Cynthia Wright

Interaction of HLA-B27 homodimers with KIR3DL1 and KIR3DL2, unlike HLA-B27 heterotrimers, is independent of the sequence of bound peptide.

HLA‐B27 can form beta‐2 microglobulin (β2m)‐associated heterotrimers (HLA‐B27) and β2m‐free homodimers (B272). Here, we study the role of complexed peptide in the interaction of these forms of B27 with the killer cell immunoglobulin (Ig)‐like receptors KIR3DL1 and KIR3DL2 and with Ig‐like transcripts LILRB1 and LILRB2. HLA‐B27 tetramers complexed with three of five different naturally processed self peptides and three of seven pathogen‐derived epitopes bound to KIR3DL1‐expressing transfectants and NK cells. Heterotrimeric complexes containing peptides with charged amino acids at position 8 did not bind to KIR3DL1; however, studies with analogue peptides demonstrated that these are not the only peptide residues involved in binding. KIR3DL1 ligation by HLA‐B27 inhibited NK cell IFN‐γ production in a peptide‐dependent fashion. B27 but not HLA‐A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B272 bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B272 inhibited NK and T cell IFN‐γ production. By contrast with HLA heterotrimers, B272 binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B272 could be involved in the pathogenesis of spondyloarthritis.

Discovery of Candidate Serum Proteomic and Metabolomic Biomarkers in Ankylosing Spondylitis

Ankylosing Spondylitis (AS) is a common inflammatory rheumatic disease with a predilection for the axial skeleton, affecting 0.2% of the population. Current diagnostic criteria rely on a composite of clinical and radiological changes, with a mean time to diagnosis of 5 to 10 years. In this study we employed nano liquid-chromatography mass spectrometry analysis to detect and quantify proteins and small compounds including endogenous peptides and metabolites in serum from 18 AS patients and nine healthy individuals. We identified a total of 316 proteins in serum, of which 22 showed significant up- or down-regulation (p < 0.05) in AS patients. Receiver operating characteristic analysis of combined levels of serum amyloid P component and inter-α-trypsin inhibitor heavy chain 1 revealed high diagnostic value for Ankylosing Spondylitis (area under the curve = 0.98). We also depleted individual sera of proteins to analyze endogenous peptides and metabolic compounds. We detected more than 7000 molecular features in patients and healthy individuals. Quantitative MS analysis revealed compound profiles that correlate with the clinical assessment of disease activity. One molecular feature identified as a Vitamin D3 metabolite—(23S,25R)-25-hydroxyvitamin D3 26,23-peroxylactone—was down-regulated in AS. The ratio of this vitamin D metabolite versus vitamin D binding protein serum levels was also altered in AS as compared with controls. These changes may contribute to pathological skeletal changes in AS. Our study is the first example of an integration of proteomic and metabolomic techniques to find new biomarker candidates for the diagnosis of Ankylosing Spondylitis.

Detection of Multiple Autoantibodies in Patients with Ankylosing Spondylitis Using Nucleic Acid Programmable Protein Arrays

Ankylosing spondylitis (AS) is a common, inflammatory rheumatic disease that primarily affects the axial skeleton and is associated with sacroiliitis, uveitis, and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine whether plasma from patients with AS contained autoantibodies and, if so, characterize and quantify this response in comparison to patients with rheumatoid arthritis (RA) and healthy controls. Two high density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA, and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis to determine the patterns of signaling cascades or tissue origin. 44% of patients with ankylosing spondylitis demonstrated a broad autoantibody response, as compared with 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The autoantibody responses in the AS patients were targeted toward connective, skeletal, and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic acid programmable protein arrays constitute a powerful tool to study autoimmune diseases.

