Glycolysis: An early marker for vancomycin-specific T-cell activation.

BACKGROUND: Vancomycin, a glycopeptide antibiotic used for Gram-positive bacterial infections, has been linked with drug reaction with eosinophilia and systemic symptoms (DRESS) in HLA-A*32:01-expressing individuals. This is associated with activation of T lymphocytes, for which glycolysis has been isolated as a fuel pathway following antigenic stimulation. However, the metabolic processes that underpin drug-reactive T-cell activation are currently undefined and may shed light on the energetic conditions needed for the elicitation of drug hypersensitivity or tolerogenic pathways. Here, we sought to characterise the immunological and metabolic pathways involved in drug-specific T-cell activation within the context of DRESS pathogenesis using vancomycin as model compound and drug-reactive T-cell clones (TCCs) generated from healthy donors and vancomycin-hypersensitive patients. METHODS: CD4+ and CD8+ vancomycin-responsive TCCs were generated by serial dilution. The Seahorse XFe96 Analyzer was used to measure the extracellular acidification rate (ECAR) as an indicator of glycolytic function. Additionally, T-cell proliferation and cytokine release (IFN-γ) assay were utilised to correlate the bioenergetic characteristics of T-cell activation with in vitro assays. RESULTS: Model T-cell stimulants induced non-specific T-cell activation, characterised by immediate augmentation of ECAR and rate of ATP production (JATPglyc). There was a dose-dependent and drug-specific glycolytic shift when vancomycin-reactive TCCs were exposed to the drug. Vancomycin-reactive TCCs did not exhibit T-cell cross-reactivity with structurally similar compounds within proliferative and cytokine readouts. However, cross-reactivity was observed when analysing energetic responses; TCCs with prior specificity for vancomycin were also found to exhibit glycolytic switching after exposure to teicoplanin. Glycolytic activation of TCC was HLA restricted, as exposure to HLA blockade attenuated the glycolytic induction. CONCLUSION: These studies describe the glycolytic shift of CD4+ and CD8+ T cells following vancomycin exposure. Since similar glycolytic switching is observed with teicoplanin, which did not activate T cells, it is possible the master switch for T-cell activation is located upstream of metabolic signalling.

Patients with naproxen-induced liver injury display T-cell memory responses toward an oxidative (S)-O-desmethyl naproxen metabolite but not the acyl glucuronide.

BACKGROUND: Exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen (IBU) and naproxen (NAP) is associated with idiosyncratic drug-induced liver injury (DILI). Carboxylate bioactivation into reactive metabolites (e.g., acyl glucuronides, AG) and resulting T-cell activation is hypothesized as causal for this adverse event. However, conclusive evidence supporting this is lacking. METHODS: In this work, we identify CD4+ and CD8+ T-cell hepatic infiltration in a biopsy from an IBU DILI patient. Lymphocyte transformation test and IFN-γ ELIspot, conducted on peripheral blood mononuclear cells (PBMCs) of patients with NAP-DILI, were used to explore drug-specific T-cell activation. T-cell clones (TCC) were generated and tested for drug specificity, phenotype/function, and pathways of T-cell activation. Cells were exposed to NAP, its oxidative metabolite 6-O-desmethyl NAP (DM-NAP), its AG or synthesized NAP-AG human-serum albumin adducts (NAP-AG adduct). RESULTS: CD4+ and CD8+ T-cells from patients expressing a range of different Vß receptors were stimulated to proliferate and secrete IFN-γ and IL-22 when exposed to DM-NAP, but not NAP, NAP-AG or the NAP-AG adduct. Activation of the CD4+ TCC was HLA-DQ-restricted and dependent on antigen presenting cells (APC); most TCC were activated with DM-NAP-pulsed APC, while fixation of APC blocked the T-cell response. Cross-reactivity was not observed with structurally-related drugs. CONCLUSION: Our results confirm hepatic T-cell infiltrations in NSAID-induced DILI, and show a T-cell memory response toward DM-NAP indicating an immune-mediated basis for the adverse event. Whilst bioactivation at the carboxylate group is widely hypothesized to be pathogenic for NSAID associated DILI, we found no evidence of this with NAP.

Lymphocyte transformation tests predict delayed-type allergy to piperacillin/tazobactam in patients with cystic fibrosis.

