revealed that GCIs consist of 11.7% α-synuclein, 1.9% α-β-crystallin and 2.3% 14-3-3 proteins compared to 8.5%, 2.0% and 1.5% in LBs, respectively. Conclusions This study has generated an improved method for the purification of α-synuclein-containing inclusions with a yield sufficient for multiple forms of analysis.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

SummaryAnti-double-stranded (ds)DNA autoantibodies are prototypical serological markers of systemic lupus erythematosus (SLE), but little is known about their immunoglobulin variable (IgV) region composition at the level of the secreted (serum) proteome. Here, we use a novel proteomic workflow based on de novo mass spectrometric sequencing of anti-dsDNA precipitins to analyse IgV subfamily expression and mutational signatures of high-affinity, precipitating anti-dsDNA responses. Serum anti-dsDNA proteomes were oligoclonal with shared (public) expression of immunoglobulin (Ig)G heavy chain variable region (IGHV) and kappa chain variable region (IGKV) subfamilies. IgV peptide maps from eight subjects showed extensive public and random (private) amino acid replacement mutations with prominent arginine substitutions across heavy (H)- and light (L)-chains. Shared sets of L-chain complementarity determining region 3 (CDR3) peptides specified by arginine substitutions were sequenced from the dominantly expressed IGKV3-20 subfamily, with changes in expression levels of a clonal L-chain CDR3 peptide by quantitative multiple reaction monitoring (MRM) paralleling the rise and fall of anti-dsDNA levels by Farr radioimmunoassays (RIA). The heavily mutated IgV peptide signatures of precipitating anti-dsDNA autoantibody proteomes reflect the strong selective forces that shape humoral anti-dsDNA responses in germinal centres. Direct sequencing of agarose gel precipitins using microlitre volumes of stored sera streamlines the antibody sequencing workflow and is generalizable to other precipitating serum antibodies.

Background Altered neuronal connectivity in peri-infarct tissue is an important contributor to both the spontaneous recovery of neurological function that commonly develops after stroke and improvements in recovery that have been induced by experimental treatments in animal models. Microglia and astrocytes are primary determinants of the environment in peri-infarct tissue and hence strongly influence the potential for neuronal plasticity. However, the specific roles of these cells and the timing of critical changes in their function are not well understood. Minocycline can protect against ischemic damage and promote recovery. These effects are usually attributed, at least partially, to the ability of this drug to suppress microglial activation. This study tested the ability of minocycline treatment early after stroke to modify reactive responses in microglia and astrocytes and improve recovery. Methods Stroke was induced by photothrombosis in the forelimb sensorimotor cortex of Sprague-Dawley rats. Minocycline was administered for 2 days after stroke induction and the effects on forelimb function assessed up to 28 days. The responses of peri-infarct Iba1-positive cells and astrocytes were evaluated using immunohistochemistry and Western blots. Results Initial characterization showed that the numbers of Iba1-positive microglia and macrophages decreased in peri-infarct tissue at 24 h then increased markedly over the next few days. Morphological changes characteristic of activation were readily apparent by 3 h and increased by 24 h. Minocycline treatment improved the rate of recovery of motor function as measured by a forelimb placing test but did not alter infarct volume. At 3 days, there were only minor effects on core features of peri-infarct microglial reactivity including the morphological changes and increased density of Iba1-positive cells. The treatment caused a decrease of 57% in the small subpopulation of cells that expressed CD68, a marker of phagocytosis. At 7 days, the expression of glial fibrillary acidic protein and vimentin was markedly increased by minocycline treatment, indicating enhanced reactive astrogliosis. Conclusions Early post-stroke treatment with minocycline improved recovery but had little effect on key features of microglial activation. Both the decrease in CD68-positive cells and the increased activation of astrogliosis could influence neuronal plasticity and contribute to the improved recovery. Electronic supplementary material The online version of this article (10.1186/s12974-018-1379-y) contains supplementary material, which is available to authorized users.

