Identification of autoantigens and their potential post-translational modification in EGPA and severe eosinophilic asthma.

Background: The chronic airway inflammation in severe eosinophilic asthma (SEA) suggests potential autoimmune aetiology with unidentified autoantibodies analogous to myeloperoxidase (MPO) in ANCA-positive EGPA (eosinophilic granulomatosis with polyangiitis). Previous research has shown that oxidative post-translational modification (oxPTM) of proteins is an important mechanism by which autoantibody responses may escape immune tolerance. Autoantibodies to oxPTM autoantigens in SEA have not previously been studied. Methods: Patients with EGPA and SEA were recruited as well as healthy control participants. Autoantigen agnostic approach: Participant serum was incubated with slides of unstimulated and PMA-stimulated neutrophils and eosinophils, and autoantibodies to granulocytes were identified by immunofluorescence with anti-human IgG FITC antibody. Target autoantigen approach: Candidate proteins were identified from previous literature and FANTOM5 gene set analysis for eosinophil expressed proteins. Serum IgG autoantibodies to these proteins, in native and oxPTM form, were detected by indirect ELISA. Results: Immunofluorescence studies showed that serum from patients with known ANCA stained for IgG against neutrophils as expected. In addition, serum from 9 of 17 tested SEA patients stained for IgG to PMA-stimulated neutrophils undergoing NETosis. Immunofluorescent staining of eosinophil slides was evident with serum from all participants (healthy and with eosinophilic disease) with diffuse cytoplasmic staining except for one SEA individual in whom subtle nuclear staining was evident. FANTOM5 gene set analysis identified TREM1 (triggering receptor expressed on myeloid cells 1) and IL-1 receptor 2 (IL1R2) as eosinophil-specific targets to test for autoantibody responses in addition to MPO, eosinophil peroxidase (EPX), and Collagen-V identified from previous literature. Indirect ELISAs found high concentrations of serum autoantibodies to Collagen-V, MPO, and TREM1 in a higher proportion of SEA patients than healthy controls. High concentrations of serum autoantibodies to EPX were evident in serum from both healthy and SEA participants. The proportion of patients with positive autoantibody ELISAs was not increased when examining oxPTM compared to native proteins. Discussion: Although none of the target proteins studied showed high sensitivity for SEA, the high proportion of patients positive for at least one serum autoantibody shows the potential of more research on autoantibody serology to improve diagnostic testing for severe asthma. Clinical trial registration: ClinicalTrials.gov, identifier, NCT04671446.

Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes.

AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. CONCLUSIONS/INTERPRETATION: Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.

Early detection of osteoarthritis in the rat with an antibody specific to type II collagen modified by reactive oxygen species.

BACKGROUND: Osteoarthritis (OA) is a disease of the whole joint, with articular cartilage breakdown as a major characteristic. Inflammatory mediators, proteases, and oxidants produced by chondrocytes are known to be responsible for driving cartilage degradation. Nevertheless, the early pathogenic events are still unclear. To investigate this, we employed an antibody that is specific to oxidative post-translationally modified collagen type II (anti-oxPTM-CII) to detect early cartilage pathogenic changes in two rat models of OA. METHODS: The animals underwent surgery for destabilization of the medial meniscus (DMM) and were sacrificed after 3, 5, 7, 14, and 28 days. Alternatively, anterior cruciate ligament transection with partial meniscectomy (ACLT+pMx) was performed and animals were sacrificed after 1, 3, 5, 7, and 14 days. Joints were stained with toluidine blue and saffron du Gatinais for histological scoring, anti-oxPTM-CII, and anti-collagen type X antibodies (anti-CX). RESULTS: We observed positive oxPTM-CII staining as early as 1 or 3 days after ACLT+pMx or DMM surgeries, respectively, before overt cartilage lesions were visible. oxPTM-CII was located mostly in the deep zone of the medial tibial cartilage, in the pericellular and territorial matrix of hypertrophic chondrocytes, and co-localized with CX staining. Staining was weak or absent for the lateral compartment or the contralateral knees except at later time points. CONCLUSION: The results demonstrate that oxidant production and chondrocyte hypertrophy occur very early in the onset of OA, possibly initiating the pathogenic events of OA. We propose to use anti-oxPTM-CII as an early biomarker for OA ahead of radiographic changes.

