Morphological and Mechanistic Aspects of Thiourea-Induced Acute Lung Injury and Tolerance in the Rat.

Thiourea-based molecules cause pulmonary edema when administered to rats at relatively low doses. However, rats survive normally lethal doses after prior exposure to a lower, nonlethal dose; this phenomenon is known as tolerance. The present study investigated the morphological and functional aspects of acute lung injury (ALI) induced by methylphenylthiourea (MPTU) in the Wistar rat and the pulmonary response involved in prevention of the injury. We identified pulmonary endothelial cells as the main target of acute MPTU injury; they exhibited ultrastructural alterations that can result in increased vascular permeability. In tolerant rats, the lungs showed only transient endothelial changes, at 24-hour post dosing, and mild type II pneumocyte hyperplasia on day 7 post dosing. They exhibited glutathione levels similar to the controls and increased expression of flavin-containing monooxygenase 1 (FMO1), the enzyme responsible for bioactivation of small thioureas in the laboratory rat. Incubation of rat pulmonary microsomal preparations with MPTU inhibited FMO activity, indicating that tolerance is related to irreversible inhibition of FMOs. The rat model of thiourea-induced pulmonary toxicity and tolerance represents an interesting approach to investigate certain aspects of the pathogenesis of ALI and therapeutic approaches to lung diseases, such as acute respiratory distress syndrome.

Identification of Flucloxacillin-Haptenated HLA-B*57:01 Ligands: Evidence of Antigen Processing and Presentation.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver reactions. Although expression of human leukocyte antigen (HLA)-B*57:01 increases 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 modification of peptides that are presented by the risk allele. In this study, the binding of flucloxacillin to immune cells was characterized and the nature of the peptides presented by HLA-B*57:01 was analyzed using mass spectrometric-based immunopeptidomics methods. Flucloxacillin modification of multiple proteins was observed, providing a potential source of neoantigens for HLA presentation. Of the peptides eluted from flucloxacillin-treated C1R-B*57:01 cells, 6 putative peptides were annotated as flucloxacillin-modified HLA-B*57:01 peptide ligands (data are available via ProteomeXchange with identifier PXD020137). To conclude, we have characterized naturally processed drug-haptenated HLA ligands presented on the surface of antigen presenting cells that may drive drug-specific CD8+ T-cell responses.

Development of an Improved T-cell Assay to Assess the Intrinsic Immunogenicity of Haptenic Compounds.

The prediction of drug hypersensitivity is difficult due to the lack of appropriate models and known risk factors. In vitro naïve T-cell priming assays that assess immunogenicity have been developed. However, their application is limited due requirements for 2 batches of autologous dendritic cells (DC) and inconsistent results; a consequence of single well readouts when exploring reactions where compound-specific T-cell frequency is undefined. Hence, we aimed to develop an improved, but simplified assay, termed the T-cell multiple well assay (T-MWA), that permits assessment of drug-specific activation of naïve T cells, alongside analysis of the strength of the induced response and the number of cultures that respond. DC naïve T-cell coculture, depleted of regulatory T cells (Tregs), was conducted in up to 48 wells for 2 weeks with model haptens (nitroso sulfamethoxazole [SMX-NO], Bandrowski's base [BB], or piperacillin [PIP]). Cultures were rechallenged with hapten and T-cell proliferation was measured using [3H]-thymidine incorporation. Priming of naïve T cells was observed with SMX-NO, with no requirement for DC during restimulation. Greater than 65% of cultures were activated with SMX-NO; with 8.0%, 30.8%, and 27.2% characterized as weak (stimulation index [SI] =1.5-1.9), moderate (SI = 2-3.9), and strong responses (SI > 4), respectively. The number of responding cultures and strength of the response was reproducible when separate blood donations were compared. Coinhibitory checkpoint blockade increased the strength of the proliferative response, but not the number of responding cultures. Moderate to strong priming responses were detected with BB, whereas PIP stimulated only a small number of cultures to proliferate weakly. In drug-responsive cultures inducible CD4+CD25+FoxP3+CD127low Tregs were also identified. To conclude, the T-MWA offers improvements over existing assays and with development it could be used to study multiple HLA-typed donors in a single plate format.

