Unrepresentative big surveys significantly overestimated US vaccine uptake.

Surveys are a crucial tool for understanding public opinion and behaviour, and their accuracy depends on maintaining statistical representativeness of their target populations by minimizing biases from all sources. Increasing data size shrinks confidence intervals but magnifies the effect of survey bias: an instance of the Big Data Paradox1. Here we demonstrate this paradox in estimates of first-dose COVID-19 vaccine uptake in US adults from 9 January to 19 May 2021 from two large surveys: Delphi-Facebook2,3 (about 250,000 responses per week) and Census Household Pulse4 (about 75,000 every two weeks). In May 2021, Delphi-Facebook overestimated uptake by 17 percentage points (14-20 percentage points with 5% benchmark imprecision) and Census Household Pulse by 14 (11-17 percentage points with 5% benchmark imprecision), compared to a retroactively updated benchmark the Centers for Disease Control and Prevention published on 26 May 2021. Moreover, their large sample sizes led to miniscule margins of error on the incorrect estimates. By contrast, an Axios-Ipsos online panel5 with about 1,000 responses per week following survey research best practices6 provided reliable estimates and uncertainty quantification. We decompose observed error using a recent analytic framework1 to explain the inaccuracy in the three surveys. We then analyse the implications for vaccine hesitancy and willingness. We show how a survey of 250,000 respondents can produce an estimate of the population mean that is no more accurate than an estimate from a simple random sample of size 10. Our central message is that data quality matters more than data quantity, and that compensating the former with the latter is a mathematically provable losing proposition.

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.

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.

Protein Haptenation and Its Role in Allergy.

Humans are exposed to numerous electrophilic chemicals either as medicines, in the workplace, in nature, or through use of many common cosmetic and household products. Covalent modification of human proteins by such chemicals, or protein haptenation, is a common occurrence in cells and may result in generation of antigenic species, leading to development of hypersensitivity reactions. Ranging in severity of symptoms from local cutaneous reactions and rhinitis to potentially life-threatening anaphylaxis and severe hypersensitivity reactions such as Stephen-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), all these reactions have the same Molecular Initiating Event (MIE), i.e. haptenation. However, not all individuals who are exposed to electrophilic chemicals develop symptoms of hypersensitivity. In the present review, we examine common chemistry behind the haptenation reactions leading to formation of neoantigens. We explore simple reactions involving single molecule additions to a nucleophilic side chain of proteins and complex reactions involving multiple electrophilic centers on a single molecule or involving more than one electrophilic molecule as well as the generation of reactive molecules from the interaction with cellular detoxification mechanisms. Besides generation of antigenic species and enabling activation of the immune system, we explore additional events which result directly from the presence of electrophilic chemicals in cells, including activation of key defense mechanisms and immediate consequences of those reactions, and explore their potential effects. We discuss the factors that work in concert with haptenation leading to the development of hypersensitivity reactions and those that may act to prevent it from developing. We also review the potential harnessing of the specificity of haptenation in the design of potent covalent therapeutic inhibitors.

Selective HLA Class II Allele-Restricted Activation of Atabecestat Metabolite-Specific Human T-Cells.

Elevations in hepatic enzymes were detected in several trial patients exposed to the Alzheimer's drug atabecestat, which resulted in termination of the drug development program. Characterization of hepatic T-lymphocyte infiltrates and diaminothiazine (DIAT) metabolite-responsive, human leukocyte antigen (HLA)-DR-restricted, CD4+ T-lymphocytes in the blood of patients confirmed an immune pathogenesis. Patients with immune-mediated liver injury expressed a restricted panel of HLA-DRB1 alleles including HLA-DRB1*12:01, HLA-DRB1*13:02, and HLA-DRB1*15:01. Thus, the objectives of this study were to (i) generate DIAT-responsive T-cell clones from HLA-genotyped drug-naive donors, (ii) characterize pathways of DIAT-specific T-cell activation, and (iii) assess HLA allele restriction of the DIAT-specific T-cell response. Sixteen drug-naive donors expressing the HLA-DR molecules outlined above were recruited, and T-cell clones were generated. Cellular phenotype, function, and HLA-allele restriction were assessed using culture assays. Peptides displayed by HLA class II molecules in the presence and absence of atabecestat were analyzed by mass spectrometry. Several DIAT-responsive CD4+ clones, displaying no reactivity toward the parent drug, were successfully generated from donors expressing HLA-DRB1*12:01, HLA-DRB1*13:02, and HLA-DRB1*15:01 but not from other donors expressing other HLA-DRB1 alleles. T-cell clones were activated following direct binding of DIAT to HLA-DR proteins expressed on the surface of antigen presenting cells. DIAT binding did not alter the HLA-DRB1 peptide binding repertoire, indicative of a binding interaction with the HLA-associated peptide rather than with the HLA protein itself. DIAT-specific T-cell responses displayed HLA-DRB1*12:01, HLA-DRB1*13:02, and HLA-DRB1*15:01 restriction. These data demonstrate that DIAT displays a degree of selectivity toward HLA protein and associated peptides, with expression of certain alleles increasing and that of others decreasing, the likelihood that a drug-specific T-cell response develops.