Targeting Extracellular Vesicles to the Arthritic Joint Using a Damaged Cartilage-Specific Antibody.

The targeted delivery of therapies to diseased tissues offers a safe opportunity to achieve optimal efficacy while limiting systemic exposure. These considerations apply to many disease indications but are especially relevant for rheumatoid arthritis (RA), as RA is a systemic autoimmune disease which affects multiple joints. We have identified an antibody that is specific to damaged arthritic cartilage (anti-ROS-CII) that can be used to deliver treatments specifically to arthritic joints, yielding augmented efficacy in experimental arthritis. In the current study, we demonstrate that scaffolds enriched with bioactive payloads can be delivered precisely to an inflamed joint and achieve superior efficacy outcomes consistent with the pharmacological properties of these payloads. As a scaffold, we have used extracellular vesicles (EVs) prepared from human neutrophils (PMNs), which possess intrinsic anti-inflammatory properties and the ability to penetrate inflamed arthritic cartilage. EV fortified with anti-ROS-CII (EV/anti-ROS-CII) retained anti-ROS-CII specificity and bound exclusively to the damaged cartilage. Following systemic administration, EV/anti-ROS-CII (a) exhibited the ability to localize specifically in the arthritic joint in vivo and (b) was able to specifically target single (viral IL-10 or anti-TNF) or combined (viral IL-10 and anti-TNF) anti-inflammatory treatments to the arthritic joint, which accelerated attenuation of clinical and synovial inflammation. Overall, this study demonstrates the attainability of targeting a pro-resolving biological scaffold to the arthritic joint. The potential of targeting scaffolds such as EV, nanoparticles, or a combination thereof alongside combined therapeutics is paramount for designing systemically administered broad-spectrum of anti-inflammatory treatments.

Antibodies to oxidized insulin improve prediction of type 1 diabetes in children with positive standard islet autoantibodies.

BACKGROUND: Antibodies to posttranslationally modified insulin (oxPTM-INS-Ab) are a novel biomarker of type 1 diabetes (T1D). Here, we evaluated whether oxPTM-INS-Ab can improve T1D prediction in children with positive standard islet autoantibodies (AAB). METHODS: We evaluated sensitivity, specificity, accuracy, and risk for progression to T1D associated with oxPTM-INS-Ab and the standard islet AAB that include insulin (IAA), GAD (GADA), and tyrosine phosphatase 2 (IA-2A) in a cohort of islet AAB-positive (AAB+ ) children from the general population (median follow-up 8.8 years). RESULTS: oxPTM-INS-Ab was the most sensitive and specific autoantibody biomarker (74% sensitivity, 91% specificity), followed by IA-2A (71% sensitivity, 91% specificity). GADA and IAA showed lower sensitivity (65% and 50%, respectively) and specificity (66% and 68%, respectively). Accuracy (AUC of ROC) of oxPTM-INS-Ab was higher than GADA and IAA (P = 0.003 and P = 0.017, respectively), and similar to IA-2A (P = 0.896). oxPTM-INS-Ab and IA-2A were more effective than IAA for detecting progr-T1D when used as second-line biomarker in GADA+ children. Risk for diabetes was higher (P = 0.03) among multiple AAB+ who were also oxPTM-INS-Ab+ compared with those who were oxPTM-INS-Ab- . Importantly, when replacing IAA with oxPTM-INS-Ab, diabetes risk increased to 100% in children with oxPTM-INS-Ab+ in combination with GADA+ and IA-2A+ , compared with 84.37% in those with IAA+ , GADA+ , and IA-2A+ (P = 0.04). CONCLUSIONS: Antibodies to oxidized insulin (oxPTM-INS-Ab), compared with IAA which measure autoantibodies to native insulin, improve T1D risk assessment and prediction accuracy in AAB+ children.