BACKGROUND: Antibiotic treatment is crucial for patients with chronic bacterial infections. Suspected drug allergies often lead to inconsistent therapies and challenging clinical management for patients and caregivers. The objective of this study was to evaluate the value of lymphocyte transformation tests in comparison to skin tests for the prediction of delayed-type allergic reactions. METHODS: This prospective, observational study tested the diagnostic value of skin prick tests, intradermal tests (reading: 15 min and 72 h) and lymphocyte transformations tests for the prediction of allergic reactions in CF patients with physician reported allergy to piperacillin/tazobactam, meropenem and ceftazidime. The tests were performed directly before a 14d intravenous drug challenge. RESULTS: We performed 33 drug challenges in 29 subjects. 21 drug challenges were negative (63 %); 12 lead to a reaction (37 %), of those 2 were immediate and 10 were delayed-type. 100 % of the skin prick tests were negative. 97 % (33/34) of the intradermal tests with early reading and 100 % of the intradermal tests with late reading yielded negative results. 5/11 patients who experienced a delayed-type reaction during the drug challenge had a positive lymphocyte transformations test. All 17 patients who did not react had a negative lymphocyte transformations test. For piperacillin/tazobactam, 4/5 patients who experienced a delayed-type reaction during the drug challenge had positive lymphocyte transformations tests. Hence, for piperacillin/tazobactam, the sensitivity of the lymphocyte transformation test for prediction of reactions was 80.0 % and the specificity 100 %. CONCLUSION: We demonstrate that the lymphocyte transformation test predicts delayed-type allergy to piperacillin/tazobactam in contrast to skin tests.

Investigating the Immune Basis of Green Tea Extract Induced Liver Injury in Healthy Donors Expressing HLA-B*35:01.

Epigallocatechin-3-O-gallate (EGCG) is the major component of green tea extract, commonly found in dietary supplements, and has been associated with immune-mediated liver injury. The purpose of this study was to investigate the immunogenicity of EGCG in healthy donors expressing HLA-B*35:01, and characterize EGCG responsive T-cell clones. We have shown that EGCG can prime peripheral blood mononuclear cells and T-cells from donors with and without the HLA-B*35:01 allele. T-cell clones were CD4+ve and capable of secreting Th1, Th2, and cytolytic molecules. These data demonstrate that EGCG can activate T-cells in vitro, suggesting a significant role in the pathogenesis of green tea extract induced liver injury.

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment.

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilized immune cells from drug-naïve donors, recently established immune cell coculture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and 5 training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base, and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell coculture; however, CD4+ clones displaying reactivity were detected toward 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients.

Why drug exposure is frequently associated with T-cell mediated cutaneous hypersensitivity reactions.

Cutaneous hypersensitivity reactions represent the most common manifestation of drug allergy seen in the clinic, with 25% of all adverse drug reactions appearing in the skin. The severity of cutaneous eruptions can vastly differ depending on the cellular mechanisms involved from a minor, self-resolving maculopapular rash to major, life-threatening pathologies such as the T-cell mediated bullous eruptions, i.e., Stevens Johnson syndrome/toxic epidermal necrolysis. It remains a significant question as to why these reactions are so frequently associated with the skin and what factors polarise these reactions towards more serious disease states. The barrier function which the skin performs means it is constantly subject to a barrage of danger signals, creating an environment that favors elicitation. Therefore, a critical question is what drives the expansion of cutaneous lymphocyte antigen positive, skin homing, T-cell sub-populations in draining lymph nodes. One answer could be the heterologous immunity hypothesis whereby tissue resident memory T-cells that express T-cell receptors (TCRs) for pathogen derived antigens cross-react with drug antigen. A significant amount of research has been conducted on skin immunity in the context of contact allergy and the role of tissue specific antigen presenting cells in presenting drug antigen to T-cells, but it is unclear how this relates to epitopes derived from circulation. Studies have shown that the skin is a metabolically active organ, capable of generating reactive drug metabolites. However, we know that drug antigens are displayed systemically so what factors permit tolerance in one part of the body, but reactivity in the skin. Most adverse drug reactions are mild, and skin eruptions tend to be visible to the patient, whereas minor organ injury such as transient transaminase elevation is often not apparent. Systemic hypersensitivity reactions tend to have early cutaneous manifestations, the progression of which is halted by early diagnosis and treatment. It is apparent that the preference for cutaneous involvement of drug hypersensitivity reactions is multi-faceted, therefore this review aims to abridge the findings from literature on the current state of the field and provide insight into the cellular and metabolic mechanisms which may contribute to severe cutaneous adverse reactions.