Chronic lymphocytic leukaemia (CLL) remains the most common incurable malignancy of B cells in the western world. Patient outcomes are heterogeneous and can be difficult to predict with current prognostic markers. Here, we used a quantitative label-free proteomic technique to ascertain differences in the B-cell proteome from healthy donors and CLL patients with either mutated (M-CLL) or unmutated (UM-CLL) IGHV to identify new prognostic markers. In peripheral B-CLL cells, 349 (22%) proteins were differentially expressed between normal B cells and B-CLL cells and 189 (12%) were differentially expressed between M-CLL and UM-CLL. We also examined the proteome of proliferating CLL cells in the lymph nodes, and identified 76 (~8%) differentially expressed proteins between healthy and CLL lymph nodes. B-CLL cells show over-expression of proteins involved in lipid and cholesterol metabolism. A comprehensive lipidomic analysis highlighted large differences in glycolipids and sphingolipids. A shift was observed from the pro-apoptotic lipid ceramide towards the anti-apoptotic/chemoresistant lipid, glucosylceramide, which was more evident in patients with aggressive disease (UM-CLL). This study details a novel quantitative proteomic technique applied for the first time to primary patient samples in CLL and highlights that primary CLL lymphocytes display markers of a metabolic shift towards lipid synthesis and breakdown.

Analysis of the anti-haemagglutinin serum antibody proteome from six H1N1pdm09 influenza A vaccinated subjects demonstrated restricted IgG1 heavy chain species encoded by IGHV5-51 and IGHV3-7 gene families in 2 subjects and either IGHV5-51 or IGHV3-7 in 4 individuals. All subjects exhibited a dominant IGKV3-20 light chain, however 5 subjects also exhibited IGKV3-11 and IGKV4-1 families. Sequences were closely aligned with the matched germline sequence, with few shared mutations. This study illustrates the feasibility of using a proteomic approach to determine the expressed V region signatures of serum antibodies induced by vaccination.

Background and aims Autoantibodies directed against the 60-kD Ro (Ro60)/SSA ribonucleoprotein particle are the major target of humoral autoimmunity in patients with systemic lupus erythematosus (SLE) and primary Sjogren’s syndrome (SS). However, little is known of the anti-Ro60 immunoglobulin variable-region (IgV) repertoire in terms of clonality and IgV gene usage at the level of the serum proteome. Methods We used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with SLE and primary SS and compare IgV peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Additionally, Ro60 precipitins from counterimmunoelectrophoresis gels were excised, digested and sequenced directly by mass spectrometry. Results Anti-Ro60 Igs purified from ELISA plates and Ro60 precipitins were comprised dominant public sets of IgG1 kappa and lambda restricted heavy and light chains (with sharing of IGHV3-23, IGHV3-74 and IGHV1-18; IGKV3-20, IGKV1-5 and IGLV3-19). Significantly, mass spectrometric sequencing of purified anti-Ro60 IgGs from SLE patients showed the same convergence of autoantibody repertoires as primary SS, apart from one SLE patient who lacked IGHV3-74, suggesting that humoral anti-Ro60 molecular signatures are conserved across these two systemic autoimmune diseases. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60 plus anti-La responses, providing a molecular fingerprint of Ro60/La determinant spreading. Conclusions Unique anti-Ro60 IgV peptide signatures provide insights in to mechanisms of autoantibody production as well as holding promise as serum-based molecular markers for clinical syndromes linked to Ro60 autoimmunity.