Oxidative stress in autoimmune rheumatic diseases.

The management of patients with autoimmune rheumatic diseases such as rheumatoid arthritis (RA) remains a significant challenge. Often the rheumatologist is restricted to treating and relieving the symptoms and consequences and not the underlying cause of the disease. Oxidative stress occurs in many autoimmune diseases, along with the excess production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The sources of such reactive species include NADPH oxidases (NOXs), the mitochondrial electron transport chain, nitric oxide synthases, nitrite reductases, and the hydrogen sulfide producing enzymes cystathionine-ß synthase and cystathionine-γ lyase. Superoxide undergoes a dismutation reaction to generate hydrogen peroxide which, in the presence of transition metal ions (e.g. ferrous ions), forms the hydroxyl radical. The enzyme myeloperoxidase, present in inflammatory cells, produces hypochlorous acid, and in healthy individuals ROS and RNS production by phagocytic cells is important in microbial killing. Both low molecular weight antioxidant molecules and antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and peroxiredoxin remove ROS. However, when ROS production exceeds the antioxidant protection, oxidative stress occurs. Oxidative post-translational modifications of proteins then occur. Sometimes protein modifications may give rise to neoepitopes that are recognized by the immune system as 'non-self' and result in the formation of autoantibodies. The detection of autoantibodies against specific antigens, might improve both early diagnosis and monitoring of disease activity. Promising diagnostic autoantibodies include anti-carbamylated proteins and anti-oxidized type II collagen antibodies. Some of the most promising future strategies for redox-based therapeutic compounds are the activation of endogenous cellular antioxidant systems (e.g. Nrf2-dependent pathways), inhibition of disease-relevant sources of ROS/RNS (e.g. isoform-specific NOX inhibitors), or perhaps specifically scavenging disease-related ROS/RNS via site-specific antioxidants.

Antibodies to post-translationally modified insulin as a novel biomarker for prediction of type 1 diabetes in children.

AIMS/HYPOTHESIS: We have shown that autoimmunity to insulin in type 1 diabetes may result from neoepitopes induced by oxidative post-translational modifications (oxPTM). Antibodies specific to oxPTM-insulin (oxPTM-INS-Ab) are present in most newly diagnosed individuals with type 1 diabetes and are more common than autoantibodies to native insulin. In this study, we investigated whether oxPTM-INS-Ab are present before clinical onset of type 1 diabetes, and evaluated the ability of oxPTM-INS-Ab to identify children progressing to type 1 diabetes. METHODS: We used serum samples collected longitudinally from the 'All Babies in Southeast Sweden (ABIS)' cohort tested for the gold standard islet autoantibodies to insulin (IAA), GAD (GADA), tyrosine phosphatase 2 (IA-2A) and zinc transporter 8 (ZnT8A). We studied 23 children who progressed to type 1 diabetes (progr-T1D) and 63 children who did not progress to type 1 diabetes (NP) after a median follow-up of 10.8 years (interquartile range 7.7-12.8). Of the latter group, 32 were positive for one or more islet autoantibodies (NP-AAB+). oxPTM-INS-Ab to insulin modified by •OH or HOCl were measured by our developed ELISA platform. RESULTS: Antibodies to at least one oxPTM-INS were present in 91.3% of progr-T1D children. oxPTM-INS-Ab co-existed with GADA, IA-2A, IAA or ZnT8A in 65.2%, 56.5%, 38.9% and 33.3% progr-T1D children, respectively. In addition, oxPTM-INS-Ab were present in 17.4%, 26.1%, 38.9% and 41.6% of progr-T1D children who were negative for GADA, IA-2A, IAA and ZnT8A, respectively. •OH-INS-Ab were more common in progr-T1D children than in NP-AAB+ children (82.6% vs 19%; p < 0.001) and allowed discrimination between progr-T1D and NP-AAB+ children with 74% sensitivity and 91% specificity. None of the NP-AAB- children were positive for oxPTM-INS-Ab. CONCLUSIONS/INTERPRETATION: oxPTM-INS-Ab are present before the clinical onset of type 1 diabetes and can identify children progressing to type 1 diabetes.