Quantitative Systems Toxicology Modeling To Address Key Safety Questions in Drug Development: A Focus of the TransQST Consortium.

Omics data have been increasingly generated with limited demonstrated value in drug safety assessment. The TransQST consortium was launched to use omics and other data in mechanistic-based quantitative systems toxicology (QST) models to evaluate their potential use in species translation.

Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy.

Approximately 30% of epilepsy patients do not respond to antiepileptic drugs, representing an unmet medical need. There is evidence that neuroinflammation plays a pathogenic role in drug-resistant epilepsy. The high-mobility group box 1 (HMGB1)/TLR4 axis is a key initiator of neuroinflammation following epileptogenic injuries, and its activation contributes to seizure generation in animal models. However, further work is required to understand the role of HMGB1 and its isoforms in epileptogenesis and drug resistance. Using a combination of animal models and sera from clinically well-characterized patients, we have demonstrated that there are dynamic changes in HMGB1 isoforms in the brain and blood of animals undergoing epileptogenesis. The pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset and prospectively identified animals that developed the disease. Consistent with animal data, we observed early expression of disulfide HMGB1 in patients with newly diagnosed epilepsy, and its persistence was associated with subsequent seizures. In contrast with patients with well-controlled epilepsy, patients with chronic, drug-refractory epilepsy persistently expressed the acetylated, disulfide HMGB1 isoforms. Moreover, treatment of animals with antiinflammatory drugs during epileptogenesis prevented both disease progression and blood increase in HMGB1 isoforms. Our data suggest that HMGB1 isoforms are mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy in humans, necessitating evaluation in larger-scale prospective studies.

Characterization of Drug-Specific Signaling Between Primary Human Hepatocytes and Immune Cells.

It is now apparent that antigen-specific T-cells are activated in certain patients with drug-induced liver injury (DILI). Since cross-talk between hepatocytes and immune cells is likely to be critical in determining the outcome of drug exposure, the aim of this study was to profile the signals released by drug-treated hepatocytes and to characterize the impact of these molecules on dendritic cells. Human hepatocytes were exposed to 3 drugs (flucloxacillin, amoxicillin, and isoniazid) associated with DILI potentially mediated by the adaptive immune system as drug-specific T-cells have been isolated from DILI patients, and the metabolite nitroso-sulfamethoxazole (SMX-NO). Hepatocyte toxicity, cytokine release and activation of oxidative stress pathways were measured. Supernatants were transferred to monocyte-derived dendritic cells and cell phenotype and function were assessed. High-mobility group box 1 protein (HMGB1) and lactate dehydrogenase release as well as adenosine triphosphate depletion occurred in a drug-, time-, and concentration-dependent manner with SMX-NO and flucloxacillin, whereas isoniazid and amoxicillin were nontoxic. Furthermore, drug-induced activation of nuclear factor (erythroid-derived 2)-like 2 marker genes was observed when hepatocytes were exposed to test drugs. The disulfide isoform of HMGB1 stimulated dendritic cell cytokine release and enhanced the priming of naive T-cells. Incubation of dendritic cells with supernatant from drug-treated hepatocytes resulted in 2 distinct cytokine profiles. SMX-NO/flucloxacillin stimulated secretion of TNF-α, IL-6, IL-1α, and IL-1-ß. Isoniazid which did not induce significant hepatocyte toxicity, compared with SMX-NO and flucloxacillin, stimulated the release of a panel of cytokines including the above and IFN-γ, IL-12, IL-17A, IP-10, and IL-10. Collectively, our study identifies drug-specific signaling pathways between hepatocytes and immune cells that could influence whether drug exposure will result in an immune response and tissue injury.