HLA-C*15:02 and epidermal growth factor receptor inhibitor-induced erosive pustular dermatosis of the scalp.

Epidermal growth factor receptor inhibitors (EGFRIs) are widely used to treat various types of malignancies. One of the common adverse reactions is cutaneous toxicity, mostly presenting as acneiform eruptions, paronychia and xerosis. Erosive pustular dermatosis of the scalp (EPDS) is a rare cutaneous adverse reaction that develops during treatment with EGFRIs. The pathogenesis of EGFRI-induced EPDS is poorly understood. Here we present three cases of EPDS induced by EGFRIs. The proteins LTA4H (leukotriene A-4 hydrolase), METAP1 (methionine aminopeptidase 1), BID (BH3-interacting domain death agonist), SMAD1 (mothers against decapentaplegic homologue), PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A), YES1 (tyrosine-protein kinase Yes) and EGFL7 (epidermal growth factor-like protein 7) were significantly upregulated in EGFRI-stimulated peripheral blood mononuclear cell cultures, and validated in the lesions. All of the proteins colocalized with CD4+ and CD8+ T-cell expression. Next-generation-based human leucocyte antigen (HLA) typing showed all patients carried HLA-C*15:02, and modelling studies showed that afatinib and erlotinib bound well within the E/F binding pockets of HLA-C*15:02. Moreover, T cells were preferentially activated by EGFRIs in individuals carrying HLA-C*15:02. The case series revealed that EGFRI-induced EPDS may be mediated by drug-specific T cells.

Characterization of Drug-Specific CD4+ T-Cells Reveals Possible Roles of HLA Class II in the Pathogenesis of Carbamazepine Hypersensitivity Reactions.

Carbamazepine (CBZ) is an aromatic anticonvulsant known to cause drug hypersensitivity reactions, which range in severity from relatively mild maculopapular exanthema to potentially fatal Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS-TEN). These reactions are known to be associated with human leukocyte antigen (HLA) class I alleles, and CBZ interacts preferentially with the related HLA proteins to activate CD8+ T-cells. This study aimed to evaluate the contribution of HLA class II in the effector mechanism(s) of CBZ hypersensitivity. CBZ-specific T-cells clones were generated from two healthy donors and two hypersensitive patients with high-risk HLA class I markers. Phenotype, function, HLA allele restriction, response pathways, and cross-reactivity of CBZ-specific T-cells were assessed using flow cytometry, proliferation analysis, enzyme-linked immunosorbent spot, and enzyme-linked immunosorbent assay. The association between HLA class II allele restriction and CBZ hypersensitivity was reviewed using Allele Frequency Net Database. Forty-four polyclonal CD4+ CBZ-specific T-cell clones were generated and found to be restricted to HLA-DR, particularly HLA-DRB1*07:01. This CD4+-mediated response proceeded through a direct pharmacological interaction between CBZ and HLA-DR molecules. Similar to the CD8+ response, CBZ-stimulated CD4+ clones secreted granulysin, a key mediator of SJS-TEN. Our database review revealed an association between HLA-DRB1*07:01 and CBZ-induced SJS-TEN. These findings implicate HLA class II antigen presentation as an additional pathogenic factor for CBZ hypersensitivity reactions. Both HLA class II molecules and drug-responsive CD4+ T-cells should be evaluated further to gain better insights into the pathogenesis of drug hypersensitivity reactions.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Identification of flucloxacillin-modified hepatocellular proteins: implications in flucloxacillin-induced liver injury.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver injury. Although expression of HLA-B*57:01 is associated with increased susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the provision of flucloxacillin-modified peptides that are presented to T-cells by the protein encoded by the risk allele. In this study, we have shown that flucloxacillin binds to multiple proteins within human primary hepatocytes, including major hepatocellular proteins (hemoglobin and albumin) and mitochondrial proteins. Inhibition of membrane transporters multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) appeared to reduce the levels of covalent binding. A diverse range of proteins with different functions was found to be targeted by flucloxacillin, including adaptor proteins (14-3-3), proteins with catalytic activities (liver carboxylesterase 1, tRNA-splicing endonuclease subunit Sen2, All-trans-retinol dehydrogenase ADH1B, Glutamate dehydrogenase 1 mitochondrial, Carbamoyl-phosphate synthase [ammonia] mitochondrial), and transporters (hemoglobin, albumin, and UTP-glucose-1-phosphate uridylyltransferase). These flucloxacillin-modified intracellular proteins could provide a potential source of neoantigens for HLA-B*57:01 presentation by hepatocytes. More importantly, covalent binding to critical cellular proteins could be the molecular initiating events that lead to flucloxacillin-induced cholestasis Data are available via ProteomeXchange with identifier PXD038581.