Autoantibodies to post-translationally modified type I and II collagen in Charcot neuroarthropathy in subjects with type 2 diabetes mellitus.

AIMS: Charcot neuroarthropathy (CN) is a disabling complication, culminating in bone destruction and involving joints and articular cartilage with high inflammatory environment. Its real pathogenesis is as yet unknown. In autoinflammatory diseases, such as rheumatoid arthritis, characterized by inflammation and joint involvement, autoantibodies against oxidative post-translationally modified (oxPTM) collagen type I (CI) and type II (CII) were detected. Therefore, the aim of our study was to assess the potential involvement of autoimmunity in charcot neuroarthropathy, investigating the presence of autoantibodies oxPTM-CI and oxPTM-CII, in participants with charcot neuroarthropathy. METHODS: In this case-control study, we enrolled 124 participants with type 2 diabetes mellitus (47 with charcot neuroarthropathy, 37 with diabetic peripheral neuropathy without charcot neuroarthropathy, and 40 with uncomplicated diabetes), and 32 healthy controls. The CI and CII were modified with ribose and other oxidant species, and the modifications were evaluated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of sera from the participants was analyzed with enzyme-linked immunosorbent assay. RESULTS: Age, body mass index, waist and hip circumferences, and lipid profile were similar across the 4 groups, as well as glycated hemoglobin and duration of diabetes among people with diabetes. An increased binding to both native and all oxidation-modified forms of CII was found in participants with CN and diabetic neuropathy. Conversely, for CI, an aspecific increased reactivity was noted. CONCLUSIONS: Our results detected the presence of autoantibodies against oxidative post-translational modified collagen, particularly type 2 collagen, in participants with charcot neuroarthropathy and diabetic neuropathy, suggesting the possible involvement of autoimmunity. Further studies are required to understand the role of autoimmunity in the pathogenesis of charcot neuroarthropathy.

Progress and clinical potential of antibody-targeted therapy for arthritic damage.

INTRODUCTION: The advent of biologic drugs like infliximab, Etanercept, rituximab and tocilizumab has greatly improved the treatment of rheumatoid arthritis, however, increased risk of infection and high cost still remain unmet needs. A new generation of targeted therapeutics is being developed to target payload drug specifically to arthritic tissue; to concentrate the drug in the disease area and limit the off target systemic exposure. This might also reduce total effective dose. AREAS COVERED: This article summarizes the properties and progress of targeted therapies that have been published on PubMed, and addresses their clinical potential. Expert commentary: Incredible progress with targeted therapies has already been made in the short time since the principle was first proven in animal models in 2007 when targeting payload drug to overexpressed oncofetal domain of fibronectin in inflamed arthritic joints.

In vivo optical imaging of early osteoarthritis using an antibody specific to damaged arthritic cartilage.