Clinical and genomic features in patients with second primary glioblastoma following first primary renal cell carcinoma.

PURPOSE: To explore the potential pathogenesis and clinical features of second primary glioblastoma (spGBM) following first primary renal cell carcinoma (fpRCC). METHODS: Patients with spGBM after fpRCC were enrolled from our institution and the SEER dataset. Sanger sequencing, whole genome sequencing, and immunehistochemistry were used to detect molecular biomarkers. RESULTS: Four and 122 cases from our institution and the SEER dataset, respectively, were collected with an overall median age of 69 years at spGBM diagnosis following fpRCC. The median interval time between fpRCC and spGBM was 50.7 months and 4 years, for the four and 122 cases respectively. The median overall survival time was 11.2 and 6.0 months for the two datasets. In addition, spGBM patients of younger age (< 75 years) or shorter interval time (< 1 year) had favorable prognosis (p = 0.081 and 0.05, respectively). Moreover, the spGBM cases were molecularly classified as TERT only paired with TP53 mutation, PIK3CA mutation, EGFR alteration, low tumor mutation burden, and stable microsatellite status. CONCLUSIONS: This is the first study to investigate the pathogenesis and clinical features of spGBM following spRCC. We found that spGBMs are old-age related, highly malignant, and have short survival time. Moreover, they might be misdiagnosed and treated as brain metastases from RCC. Thus, the incidence of spGBMs after fpRCC is underestimated. Further studies are needed to investigate the underlying molecular mechanisms and clinical biomarkers for the development of spGBM following fpRCC.

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.

Head-to-Head Comparison of 68Ga-PSMA-11 with 68Ga-P137 in Patients with Suspected Prostate Cancer.

P137 is a novel oxalyldiaminopropionic acid-urea-based prostate-specific membrane antigen (PSMA) targeting agent. This study compared the uptake patterns of 68Ga-P137 and the FDA-approved PET tracer 68Ga-PSMA-11 for diagnosing prostate cancer (PCa). Sixteen patients suspected of PCa were scanned by 68Ga-PSMA-11 and 68Ga-P137 PET/CT, respectively, followed by prospective analysis. The tumor-to-background ratio was calculated using normal prostate tissue, blood pool, muscle, and urine as backgrounds. Pathology or follow-up results were used to analyze uptake patterns of benign/malignant lesions and various organs. Thirteen patients were diagnosed with PCa and three with benign prostate diseases (BPD). The number and location of primary lesions, lymph node metastasis (LNM) (n = 25), bone metastasis (n = 30), and liver metastasis (n = 3) detected by the two tracers were identical. Maximum standardized uptake value (SUVmax), tumor/normal prostate ratio, as well as semiquantitative miPSMA-ES and PRIMARY diagnostic scores (P all >0.05) showed similar uptake levels of primary lesions between 68Ga-P137 and 68Ga-PSMA-11. Compared to 68Ga-P137, the SUVmax of 68Ga-PSMA-11 was significantly higher for bone metastasis, LNM, and liver metastasis (14.9 ± 7.2 vs 9.1 ± 4.4, 14.4 ± 5.0 vs 7.5 ± 2.4, 13.9 ± 2.0 vs 8.8 ± 2.4, P all <0.05). One-hour postinjection, SUVmax of the duodenum (9.4 ± 2.1 vs 16.2 ± 6.1), kidney (19.4 ± 4.3 vs 45.6 ± 20.9), and urine (14.1 ± 7.1 vs 42.1 ± 25.9) were significantly lower for 68Ga-P137 than for 68Ga-PSMA-11 (P all <0.05), whereas the radioactivity accumulation of blood pool and muscle (3.9 ± 0.5 vs 1.6 ± 0.4, 1.0 ± 0.1 vs 0.6 ± 0.1, P all <0.05) of 68Ga-P137 was significantly higher than 68Ga-PSMA-11. The uptake level of 68Ga-P137 has no significant difference from that of 68Ga-PSMA-11 in prostate primary lesions, and their imaging performances are visually equivalent for both primary and metastatic lesions, despite a higher blood pool and muscle background and a lower uptake in metastatic lesions. Due to the lower urine excretion of 68Ga-P137, primary prostate lesions near the urine can potentially be displayed clearer than 68Ga-PSMA-11.