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1−/− cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Refereed/Peer-reviewed

Sea cucumbers are prolific producers of a wide range of bioactive compounds. This study aimed to purify and characterize one class of compound, the saponins, from the viscera of the Australian sea cucumber Holothuria lessoni. The saponins were obtained by ethanolic extraction of the viscera and enriched by a liquid-liquid partition process and adsorption column chromatography. A high performance centrifugal partition chromatography (HPCPC) was applied to the saponin-enriched mixture to obtain saponins with high purity. The resultant purified saponins were profiled using MALDI-MS/MS and ESI-MS/MS which revealed the structure of isomeric saponins to contain multiple aglycones and/or sugar residues. We have elucidated the structure of five novel saponins, Holothurins D/E and Holothurinosides X/Y/Z, along with seven reported triterpene glycosides, including sulfated and non-sulfated saponins containing a range of aglycones and sugar moieties, from the viscera of H. lessoni. The abundance of novel compounds from this species holds promise for biotechnological applications.

Chronic lymphocytic leukemia (CLL) remains the most common leukemia in the Western world. Whilst its disease course is extremely heterogeneous (ranging from indolent to aggressive), current methods are unable to accurately predict the clinical journey of each patient. There is clearly a pressing need for both improved prognostication and treatment options for patients with this disease. Whilst molecular studies have analyzed both genetic mutations and gene expression profiles of these malignant B-cells, and as a result have shed light on the pathogenesis of CLL, proteomic studies have been largely overlooked to date. This review summarizes our current knowledge of the proteomics of CLL, and discusses some of the issues in CLL proteomic research, such as reproducibility and data interpretation. In addition, we look ahead to how proteomics may significantly help in the development of a successful treatment for this currently incurable disease.

Type 2 diabetes (T2D) increases the risk of death associated with cardiovascular complications. However, a complete understanding of protein changes within the diabetic vasculature is still lacking. Herein, we utilized mass spectrometry to perform vascular and urinary proteome analysis using a rat model of high-fat feeding and low-dose streptozotocin to simulate late-stage T2D. The purpose of this study was to identify aortic and urine proteins that are differentially expressed in normal and T2D rats. High-fat feeding and low-dose streptozotocin resulted in hyperglycemia, hypoinsulinemia and high levels of circulating free fatty acids. Using a shotgun proteomic approach, high-mobility-group protein B1 and spondin-1 were significantly increased in T2D aorta compared to control aorta, suggesting vascular inflammation and smooth muscle proliferation, respectively. However, the majority of differentially expressed aortic proteins were downregulated in T2D, including proteins associated with coagulation, cell differentiation and redox homeostasis. Strikingly, we report a significant downregulation of commonly used cytoskeletal housekeeping proteins in T2D aorta. Urine from T2D rats displayed increased expression of proteins involved in inflammation and oxidative stress and decreased expression of proteins associated with lipid metabolism and cell adhesion. A number of differentially expressed proteins in urine of T2D rats have previously been reported in human T2D, thereby supporting this animal model as a good representation of human T2D. Our data offer new information regarding key pathways that could be therapeutically targeted to combat the cardiovascular complications of T2D.

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

To evaluate urinary neurotrophin receptor p75 extracellular domain (p75The population in this study comprised 45 healthy controls and 54 people with ALS, 31 of whom were sampled longitudinally. Urinary p75Confirming our previous findings, p75The assay for urinary p75

We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjogren's syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity.

Background: Current peanut oral immunotherapy is hampered by frequent adverse events. It has been shown that boiling can reduce peanut allergenicity. Hypoallergenic peanut products have the potential to reduce treatment-related reactions during desensitization. Objective: To show that extended boiling (for up to 12 h) can progressively reduce peanut allergenicity while retaining T cell reactivity. Methods: Raw peanuts were boiled for half, 1, 2, 4 and 12 h in deionized water. After dehydration, boiled and raw peanuts were ground, defatted and soluble proteins extracted in PBS and cooking water (leachate) retained. SDS-PAGE, Western blot, inhibition ELISA, mass spectrometry and skin prick test were used to characterize changes to peanut allergens and human IgE reactivity. T cell responses to raw and boiled peanut extracts were determined by proliferation of CD4+/CD25+/CD134+ T cells in peanut-allergic and non-allergic individuals. Results: Extended boiling progressively reduced peanut allergenicity through a combination of leaching of allergens into cooking water, fragmentation of allergens and denaturation of conformational epitopes. Two-hour boiling led to an eightfold reduction in IgE binding capacity of boiled peanuts as determined by inhibition ELISA, while 12-h boiling led to a 19-fold reduction. Mass spectrometry revealed an increasing number of unique allergen peptides with longer boiling times. Raw, 2- and 12-h boiled peanut extracts were equivalent in their ability to stimulate T cell activation and proliferation. Conclusion and Clinical Relevance: Progressive reduction in peanut allergenicity with extended boiling does not affect T cell reactivity. Boiled peanuts may be a candidate for oral immunotherapy. Refereed/Peer-reviewed

SummaryLupus-specific anti-ribosomal P (anti-Rib-P) autoantibodies have been implicated in the pathogenesis of neurological complications in systemic lupus erythematosus (SLE). The aim of the present study was to determine variable (V)-region signatures of secreted autoantibody proteomes specific for the Rib-P heterocomplex and investigate the molecular basis of the reported cross-reactivity with Sm autoantigen. Anti-Rib-P immunoglobulins (IgGs) were purified from six anti-Rib-P-positive sera by elution from enzyme-linked immunosorbent assay (ELISA) plates coated with either native Rib-P proteins or an 11-amino acid peptide (11-C peptide) representing the conserved COOH-terminal P epitope. Rib-P- and 11-C peptide-specific IgGs were analysed for heavy (H) and light (L) chain clonality and V-region expression using an electrophoretic and de-novo and database-driven mass spectrometric sequencing workflow. Purified anti-Rib-P and anti-SmD IgGs were tested for cross-reactivity on ELISA and their proteome data sets analysed for shared clonotypes. Anti-Rib-P autoantibody proteomes were IgG1 kappa-restricted and comprised two public clonotypes defined by unique H/L chain pairings. The major clonotypic population was specific for the common COOH-terminal epitope, while the second shared the same pairing signature as a recently reported anti-SmD clonotype, accounting for two-way immunoassay cross-reactivity between these lupus autoantibodies. Sequence convergence of anti-Rib-P proteomes suggests common molecular pathways of autoantibody production and identifies stereotyped clonal populations that are thought to play a pathogenic role in neuropsychiatric lupus. Shared clonotypic structures for anti-Rib-P and anti-Sm responses suggest a common B cell clonal origin for subsets of these lupus-specific autoantibodies.

The structures of epitopes bound by autoantibodies against RNA-protein complexes have been well-defined over several decades, but little is known of the clonality, immunoglobulin (Ig) variable (V) gene usage and mutational status of the autoantibodies themselves at the level of the secreted (serum) proteome. A novel proteomic workflow is presented based on affinity purification of specific Igs from serum, high-resolution two-dimensional gel electrophoresis, and de novo and database-driven sequencing of V-region proteins by mass spectrometry. Analysis of anti-Ro52/Ro60/La proteomes in primary Sjogren's syndrome (SS) and anti-Sm and anti-ribosomal P proteomes in systemic lupus erythematosus (SLE) has revealed that these antibody responses are dominated by restricted sets of public (shared) clonotypes, consistent with common pathways of production across unrelated individuals. The discovery of shared sets of specific V-region peptides can be exploited for diagnostic biomarkers in targeted mass spectrometry platforms and for tracking and removal of pathogenic clones.

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1−/− cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1−/− brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. (Figure presented.) The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling. Refereed/Peer-reviewed

Objective This study was undertaken to determine the molecular characteristics of clonotypic autoantibodies in the sera of patients with primary Sjogren's syndrome (SS). This characterization is hampered by the presence of mixed anti-Ro/La specificities that may conceal clonotypic species. In order to narrow clonotypic diversity, a positive selection step was performed on a peg-like determinant of Ro 60 (termed Ro 60–peg) prior to analysis of the autoantibody proteome. Methods Monospecific anti–Ro 60–peg IgG were isolated by affinity purification from the sera of 7 patients with primary SS and anti-Ro/La and subjected to 2-dimensional gel electrophoresis and high-resolution orbitrap mass spectrometric sequencing. V regions of heavy and light chains were analyzed by combined database and de novo amino acid sequencing. Results Proteomic analysis revealed a Ro 60–peg–specific IgG1κ-restricted monoclonal autoantibody that was present in the sera of all patients and specified by a VH3–23 heavy chain paired with a Vκ3–20 light chain. The public anti–Ro 60–peg clonotype was specified further by common mutations in the heavy-chain and light-chain complementarity-determining regions. Titers and relative affinities of clonotypic IgG did not vary over the course of the disease. Conclusion The expression of a Ro 60–reactive public B cell clonotype in a subset of patients with primary SS as a long-lived, class-switched circulating autoantibody implies a common breach of B cell tolerance checkpoints in these patients. The unique heavy chain/light chain signature opens the possibility of tracking the development of a “forbidden” clone against a bona fide systemic autoantigen in human disease.

Allergy to Myrmecia pilosula (Jack Jumper Ant) venom is common in Australia, affecting ~2.7% of some communities. Venom immunotherapy is a highly effective treatment, but for the venom to be widely distributed for clinical use, the stability and shelf-life of formulated Jack Jumper Ant venom must be demonstrated. HPLC–UV, ELISA Inhibition, SDS-PAGE and SDS-PAGE Immunoblot were used to assess venom stability under conditions of varying temperature, pH and in the presence of various stabilising agents. Optimal stability occurred between pH 8 and 10, however the presence of benzyl alcohol within this pH range resulted in a cloudy appearance within 3 days, so a pHof 6 was used. Increasing polysorbate 80 concentrations accelerated the degradation of allergenic peptides in 100 g/mL venom, but improved stability at concentrations of 1 g/mL or less. Sucrose reduced degradation of allergens Myr p 1 and Myr p 3, whilst glycerol was destabilising. In the presence of 22% sucrose, 1.1mg/mL Jack Jumper Ant venom was stable at -18 ?C and 4 ?C for 12 months; following dilution to 100 g/mL with 0.9% sodium chloride, 10mM phosphate (pH 6), 0.05% polysorbate 80 and 0.9% benzyl alcohol (giving 2% sucrose), venom was stable for 7 days when stored at 4 ?C. Concentrated Jack Jumper Ant venom can be stored in 22% sucrose for 12 months, and after dilution to 100 g/mL for clinical use, it should be discarded after 7 days. Refereed/Peer-reviewed

Although the water channel protein aquaporin-1 (AQP1) is widely observed outside the rat brain in continuous, but not fenestrated, vascular endothelia, it has not previously been observed in any endothelia within the normal rat brain and only to a limited extent in the human brain. In this immunohistochemical study of rat brain, AQP1 has also been found in microvessel endothelia, probably of the fenestrated type, in all circumventricular organs (except the subcommissural organ and the vascular organ of the lamina terminalis): in the median eminence, pineal, subfornical organ, area postrema and choroid plexus. The majority of microvessels in the median eminence, pineal and choroid plexus, known to be exclusively fenestrated, are shown to be AQP1-immunoreactive. In the subfornical organ and area postrema in which many, but not all, microvessels are fenestrated, not all microvessels are AQP1-immunoreactive. In the AQP1-immunoreactive microvessels, the AQP1 probably facilitates water movement between blood and interstitium as one component of the normal fluxes that occur in these specialised sensory and secretory areas. AQP1-immunoreactive endothelia have also been seen in a small population of blood vessels in the cerebral parenchyma outside the circumventricular organs, similar to other observations in human brain. The proposed development of AQP1 modulators to treat various brain pathologies in which AQP1 plays a deleterious role will necessitate further work to determine the effect of such modulators on the normal function of the circumventricular organs.