BACKGROUND: The lack of specific and sensitive serum and radiographic biomarkers for early diagnosis of osteoarthritis (OA) as well as for monitoring subtle changes in disease activity in clinical trials has hampered the development of treatments for OA. We previously showed that 1-11E, a human single chain fragment variable (scFv) specific to collagen type II that has been post-translationally modified by reactive oxidants (ROS-CII), binds exclusively to arthritic cartilage. Here we test the validity of 1-11E as a radiographic biomarker for early disease in experimental OA. METHODS: Murine OA was induced by destabilisation of the medial meniscus (DMM) in adult male mice. Immunohistochemistry of destabilised or sham-operated knees was performed from 2 to 8 weeks post-surgery with Cy5.5-labelled 1-11E and negative control scFv, C7. Prospective in vivo optical images were taken 4 and 8 weeks post-DMM following intra-articular injection of Cy5.5-labelled scFvs, or intravenous injection of Cy5.5-labelled full length monoclonal antibodies (mAbs). RESULTS: Specific cartilage staining with 1-11E was apparent as early as 4 weeks post-DMM at the time of earlier cartilage degradation assessed by histology. Prospective in vivo optical images taken 4 and 8 weeks post-DMM following local intra-articular injection of Cy5.5-labelled scFv (n = 7) showed specific in vivo retention of Cys5.5-1-11E scFv following local administration into the knee joint (tissue half-life >78 hours, n = 7, signal to noise ratio (SNR) > 2.1). Specific localization of Cys-5.5-1-11E-mAb to DMM knees (SNR >1.65) was also observed (p < 0.01, n = 8, SNR >1.65). In both cases the SNR increased with time post-DMM. CONCLUSIONS: 1-11E binds specifically to early osteoarthritic cartilage and can be used as a radiographic biomarker following local or systemic delivery to facilitate early diagnosis and monitor disease progression in OA.

Antibodies to post-translationally modified insulin in type 1 diabetes.

AIM/HYPOTHESIS: Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. METHODS: oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAA was measured using the gold-standard radiobinding assay (RBA). RESULTS: MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; and glycation of Lys29 and Phe1 in chain B. Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin. CONCLUSION/INTERPRETATION: These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.

Serum neurofilament light chain is a biomarker of human spinal cord injury severity and outcome.

BACKGROUND: Neurofilaments (Nf) are major structural proteins that occur exclusively in neurons. In spinal cord injury (SCI), the severity of disease is quantified by clinical measures that have limited sensitivity and reliability, and no blood-based biomarker has been established to further stratify the degree of injury. We aimed to examine a serum-based NfL immunoassay as predictor of the clinical outcome in SCI. METHODS: Longitudinal measurement of serum NfL was performed in patients with central cord syndrome (CCS, n=4), motor-incomplete SCI (iSCI, n=10), motor-complete SCI (cSCI, n=13) and healthy controls (HC, n=67), and correlated with clinical severity, neurological outcome, and neuroprotective effect of the drug minocycline. RESULTS: Baseline NfL levels were higher in iSCI (21 pg/mL) and cSCI (70 pg/mL) than in HC (5 pg/mL, p=0.006 and p<0.001) and CCS (6 pg/mL, p=0.025 and p=0.010). Levels increased over time (p<0.001) and remained higher in cSCI versus iSCI (p=0.011) and than in CCS (p<0.001). NfL levels correlated with American Spinal Injury Association (ASIA) motor score at baseline (r=-0.53, p=0.004) and after 24 h (r=-0.69, p<0.001) and 3-12-month motor outcome (baseline NfL: r=-0.43, p=0.026 and 24 h NfL: r=-0.72, p<0.001). Minocycline treatment showed decreased NfL levels in the subgroup of cSCI patients. CONCLUSIONS: Serum NfL concentrations in SCI patients show a close correlation with acute severity and neurological outcome. Our data provide evidence that serum NfL is of prognostic value in SCI patients for the first time. Further, blood NfL levels may qualify as drug response markers in SCI.

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency.

INTRODUCTION: We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis. METHODS: Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis. RESULTS: 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10). CONCLUSIONS: Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

Oxidative post-translational modifications and their involvement in the pathogenesis of autoimmune diseases.