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.

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.

Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.

Drug hapten-specific T-cell activation: Current status and unanswered questions.

Drug haptens are formed from the irreversible, covalent binding of drugs to nucleophilic moieties on proteins, which can warrant adverse reactions in the body including severe delayed-type, T-cell mediated, drug hypersensitivity reactions (DHRs). While three main pathways exist for the activation of T-cells in DHRs, namely the hapten model, the pharmacological interaction model and the altered peptide repertoire model, the exact antigenic determinants responsible have not yet been defined. In recent years, progress has been made using advanced mass spectrometry-based proteomic methods to identify protein carriers and characterise the structure of drug-haptenated proteins. Since genome-wide association studies discovered a link between human leukocyte antigens (HLA) and an individual's susceptibility to DHRs, much effort has been made to define the drug-associated HLA ligands driving T-cell activation, including the elution of natural HLA peptides from HLA molecules and the generation of HLA-binding peptides. In this review, we discuss our current methodology used to design and synthesise drug-modified HLA ligands to investigate their immunogenicity using T-cell models, and thus their implication in drug hypersensitivity.

Drug-specific T-cell responses in patients with liver injury following treatment with the BACE inhibitor atabecestat.

BACKGROUND: Atabecestat is an orally administered BACE inhibitor developed to treat Alzheimer's disease. Elevations in hepatic enzymes were detected in a number of in trial patients, which resulted in termination of the drug development programme. Immunohistochemical characterization of liver tissue from an index case of atabecestat-mediated liver injury revealed an infiltration of T-lymphocytes in areas of hepatocellular damage. This coupled with the fact that liver injury had a delayed onset suggests that the adaptive immune system may be involved in the pathogenesis. The aim of this study was to generate and characterize atabecestat(metabolite)-responsive T-cell clones from patients with liver injury. METHODS: Peripheral blood mononuclear cells were cultured with atabecestat and its metabolites (diaminothiazine [DIAT], N-acetyl DIAT & epoxide) and cloning was attempted in a number of patients. Atabecestat(metabolite)-responsive clones were analysed in terms of T-cell phenotype, function, pathways of T-cell activation and cross-reactivity with structurally related compounds. RESULTS: CD4+ T-cell clones activated with the DIAT metabolite were detected in 5 out of 8 patients (up to 4.5% cloning efficiency). Lower numbers of CD4+ and CD8+ clones displayed reactivity against atabecestat. Clones proliferated and secreted IFN-γ, IL-13 and cytolytic molecules following atabecestat or DIAT stimulation. Certain atabecestat and DIAT-responsive clones cross-reacted with N-acetyl DIAT; however, no cross-reactivity was observed between atabecestat and DIAT. CD4+ clones were activated through a direct, reversible compound-HLA class II interaction with no requirement for protein processing. CONCLUSION: The detection of atabecestat metabolite-responsive T-cell clones activated via a pharmacological interactions pathway in patients with liver injury is indicative of an immune-based mechanism for the observed hepatic enzyme elevations.

Evaluation of clinical and genetic factors in the population pharmacokinetics of carbamazepine.

AIMS: Carbamazepine can cause hypersensitivity reactions in ~10% of patients. An immunogenic effect can be produced by the electrophilic 10,11-epoxide metabolite but not by carbamazepine. Hypothetically, certain single nucleotide polymorphisms might increase the formation of immunogenic metabolites, leading ultimately to hypersensitivity reactions. This study explores the role of clinical and genetic factors in the pharmacokinetics (PK) of carbamazepine and 3 metabolites known to be chemically reactive or formed through reactive intermediates. METHODS: A combination of rich and sparse PK samples were collected from healthy volunteers and epilepsy patients. All subjects were genotyped for 20 single nucleotide polymorphisms in 11 genes known to be involved in the metabolism or transport of carbamazepine and carbamazepine 10,11-epoxide. Nonlinear mixed effects modelling was used to build a population-PK model. RESULTS: In total, 248 observations were collected from 80 subjects. A 1-compartment PK model with first-order absorption and elimination best described the parent carbamazepine data, with a total clearance of 1.96 L/h, central distribution volume of 164 L and absorption rate constant of 0.45 h-1 . Total daily dose and coadministration of phenytoin were significant covariates for total clearance of carbamazepine. EPHX1-416G/G genotype was a significant covariate for the clearance of carbamazepine 10,11-epoxide. CONCLUSION: Our data indicate that carbamazepine clearance was affected by total dose and phenytoin coadministration, but not by genetic factors, while carbamazepine 10,11-epoxide clearance was affected by a variant in the microsomal epoxide hydrolase gene. A much larger sample size would be required to fully evaluate the role of genetic variation in carbamazepine pharmacokinetics, and thereby predisposition to carbamazepine hypersensitivity.