Detection of Hepatic Drug Metabolite-Specific T-Cell Responses Using a Human Hepatocyte, Immune Cell Coculture System.

Drug-responsive T-cells are activated with the parent compound or metabolites, often via different pathways (pharmacological interaction and hapten). An obstacle to the investigation of drug hypersensitivity is the scarcity of reactive metabolites for functional studies and the absence of coculture systems to generate metabolites in situ. Thus, the aim of this study was to utilize dapsone metabolite-responsive T-cells from hypersensitive patients, alongside primary human hepatocytes to drive metabolite formation, and subsequent drug-specific T-cell responses. Nitroso dapsone-responsive T-cell clones were generated from hypersensitive patients and characterized in terms of cross-reactivity and pathways of T-cell activation. Primary human hepatocytes, antigen-presenting cells, and T-cell cocultures were established in various formats with the liver and immune cells separated to avoid cell contact. Cultures were exposed to dapsone, and metabolite formation and T-cell activation were measured by LC-MS and proliferation assessment, respectively. Nitroso dapsone-responsive CD4+ T-cell clones from hypersensitive patients were found to proliferate and secrete cytokines in a dose-dependent manner when exposed to the drug metabolite. Clones were activated with nitroso dapsone-pulsed antigen-presenting cells, while fixation of antigen-presenting cells or omission of antigen-presenting cells from the assay abrogated the nitroso dapsone-specific T-cell response. Importantly, clones displayed no cross-reactivity with the parent drug. Nitroso dapsone glutathione conjugates were detected in the supernatant of hepatocyte immune cell cocultures, indicating that hepatocyte-derived metabolites are formed and transferred to the immune cell compartment. Similarly, nitroso dapsone-responsive clones were stimulated to proliferate with dapsone, when hepatocytes were added to the coculture system. Collectively, our study demonstrates the use of hepatocyte immune cell coculture systems to detect in situ metabolite formation and metabolite-specific T-cell responses. Similar systems should be used in future diagnostic and predictive assays to detect metabolite-specific T-cell responses when synthetic metabolites are not available.

Activation of tolvaptan-responsive T-cell clones with the structurally-related mozavaptan.

Tolvaptan is an effective drug for the treatment of autosomal dominant polycystic kidney disease, but its use is associated with a significant risk of T-cell-mediated liver injury in a small number of patients. An important clinical conundrum following the contraindication of tolvaptan is whether administration of agents of similar pharmacological action and structure will be tolerated. Herein, we addressed this question through the exposure of tolvaptan-responsive T-cell clones to similar pharmaceutical agents. Whilst lixivaptan and conivaptan did not activate tolvaptan-responsive T-cells, mozavaptan evoked proliferative responses comparable with tolvaptan itself, indicating that there may be collateral immunological intolerance to this compound as a product of sensitization to tolvaptan.

Shared Clavulanate and Tazobactam Antigenic Determinants Activate T-Cells from Hypersensitive Patients.

ß-Lactamase inhibitors such as clavulanic acid and tazobactam were developed to overcome ß-lactam antibiotic resistance. Hypersensitivity reactions to these drugs have not been studied in detail, and the antigenic determinants that activate T-cells have not been defined. The objectives of this study were to (i) characterize clavulanate- and tazobactam-responsive T-cells from hypersensitive patients, (ii) explore clavulanate and tazobactam T-cell crossreactivity, and (iii) define the antigenic determinants that contribute to T-cell reactivity. Antigen specificity, pathways of T-cell activation, and crossreactivity with clavulanate- and tazobactam-specific T-cell clones were assessed by proliferation and cytokine release assays. Antigenic determinants were analyzed by mass spectrometry-based proteomics methods. Clavulanate- and tazobactam-responsive CD4+ T-cell clones were stimulated to proliferate and secrete IFN-γ in an MHC class II-restricted and dose-dependent manner. T-cell activation with clavulanate- and tazobactam was dependent on antigen presenting cells because their fixation prevented the T-cell response. Strong crossreactivity was observed between clavulanate- and tazobactam-T-cells; however, neither drug activated ß-lactam antibiotic-responsive T-cells. Mass spectrometric analysis revealed that both compounds form multiple antigenic determinants with lysine residues on proteins, including an overlapping aldehyde and hydrated aldehyde adduct with mass additions of 70 and 88 Da, respectively. Collectively, these data show that although clavulanate and tazobactam are structurally distinct, the antigenic determinants formed by both drugs overlap, which explains the observed T-cell cross-reactivity.

Pathology of drug hypersensitivity reactions and mechanisms of immune tolerance.

Immune-mediated type IV adverse drug reactions are idiosyncratic in nature, generally not related to the primary or secondary pharmacology of the drug. Due to their complex nature and rarity, these iatrogenic reactions are seldom predicted or encountered during preclinical/early clinical development stages, and often precipitate upon exposure to wider populations (i.e. phase III onwards). They confer a burden on the healthcare sector in both a clinical and financial sense presenting a severe impediment to the drug discovery and development process. Research over the past 50 years has improved our understanding of these reactions markedly as both in vitro and in vivo studies have placed the role of the immune system, in particular; drug-responsive T cells, firmly in the spotlight as the mediators of these reactions. Indeed, the role of different populations of T cells in adverse events and the interaction of drug molecules with HLA proteins expressed on the surface of antigen-presenting cells is of considerable interest. Herein, this review examines the pathways of immune-mediated adverse events including the various T cell subtypes implicated and the mechanisms of T cell activation. Additionally, we address the enigma of immunological tolerance and explore the role tolerance plays in determination of susceptibility to such adverse events even in individuals carrying immunogenic liabilities.

Pathology of T-cell-mediated drug hypersensitivity reactions and impact of tolerance mechanisms on patient susceptibility.

PURPOSE OF REVIEW: T-cell-mediated drug hypersensitivity is responsible for significant morbidity and mortality, and represents a substantial clinical concern. The purpose of this article is to focus on T-cell reactions and discuss recent advances in disease pathogenesis by exploring the impact of tolerance mechanisms in determining susceptibility in genetically predisposed patients. RECENT FINDINGS: Certain drugs preferentially activate pathogenic T cells that have defined pathways of effector function. Thus, a critical question is what extenuating factors influence the direction of immune activation. A large effort has been given towards identifying phenotypic (e.g., infection) or genotypic (e.g., human leukocyte antigen) associations which predispose individuals to drug hypersensitivity. However, many individuals expressing known risk factors safely tolerate drug administration. Thus, mechanistic insight is needed to determine what confers this tolerance. Herein, we discuss recent clinical/mechanistic findings which indicate that the direction in which the immune system is driven relies upon a complex interplay between co-stimulatory/co-regulatory pathways which themselves depend upon environmental inputs from the innate immune system. SUMMARY: It is becoming increasingly apparent that tolerance mechanisms impact on susceptibility to drug hypersensitivity. As the field moves forward it will be interesting to discover whether active tolerance is the primary response to drug exposure, with genetic factors such as HLA acting as a sliding scale, influencing the degree of regulation required to prevent clinical reactions in patients.

Advances in Our Understanding of the Interaction of Drugs with T-cells: Implications for the Discovery of Biomarkers in Severe Cutaneous Drug Reactions.

Drugs can activate different cells of the immune system and initiate an immune response that can lead to life-threatening diseases collectively known as severe cutaneous adverse reactions (SCARs). Antibiotics, anticonvulsants, and antiretrovirals are involved in the development of SCARs by the activation of αß naïve T-cells. However, other subsets of lymphocytes known as nonconventional T-cells with a limited T-cell receptor repertoire and innate and adaptative functions also recognize drugs and drug-like molecules, but their role in the pathogenesis of SCARs has only just begun to be explored. Despite 30 years of advances in our understanding of the mechanisms in which drugs interact with T-cells and the pathways for tissue injury seen during T-cell activation, at present, the development of useful clinical biomarkers for SCARs or predictive preclinical in vitro assays that could identify immunogenic moieties during drug discovery is an unmet goal. Therefore, the present review focuses on (i) advances in the understanding of the pathogenesis of SCARs reactions, (ii) a description of the interaction of drugs with conventional and nonconventional T-cells, and (iii) the current state of soluble blood circulating biomarker candidates for SCARs.