Tissue inflammation results in the production of numerous reactive oxygen, nitrogen and chlorine species, in addition to the products of lipid and sugar oxidation. Some of these products are capable of chemically modifying amino acids. This in turn results in changes to the structure and function of proteins. Increasing evidence demonstrates that such oxidative post-translational modifications result in the generation of neo-epitopes capable of eliciting both innate and adaptive immune responses. In this paper, we focus on how free radicals and related chemical species generated in inflammatory environments modulate the antigenicity of self-proteins, resulting in immune responses which involve the generation of autoantibodies against key autoantigens in autoimmune diseases. As examples, we will focus on Ro-60 and C1q in systemic lupus erythematosus, along with type-II collagen in rheumatoid arthritis. This review also covers some of the emerging literature which demonstrates that neo-epitopes generated by oxidation are conserved, as exemplified by the evolutionarily conserved pathogen-associated molecular patterns (PAMPs). We discuss how these observations relate to the pathogenesis of both human autoimmune diseases and inflammatory disease, such as atherosclerosis. The potential for these neo-epitopes and the immune responses against them to act as biomarkers or therapeutic targets is also discussed.

Autoantibodies to posttranslational modifications in rheumatoid arthritis.

Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell development in vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential.

Increased neurofilament light chain blood levels in neurodegenerative neurological diseases.

OBJECTIVE: Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are cytoskeletal proteins of neurons and their release into cerebrospinal fluid has shown encouraging results as a biomarker for neurodegeneration. This study aimed to validate the quantification of the Nf light chain (NfL) in blood samples, as a biofluid source easily accessible for longitudinal studies. METHODS: We developed and applied a highly sensitive electrochemiluminescence (ECL) based immunoassay for quantification of NfL in blood and CSF. RESULTS: Patients with Alzheimer's disease (AD) (30.8 pg/ml, n=20), Guillain-Barré-syndrome (GBS) (79.4 pg/ml, n=19) or amyotrophic lateral sclerosis (ALS) (95.4 pg/ml, n=46) had higher serum NfL values than a control group of neurological patients without evidence of structural CNS damage (control patients, CP) (4.4 pg/ml, n=68, p<0.0001 for each comparison, p=0.002 for AD patients) and healthy controls (HC) (3.3 pg/ml, n=67, p<0.0001). Similar differences were seen in corresponding CSF samples. CSF and serum levels correlated in AD (r=0.48, p=0.033), GBS (r=0.79, p<0.0001) and ALS (r=0.70, p<0.0001), but not in CP (r=0.11, p=0.3739). The sensitivity and specificity of serum NfL for separating ALS from healthy controls was 91.3% and 91.0%. CONCLUSIONS: We developed and validated a novel ECL based sandwich immunoassay for the NfL protein in serum (NfL(Umea47:3)); levels in ALS were more than 20-fold higher than in controls. Our data supports further longitudinal studies of serum NfL in neurodegenerative diseases as a potential biomarker of on-going disease progression, and as a potential surrogate to quantify effects of neuroprotective drugs in clinical trials.

Structural guided scaffold phage display libraries as a source of bio-therapeutics.

We have developed a structurally-guided scaffold phage display strategy for identification of ligand mimetic bio-therapeutics. As a proof of concept we used the ligand of integrin αvß6, a tumour cell surface receptor and a major new target for imaging and therapy of many types of solid cancer. NMR structure analysis showed that RGD-helix structures are optimal for αvß6 ligand-interaction, so we designed novel algorithms to generate human single chain fragment variable (scFv) libraries with synthetic VH-CDR3 encoding RGD-helix hairpins with helices of differing pitch, length and amino acid composition. Study of the lead scFv clones D25scFv and D34scFv and their corresponding VH-CDR3 derived peptides, D25p and D34p, demonstrated: specific binding to recombinant and cellular αvß6; inhibition of αvß6-dependent cell and ligand adhesion, αvß6-dependent cell internalisation; and selective retention by αvß6-expressing, but not αvß6-negative, human xenografts. NMR analysis established that both the D25p and D34p retained RGD-helix structures confirming the success of the algorithm. In conclusion, scFv libraries can be engineered based on ligand structural motifs to increase the likelihood of developing powerful bio-therapeutics.

Autoantibodies to posttranslationally modified type II collagen as potential biomarkers for rheumatoid arthritis.