Exosomal Transport of Hepatocyte-Derived Drug-Modified Proteins to the Immune System.

Idiosyncratic drug-induced liver injury (DILI) is a rare, often difficult-to-predict adverse reaction with complex pathomechanisms. However, it is now evident that certain forms of DILI are immune-mediated and may involve the activation of drug-specific T cells. Exosomes are cell-derived vesicles that carry RNA, lipids, and protein cargo from their cell of origin to distant cells, and they may play a role in immune activation. Herein, primary human hepatocytes were treated with drugs associated with a high incidence of DILI (flucloxacillin, amoxicillin, isoniazid, and nitroso-sulfamethoxazole) to characterize the proteins packaged within exosomes that are subsequently transported to dendritic cells for processing. Exosomes measured between 50 and 100 nm and expressed enriched CD63. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) identified 2,109 proteins, with 608 proteins being quantified across all exosome samples. Data are available through ProteomeXchange with identifier PXD010760. Analysis of gene ontologies revealed that exosomes mirrored whole human liver tissue in terms of the families of proteins present, regardless of drug treatment. However, exosomes from nitroso-sulfamethoxazole-treated hepatocytes selectively packaged a specific subset of proteins. LC/MS-MS also revealed the presence of hepatocyte-derived exosomal proteins covalently modified with amoxicillin, flucloxacillin, and nitroso-sulfamethoxazole. Uptake of exosomes by monocyte-derived dendritic cells occurred silently, mainly through phagocytosis, and was inhibited by latrunculin A. An amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SRY (sex determining region Y)-box 30 activated naïve T cells from human leukocyte antigen A*02:01-positive human donors. Conclusion: This study shows that exosomes have the potential to transmit drug-specific hepatocyte-derived signals to the immune system and provide a pathway for the induction of drug hapten-specific T-cell responses.

Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions.

Delayed-type, T cell-mediated, drug hypersensitivity reactions are a serious unwanted manifestation of drug exposure that develops in a small percentage of the human population. Drugs and drug metabolites are known to interact directly and indirectly (through irreversible protein binding and processing to the derived adducts) with HLA proteins that present the drug-peptide complex to T cells. Multiple forms of drug hypersensitivity are strongly linked to expression of a single HLA allele, and there is increasing evidence that drugs and peptides interact selectively with the protein encoded by the HLA allele. Despite this, many individuals expressing HLA risk alleles do not develop hypersensitivity when exposed to culprit drugs suggesting a nonlinear, multifactorial relationship in which HLA risk alleles are one factor. This has prompted a search for additional susceptibility factors. Herein, we argue that immune regulatory pathways are one key determinant of susceptibility. As expression and activity of these pathways are influenced by disease, environmental and patient factors, it is currently impossible to predict whether drug exposure will result in a health benefit, hypersensitivity or both. Thus, a concerted effort is required to investigate how immune dysregulation influences susceptibility towards drug hypersensitivity.

Characterization of amoxicillin and clavulanic acid specific T-cell clones from patients with immediate drug hypersensitivity.

BACKGROUND: Betalactam (BL) antibiotics are the most common cause of drug hypersensitivity. Amoxicillin (AX), which is often prescribed alongside clavulanic acid (Clav), is the most common elicitor. The aim of this study was to determine whether AX and Clav-responsive T-cells are detectable in patients with immediate hypersensitivity to AX-Clav, to assess whether these T-cells display the same specificity as that detected in skin and provocation testing, and to explore T-cell activation pathways. METHODS: Drug-specific T-cell clones were generated from immediate hypersensitive patients´ blood by serial dilution and repetitive mitogen stimulation. Antigen specificity was assessed by measurement of proliferation and cytokine release. CD4+ /CD8+ phenotype and chemokine receptor expression were analyzed by flow cytometry. RESULTS: 110 AX-specific and 96 Clav-specific T-cell clones were generated from seven patients with positive skin test to either AX or Clav. Proliferation of AX- and Clav-specific clones was dose-dependent, and no cross-reactivity was observed. AX- and Clav-specific clones required antigen-presenting cells to proliferate, and drugs were presented to CD4+ and CD8+ T-cells by MHC class-II and I, respectively. A higher secretion of IL-13 and IL-5 was detected in presence of the culprit drug compared with the alternative drug. Clones expressed CD69, CCR4, CXCR3, and CCR10. CONCLUSIONS: Our study details the antigen specificity and phenotype of T-cell clones generated from patients with AX-Clav-induced immediate hypersensitivity diagnosed by positive skin test. AX- and Clav-specific clones were generated from patients irrespective of whether AX or Clav was the culprit, although differences in cytokine secretion were observed.

Definition of the Chemical and Immunological Signals Involved in Drug-Induced Liver Injury.

Idiosyncratic drug-induced liver injury (iDILI), which is rare and often recognized only late in drug development, poses a major public health concern and impediment to drug development due to its high rate of morbidity and mortality. The mechanisms of DILI are not completely understood; both non-immune- and immune-mediated mechanisms have been proposed. Non-immune-mediated mechanisms including direct damage to hepatocytes, mitochondrial toxicity, interference with transporters, and alteration of bile ducts are well-known to be associated with drugs such as acetaminophen and diclofenac; whereas immune-mediated mechanisms involving activation of both adaptive and innate immune cells and the interactions of these cells with parenchymal cells have been proposed. The chemical signals involved in activation of both innate and adaptive immune responses are discussed with respect to recent scientific advances. In addition, the immunological signals including cytokine and chemokines that are involved in promoting liver injury are also reviewed. Finally, we discuss how liver tolerance and regeneration can have profound impact on the pathogenesis of iDILI. Continuous research in developing in vitro systems incorporating immune cells with liver cells and animal models with impaired liver tolerance will provide an opportunity for improved prediction and prevention of immune-mediated iDILI.

Cell Membrane Transporters Facilitate the Accumulation of Hepatocellular Flucloxacillin Protein Adducts: Implication in Flucloxacillin-Induced Liver Injury.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver reactions. Although expression of HLA-B*57:01 increases susceptibility, little is known about the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the presentation of flucloxacillin-modified peptides by the risk allele. In this study, the binding of flucloxacillin to proteins of liver-like cells was characterized. Flucloxacillin was shown to bind to proteins localized in bile canaliculi regions, coinciding with the site of clinical disease. The localization of flucloxacillin was mediated primarily by the membrane transporter multidrug resistance-associated protein 2. Modification of multiple proteins by flucloxacillin in bile canaliculi regions may provide a potential local source of neo-antigens for HLA presentation in the liver.

Tumor suppressor miR-139-5p targets Tspan3 and regulates the progression of acute myeloid leukemia through the PI3K/Akt pathway.

Dysregulation of microRNAs is closely implicated in the initiation and progression of human cancers including acute myeloid leukemia (AML). Though miR-139-5p was reported to be a potent tumor suppressor in adult AML, its underlying molecular mechanism in AML remains to be further defined. Herein, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were conducted to determine the expressions of miR-139-5p and tetraspanin3 (Tspan3) in AML patients and cells. Luciferase reporter assay, qRT-PCR, and Western blot analysis were carried out to detect the interaction between miR-139-5p and Tspan3. Cell proliferation, cell cycle distribution, invasion, and migration were evaluated by cell counting kit-8, flow cytometry, transwell invasion, and migration assays, respectively. Western blot analysis was conducted to determine phosphorylated-protein kinase B (Akt) and Akt levels. We found that a significant reduction in miR-139-5p expression and a prominent increase in Tspan3 expression were observed in AML patients and cells. Tspan3 was confirmed as a direct target of miR-139-5p and was negatively modulated by miR-139-5p. Rescue experiments showed that overexpression of miR-139-5p constrained cell proliferation, invasion and migration capabilities, and induced cell cycle arrest at the S phase in AML cells, which were partially reversed by Tspan3 overexpression. In addition, we found that miR-139-5p suppressed the phosphoinositide 3-kinase (PI3K)/Akt pathway in AML cells by targeting Tspan3. In conclusion, our study concluded that miR-139-5p suppressed the leukemogenesis in AML cells by targeting Tspan3 through inactivation of the PI3K/Akt pathway, providing a better understanding of AML progression.