Preventing Plasmon Coupling between Gold Nanorods Improves the Sensitivity of Photoacoustic Detection of Labeled Stem Cells in Vivo.

Gold nanorods are excellent contrast agents for imaging technologies which rely on near-infrared absorption such as photoacoustic imaging. For cell tracking applications, the cells of interest are labeled with the contrast agent prior to injection. However, after uptake into cells by endocytosis, the confinement and high concentration in endosomes leads to plasmon band broadening and reduced absorbance. This would limit the potential of multispectral optoacoustic tomography in terms of spectral processing and, consequently, sensitivity. Here, we show that steric hindrance provided by silica coating of the nanorods leads to the preservation of their spectral properties and improved photoacoustic sensitivity. This strategy allowed the detection and monitoring of as few as 2 × 10(4) mesenchymal stem cells in mice over a period of 15 days with a high spatial resolution. Importantly, the silica-coated nanorods did not affect the viability or differentiation potential of the transplanted mesenchymal stem cells.

Are drug metabolites able to cause T-cell-mediated hypersensitivity reactions?

INTRODUCTION: Adverse drug reactions with an immune pathogenesis are a problem in the clinic and an impediment to drug development. T lymphocytes are believed to play a role in the pathogenesis; however, the nature of the drug interaction with immune receptors remains an area of debate. AREAS COVERED: This article reviews recent advances in our understanding of drug hypersensitivity focusing specifically on the way in which drugs are displayed in MHC molecules. Most drugs associated with a high incidence of reactions have been shown to form protein-reactive metabolites. Hence, the relationship between drug metabolism and T-cell activation is discussed in detail. EXPERT OPINION: The role of metabolism in pathogenesis of immunological drug reactions has only been studied with a small number of drugs where synthetic metabolites are available for functional studies. In each case, metabolite-responsive T cells have been detected. However, the field is skewed by the fact that most research is conducted using the parent compound in metabolically inert cell systems. We propose that research efforts are directed towards the synthesis of drug metabolites and/or drug-protein conjugates. Furthermore, analytical methods need to be developed to relate metabolite exposure to the T-cell response. For now, our understanding of the chemical basis of drug hypersensitivity is incomplete.

Genetic and environmental factors determining clinical outcomes and cost of warfarin therapy: a prospective study.

BACKGROUND: In this prospective cohort study, we have undertaken a comprehensive evaluation of clinical parameters along with variation in 29 genes (including CYP2C9 and VKORC1) to identify factors determining interindividual variability in warfarin response. METHODS: Consecutive patients (n=311) were followed up prospectively for 26 weeks. Several outcomes chosen to capture both warfarin efficacy and toxicity were assessed. Univariate and multiple regression analyses were undertaken to assess the combined effect of clinical and genetic factors. RESULTS: CYP2C9 was the most important gene determining initial anticoagulant control, whereas VKORC1 was more important for stable anticoagulation. Novel associations with some clinical outcomes were found with single nucleotide polymorphisms in the cytochrome 450 genes CYP2C18 and CYP2C19, which were independent of the associations observed with CYP2C9 and in genes encoding CYP3A5, protein S and clotting factor V, although the variability explained by these genes was small. On the basis of the results of microcosting, adverse events were shown to be a significant predictor of total cost. CONCLUSION: Accurate prediction of warfarin dose requirement needs to take into account multiple genetic and environmental factors, the contributions of which vary in the induction and maintenance phases of treatment.

Effects of CYP4F2 genetic polymorphisms and haplotypes on clinical outcomes in patients initiated on warfarin therapy.

