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1.
Chem Res Toxicol ; 36(12): 1872-1875, 2023 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-38055372

RESUMEN

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.


Asunto(s)
Catequina , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Humanos , Leucocitos Mononucleares , Antioxidantes , , Antígenos HLA-B/genética , Extractos Vegetales/farmacología , Catequina/farmacología
2.
Toxicol Sci ; 197(1): 38-52, 2023 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-37788119

RESUMEN

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.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatocitos , Humanos , Células Cultivadas , Hepatocitos/metabolismo , Técnicas de Cocultivo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Medición de Riesgo
3.
Toxicol Sci ; 192(1): 106-116, 2023 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-36782357

RESUMEN

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.


Asunto(s)
Carcinoma Hepatocelular , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Neoplasias Hepáticas , Humanos , Floxacilina/toxicidad , Hígado/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Albúminas
4.
Chem Res Toxicol ; 36(3): 390-401, 2023 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-36812109

RESUMEN

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.


Asunto(s)
Hipersensibilidad a las Drogas , Humanos , Técnicas de Cocultivo , Dapsona/farmacología , Hígado , Hepatocitos , Activación de Linfocitos
5.
J Immunother Cancer ; 9(5)2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-34049931

RESUMEN

Many adverse reactions associated with immune checkpoint inhibitor (ICI) treatments are immunologically driven and may necessitate discontinuation of the ICI. Herein, we present a patient who had been administered the radio contrast media amidotrizoate multiple times without issue but who then developed a Stevens-Johnson syndrome reaction after coadministration of atezolizumab. Causality was confirmed by a positive re-challenge with amidotrizoate and laboratory investigations that implicated T cells. Importantly, the introduction of atezolizumab appears to have altered the immunologic response to amidotrizoate in terms of the tolerance-elicitation continuum. Proof of concept studies demonstrated enhancement of recall responses to a surrogate antigen panel following in-vitro (healthy donors) and in-vivo (ICI patients) administrations of ICIs. Our findings highlight the importance of considering all concomitant medications in patients on ICIs who develop immune-mediated adverse reactions. In the event of some immune-related adverse reactions, it may be critical to identify the culprit antigen-forming entity that the ICIs have altered the perception of rather than simply attribute causality to the ICI itself in order to optimize both patient safety and treatment of malignancies.


Asunto(s)
Anticuerpos Monoclonales Humanizados/efectos adversos , Carcinoma de Células Renales/tratamiento farmacológico , Medios de Contraste/efectos adversos , Diatrizoato/efectos adversos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Neoplasias Renales/tratamiento farmacológico , Síndrome de Stevens-Johnson/etiología , Linfocitos T/efectos de los fármacos , Corticoesteroides/uso terapéutico , Carcinoma de Células Renales/diagnóstico por imagen , Carcinoma de Células Renales/inmunología , Humanos , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/inmunología , Masculino , Valor Predictivo de las Pruebas , Factores de Riesgo , Síndrome de Stevens-Johnson/diagnóstico , Síndrome de Stevens-Johnson/tratamiento farmacológico , Síndrome de Stevens-Johnson/inmunología , Linfocitos T/inmunología
6.
Nat Commun ; 12(1): 2055, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33824342

RESUMEN

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.


Asunto(s)
Antivirales/farmacología , COVID-19/inmunología , COVID-19/virología , Reacciones Cruzadas/inmunología , Inmunoensayo/métodos , SARS-CoV-2/fisiología , Linfocitos T/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/epidemiología , Proliferación Celular , Citocinas/metabolismo , Células HEK293 , Personal de Salud , Humanos , Inmunoglobulina G/inmunología , Memoria Inmunológica , Interferón gamma/metabolismo , Pandemias , Péptidos/metabolismo , SARS-CoV-2/efectos de los fármacos
7.
Chem Res Toxicol ; 33(12): 2939-2943, 2020 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-33169987

RESUMEN

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.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Línea Celular , Membrana Celular/metabolismo , Floxacilina/química , Humanos , Estructura Molecular
8.
Chem Res Toxicol ; 33(1): 61-76, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31682113

RESUMEN

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.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Animales , Humanos , Tolerancia Inmunológica , Hígado/inmunología , Transducción de Señal , Linfocitos T/inmunología
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