OBJECTIVE: Type II collagen (CII) posttranslationally modified by reactive oxygen species (ROS-CII) that are present in the inflamed joint is an autoantigen in rheumatoid arthritis (RA). The aim of this study was to investigate the potential use of anti-ROS-CII autoantibodies as a biomarker of RA. METHODS: CII was exposed to oxidants that are present in the rheumatoid joint. Autoreactivity to ROS-CII was assessed by enzyme-linked immunosorbent assays in synovial fluid (SF) and serum samples obtained from patients during various phases of RA. This group included disease-modifying antirheumatic drug (DMARD)-naive patients with early RA (n = 85 serum samples) and patients with established RA (n = 80 serum and 50 SF samples), who were categorized as either DMARD responders or DMARD nonresponders. Control subjects included anti-citrullinated protein antibody (ACPA)-positive patients with arthralgia (n = 58 serum samples), patients with osteoarthritis (OA; n = 49 serum and 52 SF samples), and healthy individuals (n = 51 serum samples). RESULTS: Reactivity to ROS-CII among DMARD-naive patients with early RA was significantly higher than that among patients with ACPA-positive arthralgia, patients with OA, and healthy control subjects (P < 0.0001), with 92.9% of serum samples from the patients with early RA binding to anti-ROS-II. There was no significant difference in anti-ROS-CII reactivity between ACPA-positive and ACPA-negative patients with RA, with 93.8% and 91.6% of serum samples, respectively, binding to ROS-CII. The sensitivity and specificity of binding to ROS-CII in patients with early RA were 92% and 98%, respectively. Among patients with established RA, serum reactivity in DMARD nonresponders was significantly higher than that in DMARD responders (P < 0.01); 58.3% of serum samples from nonresponders and 7.6% of serum samples from responders bound to HOCl-ROS, while the respective values for SF were 70% and 60%. In patients with longstanding RA, autoreactivity to ROS-CII changed longitudinally. CONCLUSION: Autoantibodies to ROS-CII have the potential to become diagnostic biomarkers of RA.

A comparative study of CSF neurofilament light and heavy chain protein in MS.

BACKGROUND: There is a lack of reliable biomarkers of axonal degeneration. Neurofilaments are promising candidates to fulfil this task. We compared two highly sensitive assays to measure two subunits of the neurofilament protein (neurofilament light (NfL) and neurofilament heavy chain (NfH)). METHODS: We evaluated the analytical and clinical performance of the UmanDiagnostics NF-light(®) enzyme-linked immunosorbent assay (ELISA) in the cerebrospinal fluid (CSF) of a group of 148 patients with clinically isolated syndrome (CIS) or multiple sclerosis (MS), and 72 controls. We compared our results with referring levels of our previously-developed CSF NfH(SMI35) assay. RESULTS: Exposure to room temperature (up to 8 days) or repetitive thawing (up to 4 thaws) did not influence measurement of NfL concentrations. Values of NfL were higher in all disease stages of CIS/MS, in comparison to controls (p ≤ 0.001). NfL levels correlated with the Expanded Disability Status Scale (EDSS) score in patients with relapsing disease (r(s) = 0.31; p = 0.002), spinal cord relapses and with CSF markers of acute inflammation. The ability of NfL to distinguish patients from controls was greater than that of NfH(SMI35) in both CIS patients (p = 0.001) and all MS stages grouped together (p = 0.035). CONCLUSIONS: NfL proved to be a stable protein, an important prerequisite for a reliable biomarker, and the NF-light(®) ELISA performed better in discriminating patients from controls, compared with the ECL-NfH(SMI35) immunoassay. We confirmed and expanded upon previous findings regarding neurofilaments as quantitative markers of neurodegeneration. Our results further support the role of neurofilaments as a potential surrogate measure for neuroprotective treatment in MS studies.

Targeted delivery of cytokine therapy to rheumatoid tissue by a synovial targeting peptide.

OBJECTIVES: The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. METHODS: Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. RESULTS: In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1ß  in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. CONCLUSIONS: The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.