BACKGROUND: A variant in the CYP4F2 gene, rs2108622, has been recently shown to determine stable warfarin dose requirements. CYP4F2 has also been shown recently to metabolize vitamin K. METHODS: Three hundred and eleven patients were recruited prospectively from two UK hospitals and followed-up for 6 months. Fine mapping of the whole CYP4F2 region was performed to try and define the haplotype structure of CYP4F2. Genotyping was performed on the Sequenom platform. Univariate and multiple regression analyses were undertaken to assess the effect of CYP4F2 on predefined clinical outcomes of warfarin response. RESULTS: Fifty-nine single nucleotide polymorphisms in the CYP4F2 gene were analyzed. There was a high degree of linkage disequilibrium in the gene with two haplotype blocks. No association was found with warfarin stable dose and rs2108622 in our prospective cohort of patients even after adjustments to reduce patient heterogeneity. Interestingly, a single nucleotide polymorphism (rs2189784), which is in strong linkage disequilibrium with rs2108622, showed an association with time-to-therapeutic international normalized ratio which remained significant after the correction for multiple testing (Pc = 0.03). No association was shown with the haplotypes after false discovery rate correction. CONCLUSION: Although we were unable to demonstrate an association between rs2108622 and stable warfarin dose, our finding of an association between rs2189784 and time-to-therapeutic international normalized ratio is consistent with the recent finding that CYP4F2 plays a role in vitamin K metabolism. However, the effect of CYP4F2 is relatively small in all studies undertaken so far, and thus seems unlikely to be of clinical relevance.

Comparative preclinical drug metabolism and pharmacokinetic evaluation of novel 4-aminoquinoline anti-malarials.

The disposition of three 4-aminoquinoline leads, namely isoquine (ISO), des-ethyl isoquine (DEI) and N-tert-butyl isoquine (NTBI), were studied in a range of in vivo and in vitro assays to assist in selecting an appropriate candidate for further development. Analogous to amodiaquine (ADQ), ISO undergoes oxidative N-dealkylation to form DEI in vivo. Blood clearance of DEI was as much as 10-fold lower than that of ISO in animals and after oral administration, metabolite exposure exceeded that of parent by as much as 14-fold. Replacement of the N-ethyl with an N-tert-butyl substituent substantially reduced N-dealkylation as blood clearance of NTBI was approximately 2 to 3-fold lower than DEI in mouse, rat, dog and monkey. Mean NTBI oral bioavailability was generally higher than the other leads (>/=68%). Blood cell association was substantial for NTBI, particularly in dog and monkey, where blood to plasma concentration ratios >4 were observed. Human plasma protein binding was similar for NTBI, DEI, and des-ethyl amodiaquine (DEA). Allometric scaling predicted human blood clearance (CL) for NTBI to be low ( approximately 12% liver blood flow). All the 4-aminoquinolines inhibited recombinant human cytochrome P450 2D6 with similar potency; DEI also inhibited 1A2. On balance, NTBI appeared the most promising lead to progress towards full development.

The roles of drug metabolism in the pathogenesis of T-cell-mediated drug hypersensitivity.

PURPOSE OF REVIEW: The purpose of this review is to highlight recent studies on drug metabolism in T-cell-mediated reactions. Although the hapten theory and the danger hypothesis imply an important role of reactive metabolites in the pathogenesis of drug hypersensitivity, the more recent pi concept gives the central role to the parent drug. It is therefore important to have a broad vision on data supporting each theory to understand the potential role(s) of drug metabolism in T-cell-mediated hypersensitivity. RECENT FINDINGS: Reactive metabolites have been identified for most drugs associated with hypersensitivity. Recent studies have further characterized drug metabolism outside the liver, in key tissues such as the skin and immune cells. Localized drug metabolism is associated with oxidative stress, adduct formation and toxicity creating danger signals for antigen presenting cells, influencing whether a drug antigen will induce tolerance or immunity. SUMMARY: The involvement of metabolic drug activation in the initiation and propagation of hypersensitivity is intriguing since metabolites are generated in different quantities throughout the body, can be directly or indirectly toxic to cells, might stimulate innate immunity, and can bind to protein to generate neoantigens for cellular and humoral responses.

Activation of T-cells from allergic patients and volunteers by p-phenylenediamine and Bandrowski's base.

Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction.