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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.

Flucloxacillin is a β-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.

Purpose Our purpose was to compare the performance of prostate-specific membrane antigen (PSMA)-positron emission tomography (PET) traditional fixed threshold (FT) and newly-established Prostate Imaging Reporting and Data System (PI-RADS)-based segmented threshold (ST) for diagnosing clinically significant prostate cancer (csPCa).Methods The study retrospectively included 218 patients who underwent multiparametric magnetic resonance imaging (mpMRI) and PSMA-PET examination for suspected prostate cancer (PCa) from January 2018 to November 2021. Lesions with Gleason score (GS) ≥ 3 + 4 were diagnosed as csPCa. In PSMA-PET maximum standardized uptake value (SUVmax), the FT for all the lesions and ST for lesions with different PI-RADS score for diagnosing csPCa were determined by receiver operating characteristic (ROC) curves analysis and compared with Z test. The McNemar test was used to compare sensitivity and specificity.Results Among the 218 patients, there were 113 csPCa and 105 non-csPCa. The PSMA-PET FT was SUVmax > 5.3 (area under the curve, AUC = 0.842) and STs for PI-RADS 3/4/5 were SUVmax > 4.2/5.7/6.0 (AUCs = 0.870/0.867/0.882), respectively. The AUC of PSMA-PET ST was higher than that of PSMA-PET FT (0.872 vs. 0.842), especially for PI-RADS 3 (0.870 vs. 0.653). Multimodality diagnostic criteria combining PSMA-PET ST and PI-RADS scores of mpMRI was established and its AUC was higher than that of PSMA-PET ST (0.893 vs. 0.872), and significantly higher than that of PSMA-PET FT (0.893 vs. 0.842) with an improvement in sensitivity (93% vs. 78%, p 0.05).Conclusions For diagnosing csPCa, PI-RADS-based PSMA-PET ST achieved better performance than traditional FT, especially for PI-RADS 3 lesions.

β-Lactamase inhibitors such as clavulanic acid and tazobactam were developed to overcome β-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 β-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.

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.

The subarachnoid space width (SASw) is part of crucial neuroimaging criteria for the diagnosis of subarachnoid space enlargement in infants. In addition to indicating the presence of these diseases, SASw can be used to assess their severity. Therefore, it is important to be able to measure the SASw accurately.This study aimed to compare the accuracy of measurements made from axial and coronal T2-weighted imaging (T2WI) and to establish a consentaneous measurement scheme of SASw in infants.A total of 63 infants (31 males and 32 females) aged 4 days to 24 months were enrolled in this study. The supratentorial subarachnoid space volume (SASv) and corrected SASv (cSASv) were used as the gold standard reference. The SASw (including interhemispheric width and bilateral frontal craniocortical width) was measured on axial and coronal T2WI. The intra- and inter-observer reproducibility and agreement of the SASw were assessed by the intraclass correlation coefficient (ICC) and Bland-Altman analysis. A paired t-test was used to compare SASw measured on axial and coronal images. The accuracy of SASw measurements made from axial and coronal T2WI was evaluated by the relationships between the SASw and supratentorial SASv and between the SASw and supratentorial cSASv, and the relationships were examined by multivariate linear regression.The intra- and inter-observer ICC values of the three SASw measurements were greater on coronal T2WI than on axial T2WI. Bland-Altman analysis confirmed that the SASw values measured on coronal T2WI had better intra- and inter-observer agreement than axial T2WI. According to the multivariate linear regression results, model 4 (the SASw measured in coronal T2WI) was the best predictor of supratentorial cSASv (RThe SASw measured on coronal T2WI was more repeatable and accurate than axial T2WI and was more representative of the actual cerebrospinal fluid accumulation in the supratentorial subarachnoid space.The SASw has been found to be a simple and essential substitution for supratentorial SASv, which can be measured on both axial T2WI passing through the bodies of the bilateral ventricles and coronal T2WI at the level of the foramen of Monro. The SASw measured on coronal T2WI was more beneficial to the diagnosis and severity assessment of subarachnoid space enlargement in infants.

The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses., Ebselen is an organoselenium drug that inhibits the SARS-CoV-2 main protease (Mpro). Here, the authors co-crystallised Mpro with ebselen and an ebselen derivative and observed an enzyme bound organoselenium covalent adduct in the crystal structures, which was also confirmed by mass spectrometry analysis.

The simulation of complex power grid adopts the method of model segmentation. The system is divided into two or more subsystems. Each subsystem is calculated and solved in different computing units. Because the simulation model runs on different hardware, the time synchronization between subsystems may bring errors and stability problems. In this paper, a time synchronization method based on virtual power is proposed. It is assumed that there is a virtual load composed of resistance and inductance between different simulation subsystems, and two identical voltage sources are set at both terminals of the virtual load. When there is no time error between the subsystems, the power consumed by the virtual load should be zero, otherwise virtual power will be generated. The virtual power is sent to the PI regulator as the error signal, and the output of the regulator is used as the time error correction signal of the subsystem until the virtual power is zero, so as to realize the clock synchronization between multiple computers. In this paper, the theoretical analysis and implementation method of this time synchronization scheme are given in detail, and the simulation results show that the method is correct.

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.

Flucloxacillin is a β-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.

Amoxicillin-clavulanate is the most common cause of idiosyncratic drug-induced liver injury (DILI). Drug-specific CD4+ T cells have been detected in patients with DILI, suggestive of an immune etiology. Furthermore, genetic associations including the human leucocyte antigen (HLA) DRB1*15:01-DQB1*06:02 haplotype influence susceptibility. Amoxicillin forms protein adducts that are postulated to activate T cells, by conjugating with lysine residues. However, a role for such adducts has not been described. This study aimed to (1) investigate whether amoxicillin-modified HLA-DRB1*15:01-DQB1*06:02 binding peptides selectively activate DILI patient T cells and (2) define the nature of the T-cell response with respective to antigen structure. Peptides carrying lysine residues for amoxicillin binding in positions (KP) 2-6 and anchors for the HLA-DRB1*15:01-DQB1*06:02 haplotype were designed. The amoxicillin-modified peptides were characterized by mass spectrometry prior to culturing with patient peripheral blood mononuclear cell. T-cell clones were then tested for specificity with amoxicillin, unmodified- and amoxicillin-modified peptides, and structural variants. Amoxicillin-modified KP-2 and KP-3 peptide-specific CD4+ clones proliferated and secreted interferon gamma (IFN-γ), interleukin (IL)-10, perforin and/or IL-17/IL-22 in a dose-dependent manner and displayed no cross-reactivity with amoxicillin, unmodified peptide or with positional derivatives. The T cells response was HLA class II restricted and the amoxicillin-modified peptides bound selectively to HLA-DRB1*15:01 and/or DQB1*06:02. To conclude, we show that amoxicillin-modified peptides bind to both components of the risk haplotype to stimulate DILI patient T cells and describe the importance of the position of nucleophilic lysine residue in the HLA binding peptide sequence.

The existing methods of dam deformation detection of tailings reservoir have the problems of poor accuracy and slow speed. Therefore, a fault diagnosis algorithm based on tailing dam deformation detection method is proposed. The grey theory is used to accumulate the original feature sequence, and the first cumulative sequence is obtained. Based on this, the grey detection model is constructed, and then the concrete deformation of tailings dam body is accurately detected by precision test. Experimental results show that the method has high accuracy, high speed and practicability

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors and sulfasalazine. Thus, the aim of this study was to characterize the T-cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in-vitro platforms. A regulatory competent human dendritic cell/T-cell co-culture assay was used to model the effects of immune checkpoint inhibitors on de-novo nitroso sulfamethoxazole- and sulfapyridine (the sulfonamide component of sulfasalazine) hydroxylamine-specific priming responses. The role of T-cells in the pathogenesis of the observed reactions was explored in three patients through phenotypic characterization of sulfapyridine/sulfapyridine hydroxylamine-responsive T-cell clones, and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T-cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of sulfapyridine- and sulfapyridine hydroxylamine-responsive T-cells were isolated from all three patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B and perforin) was identified for sulfapyridine and sulfapyridine hydroxylamine responsive T-cell clones, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T-cells are effectors of hypersensitivity reactions observed in oncology patients administered immune checkpoint inhibitors and sulfasalazine. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring.

Due to the influence of load fluctuation, the power supply stability of distribution network is insufficient. Therefore, the open capacity model of medium voltage lines in the distribution network is constructed by using load data. Based on the introduction of time parameter, the load pressure of the distribution network is calculated by using the peak load of distribution network. According to the relationship between the pressure value of sustainable load of the distribution network and the pressure value under the actual power load, the open capacity model of medium voltage lines is constructed. The test results show that when the design model is used to set the open capacity of medium voltage lines, the fluctuation of the expected outage time and frequency of the distribution network system is small, which indicates that the stable power supply can be realized

Purpose The prognosis of breast cancer (BC) patients who develop into brain metastases (BMs) is very poor. Thus, it is of great significance to explore the etiology of BMs in BC and identify the key genes involved in this process to improve the survival of BC patients with BMs. Patients and methods The gene expression data and the clinical information of BC patients were downloaded from TCGA and GEO database. Differentially expressed genes (DEGs) in TCGA-BRCA and GSE12276 were overlapped to find differentially expressed metastatic genes (DEMGs). The protein-protein interaction (PPI) network of DEMGs was constructed via STRING database. ClusterProfiler R package was applied to perform the gene ontology (GO) enrichment analysis of DEMGs. The univariate Cox regression analysis and the Kaplan-Meier (K-M) curves were plotted to screen DEMGs associated with the overall survival and the metastatic recurrence survival, which were identified as the key genes associated with the BMs in BC. The immune infiltration and the expressions of immune checkpoints for BC patients with brain relapses and BC patients with other relapses were analyzed respectively. The correlations among the expressions of key genes and the differently infiltrated immune cells or the differentially expressed immune checkpoints were calculated. The gene set enrichment analysis (GSEA) of each key gene was conducted to investigate the potential mechanisms of key genes involved in BC patients with BMs. Moreover, CTD database was used to predict the drug-gene interaction network of key genes. Results A total of 154 DEGs were identified in BC patients at M0 and M1 in TCGA database. A total of 667 DEGs were identified in BC patients with brain relapses and with other relapses. By overlapping these DEGs, 17 DEMGs were identified, which were enriched in the cell proliferation related biological processes and the immune related molecular functions. The univariate Cox regression analysis and the Kaplan-Meier curves revealed that CXCL9 and GPR171 were closely associated with the overall survival and the metastatic recurrence survival and were identified as key genes associated with BMs in BC. The analyses of immune infiltration and immune checkpoint expressions showed that there was a significant difference of the immune microenvironment between brain relapses and other relapses in BC. GSEA indicated that CXCL9 and GPR171 may regulate BMs in BC via the immune-related pathways. Conclusion Our study identified the key genes associated with BMs in BC patients and explore the underlying mechanisms involved in the etiology of BMs in BC. These findings may provide a promising approach for the treatments of BC patients with BMs., Highlights • CXCL9 and GPR171, as the key genes, were closely associated with the prognosis of brain metastases in breast cancer. • There was a significant difference of the immune microenvironment between brain and other metastases in breast cancer. • We revealed candidate drugs which associated with the key genes of breast cancer patients with brain metastases. • A series of bioinformatic analysis methods were used in this article.

HLA-B∗13:01 is associated with dapsone (DDS)-induced hypersensitivity, and it has been shown that CD4+ and CD8+ T cells are activated by DDS and its nitroso metabolite (nitroso dapsone [DDS-NO]). However, there is a need to define the importance of the HLA association in the disease pathogenesis. Thus, DDS- and DDS-NO‒specific CD8+ T-cell clones (TCCs) were generated from hypersensitive patients expressing HLA-B∗13:01 and were assessed for phenotype and function, HLA allele restriction, and killing of target cells. CD8+ TCCs were stimulated to proliferate and secrete effector molecules when exposed to DDS and/or DDS-NO. DDS-responsive and several DDS-NO‒responsive TCCs expressing a variety of TCR sequences displayed HLA class-I restriction, with the drug (metabolite) interacting with multiple HLA-B alleles. However, activation of certain DDS-NO‒responsive CD8+ TCCs was inhibited with HLA class-II block, with DDS-NO binding to HLA-DQB1∗05:01. These TCCs were of different origin but expressed TCRs displaying the same amino acid sequences. They were activated through a hapten pathway; displayed CD45RO, CD28, PD-1, and CTLA-4 surface molecules; secreted the same panel of effector molecules as HLA class-I‒restricted TCCs; but displayed a lower capacity to lyse target cells. To conclude, DDS and DDS-NO interact with a number of HLA molecules to activate CD8+ TCCs, with HLA class-II‒restricted CD8+ TCCs that display hybrid CD4‒CD8 features also contributing to the promiscuous immune response that develops in patients.

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.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.

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.

Telaprevir, a protease inhibitor, was used alongside PEGylated interferon-α and ribavirin to treat hepatitis C viral infections. The triple regimen proved successful; however, the appearance of severe skin reactions alongside competition from newer drugs restricted its use. Skin reactions presented with a delayed onset indicative of a T-cell mediated reaction. Thus, the aim of this study was to investigate whether telaprevir and/or its diastereomer, which is generated in humans, activates T-cells. Telaprevir in its S-configured therapeutic form and the R-diastereomer were cultured directly with peripheral blood mononuclear cells from healthy donors prior to the generation of T-cell clones by serial dilution. Drug-specific CD4+ and CD8+ T-cell clones responsive to telaprevir and the R-diastereomer were generated and characterized in terms of phenotype and function. The clones proliferated with telaprevir and diastereomer concentrations of 5-20 µM and secreted IFN-γ, IL-13, and granzyme B. In contrast, the telaprevir M11 metabolite did not stimulate T-cells. The CD8+ T-cell response was MHC I-restricted and dependent on the presence of soluble drug. Flow cytometric analysis showed that clones expressed chemokine receptors CCR4 (skin homing) and CXCR3 (migration to peripheral tissue) and 1 of 3 distinct TCR Vβs; TCR Vβ 2, 5.1, or 22. These data show the propensity of both R- and S-forms of telaprevir to generate skin-homing cytotoxic T-cells that may induce the adverse reactions observed in human patients.

Objectives To discriminate low grade (Fuhrman I/II) and high grade (Fuhrman III/IV) clear cell renal cell carcinoma (CCRCC) by using CT-based radiomic features. Methods 161 and 99 patients diagnosed with low and high grade CCRCCs from January 2011 to May 2018 were enrolled in this study. 1029 radiomic features were extracted from corticomedullary (CMP), and nephrographic phase (NP) CT images of all patients. We used interclass correlation coefficient (ICC) and the least absolute shrinkage and selection operator (LASSO) regression method to select features, then the selected features were constructed three classification models (CMP, NP and with their combination) to discriminate high and low grades CCRCC. These three models were built by logistic regression method using 5-fold cross validation strategy, evaluated with receiver operating characteristics curve (ROC) and compared using DeLong test. Results We found 11 and 24 CMP and NP features were independently significantly associated with the Fuhrman grades. The model of CMP, NP and Combined model using radiomic feature set showed diagnostic accuracy of 0.719 (AUC [area under the curve], 0.766; 95% CI [confidence interval]: 0.709-0.816; sensitivity, 0.602; specificity, 0.838), 0.738 (AUC, 0.818; 95% CI:0.765-0.838; sensitivity, 0.693; specificity, 0.838), 0.777(AUC, 0.822; 95% CI: 0.769-0.866; sensitivity, 0.677; specificity, 0.839). There were significant differences in AUC between CMP model and Combined model (P = 0.0208), meanwhile, the differences between CMP model and NP model, NP model and Combined model reached no significant (P = 0.0844, 0.7915). Conclusions Radiomic features could be used as biomarker for the preoperative evaluation of the CCRCC Fuhrman grades.

The global emergence of SARS-CoV-2 has triggered numerous efforts to develop therapeutic options for COVID-19 pandemic. The main protease of SARS-CoV-2 (Mpro), which is a critical enzyme for transcription and replication of SARS-CoV-2, is a key target for therapeutic development against COVID-19. An organoselenium drug called ebselen has recently been demonstrated to have strong inhibition against Mpro and antiviral activity but its molecular mode of action is unknown preventing further development. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of ebselen derivatives. A free selenium atom bound with cysteine 145 of Mpro catalytic dyad has been revealed by crystallographic studies of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct, formation of a phenolic by-product is confirmed by mass spectrometry. The target engagement of these compounds with an unprecedented mechanism of SARS-CoV-2 Mpro inhibition suggests wider therapeutic applications of organo-selenium compounds in SARS-CoV-2 and other zoonotic beta-corona viruses.