Ankylosing spondylitis monocytes show upregulation of proteins involved in inflammation and the Ubiquitin Proteasome pathway

Objectives: To determine if peripheral blood monocytes from patients with ankylosing spondylitis (AS) differed in protein expression compared to rheumatoid arthritis (RA) and healthy controls (HC). Methods: Monocyte protein expression was characterised by 2D gel electrophoresis and by label-free quantitative expression profiling, using nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MSE, where E refers to low/high collision energy switching). Data sets were analysed using the Waters expression profiling system and Ingenuity pathway analysis (IPA). Results: Two-dimensional gel electrophoresis showed upregulation of proteasomal constituents in AS monocytes, including the β subunit of proteasome activator (PA)28. Monocyte expression profiling and IPA showed that significant changes in protein expression within the ubiquitin proteasome pathway (UPP) were restricted to AS monocytes. Statistically significant differences in protein expression involving the leucocyte extravasation, vascular endothelial growth factor, integrin and Toll-like receptor signalling pathways were seen in AS and RA monocytes compared to healthy controls. No evidence of upregulation of proteins involved in the endoplasmic reticulum stress response pathway was found in either AS or RA monocytes. Finally, the PA28 complex was shown to increase the generation of human leucocyte antigen (HLA)-B27 antigenic epitopes by the proteasome in vitro. Conclusions: Our proteomic analyses support the hypothesis that monocytes play an important role in the pathogenesis of AS and RA, and further suggest a specific role in AS for the UPP. Quantitative proteomic expression profiling constitutes a powerful new tool for rheumatology research.

Label-free quantitative proteomics reveals differentially regulated proteins influencing urolithiasis

Urinary proteins have been implicated as inhibitors of kidney stone formation (urolithiasis). As a proximal fluid, prefiltered by the kidneys, urine is an attractive biofluid for proteomic analysis in urologic conditions. However, it is necessary to correct for variations in urinary concentration. In our study, individual urine samples were normalized for this variation by using a total protein to creatinine ratio. Pooled urine samples were compared in two independent experiments. Differences between the urinary proteome of stone formers and nonstone-forming controls were characterized and quantified using label-free nano-ultraperformance liquid chromatography high/low collision energy switching analysis. There were 1063 proteins identified, of which 367 were unique to the stone former groups, 408 proteins were unique to the control pools, and 288 proteins were identified for comparative quantification. Proteins found to be unique in stone-formers were involved in carbohydrate metabolism pathways and associated with disease states. Thirty-four proteins demonstrated a consistent >twofold change between stone formers and controls. For ceruloplasmin, one of the proteins was shown to be more than twofold up-regulated in the stone-former pools, this observation was validated in individuals by enzyme-linked immunosorbent assay. Moreover, in vitro crystallization assays demonstrated ceruloplasmin had a dose-dependent increase on calcium oxalate crystal formation. Taken together, these results may suggest a functional role for ceruloplasmin in urolithiasis.

A quantitative proteomic analysis of long-term memory

BackgroundMemory is the ability to store, retain, and later retrieve learned information. Long-term memory (LTM) formation requires: DNA transcription, RNA translation, and the trafficking of newly synthesized proteins. Several components of these processes have already been identified. However, due to the complexity of the memory formation process, there likely remain many yet to be identified proteins involved in memory formation and persistence.ResultsHere we use a quantitative proteomic method to identify novel memory-associated proteins in neural tissue taken from animals that were trained in vivo to form a long-term memory. We identified 8 proteins that were significantly up-regulated, and 13 that were significantly down-regulated in the LTM trained animals as compared to two different control groups. In addition we found 19 proteins unique to the trained animals, and 12 unique proteins found only in the control animals.ConclusionsThese results both confirm the involvement of previously identified memory proteins such as: protein kinase C (PKC), adenylate cyclase (AC), and proteins in the mitogen-activated protein kinase (MAPK) pathway. In addition these results provide novel protein candidates (e.g. UHRF1 binding protein) on which to base future studies.

Urinary peptide profiling identifies a panel of putative biomarkers for diagnosing and staging endometriosis

OBJECTIVE To identify a potential diagnostic endometriosis marker using matrix-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based urinary proteomics. DESIGN Prospective randomized pilot study. SETTING University hospital, tertiary referral center for endometriosis. PATIENT(S) 53 women undergoing laparoscopic surgery for pain and/or infertility comprising 30 women without endometriosis and 23 with endometriosis. INTERVENTION(S) Laparoscopy and urine specimens. MAIN OUTCOME MEASURE(S) Urinary peptide profiles. RESULT(S) We observed distinct patterns of peptide profiles in the urine samples of women presenting with typical clinical symptoms of endometriosis. Six statistically significant putative peptide markers were identified (four during the periovulatory phase and two during the luteal phase) by comparing controls with moderate/severe endometriosis patients. The periovulatory peptide mass of 1,767.1 Da and the luteal peptide mass of 1,824.3 Da both showed a sensitivity of 75% and a specificity of 85% and 71%, respectively. Also detected were seven peptide markers (two during the periovulatory phase and five during the luteal phase) by comparing the urinary peptide profiles of patients with minimal/mild to moderate/severe endometriosis. The periovulatory peptide mass of 3,280.9 Da and the luteal peptide mass of 1,933.8 Da showed a sensitivity of 82% and 75% and a specificity of 88% and 75%, respectively. CONCLUSION(S) Urinary proteomic analysis may provide a novel method of diagnosing and staging endometriosis.

Detection of BK virus in urine from renal transplant subjects by mass spectrometry

BackgroundThe diagnosis and management of BK virus (BKV) reactivation following renal transplantation continues to be a significant clinical problem. Following reactivation of latent virus, impaired cellular immunity enables sustained viral replication to occur in urothelial cells, which potentially leads to the development of BKV-associated nephropathy (BKVAN). Current guidelines recommend regular surveillance for BKV reactivation through the detection of infected urothelial cells in urine (decoy cells) or viral nucleic acid in urine or blood. However, these methods have variable sensitivity and cannot routinely distinguish between different viral subtypes. We therefore asked whether mass spectrometry might be able to overcome these limitations and provide an additional non-invasive technique for the surveillance of BKV and identification of recipients at increased risk of BKVAN.ResultsHere we describe a mass spectrometry (MS)-based method for the detection of BKV derived proteins directly isolated from clinical urine samples. Peptides detected by MS derived from Viral Protein 1 (VP1) allowed differentiation between subtypes I and IV. Using this approach, we observed an association between higher decoy cell numbers and the presence of the VP1 subtype Ib-2 in urine samples derived from a cohort of 20 renal transplant recipients, consistent with the hypothesis that certain viral subtypes may be associated with more severe BKVAN.ConclusionsThis is the first study to identify BK virus proteins in clinical samples by MS and that this approach makes it possible to distinguish between different viral subtypes. Further studies are required to establish whether this information could lead to stratification of patients at risk of BKVAN, facilitate distinction between BKVAN and acute rejection (AR), and ultimately improve patient treatment and outcomes.

P17-28 LB. The antiviral efficacy of HIV-specific CD8+ T-cells to a conserved epitope is heavily dependent on the infecting HIV-1 isolate

A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef90–97 epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A–H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses.

Assessing the quality and reproducibility of a proteomic platform for clinical stroke biomarker discovery.

The aim of this study was to investigate the quality and reproducibility of mass spectra derived from a matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) platform in a patient population undergoing carotid endarterectomy. Plasma samples were either digested with trypsin or left undigested, fractionated with either C18 or weak cation exchange (WCX) columns and analysed by MALDI-TOF MS. Quality of mass spectra for each method was assessed by baseline correction (lower area under the curve ratio indicating higher quality) and signal-to-noise ratio. Mean coefficient of variation (CV%) assessed reproducibility between repeated experiments and methods. Identified mass peak intensity differences were assessed for consistency across repeated experiments. Plasma from six patients was analysed. The quality of mass spectra was significantly better when derived from digested plasma fractionated by either WCX or C18 methods compared to undigested plasma fractionated by WCX (analysis of variance, p < 0.05). Mean CV% for repeated experiments was 18% and 28% for WCX and C18 fractionated digested plasma, respectively. A small number of differences in mass peak intensities were consistently observed in repeated experiments. Repeated experiments are required to confidently identify non-random mass peak intensity differences as putative plasma biomarkers that merit further investigation.