Flucloxacillin is a synthetic penicillin used in the treatment of Staphylococcal infections. Adverse reactions to the drug are believed to arise through covalent modification of proteins, with tissue damage occurring secondary to an immune reaction. Serum proteins have been shown by adduct‐specific antibodies to be modified by flucloxacillin, but the nature and sites of modification have not been characterised. Here, in vitro studies on HSA have shown by MS that the modification of protein lysine residues occurs in a dose‐, time‐ and site‐dependent manner. Affinity, cation exchange and reversed phase chromatography prior to MS revealed in vivo modification of HSA with flucloxacillin in tolerant patients, with up to nine modified lysine residues being detected in each patient, and with modification of Lys190 and Lys212 being detected in 8/8 patients. It was also revealed for the first time that plasma proteins could be modified with the 5‐hydroxymethyl metabolite of flucloxacillin, and that essentially the same Lys residues were targeted by both the parent drug and its metabolite. This study provides a detailed characterisation of sites of chemical modification of an endogenous target and reveals candidate peptides for T‐cell and antibody assays of flucloxacillin hypersensitivity.

Covalent modification of protein by drugs may disrupt self-tolerance, leading to lymphocyte activation. Until now, determination of the threshold required for this process has not been possible. Therefore, we performed quantitative mass spectrometric analyses to define the epitopes formed in tolerant and hypersensitive patients taking the β-lactam antibiotic piperacillin and the threshold required for T cell activation. A hydrolyzed piperacillin hapten was detected on four lysine residues of human serum albumin (HSA) isolated from tolerant patients. The level of modified Lys⁵⁴¹ ranged from 2.6 to 4.8%. Analysis of plasma from hypersensitive patients revealed the same pattern and levels of modification 1–10 d after the commencement of therapy. Piperacillin-responsive skin-homing CD4⁺ clones expressing an array of Vβ receptors were activated in a dose-, time-, and processing-dependent manner; analysis of incubation medium revealed that 2.6% of Lys⁵⁴¹ in HSA was modified when T cells were activated. Piperacillin–HSA conjugates that had levels and epitopes identical to those detected in patients were shown to selectively stimulate additional CD4⁺ clones, which expressed a more restricted Vβ repertoire. To conclude, the levels of piperacillin–HSA modification that activated T cells are equivalent to the ones formed in hypersensitive and tolerant patients, which indicates that threshold levels of drug Ag are formed in all patients. Thus, the propensity to develop hypersensitivity is dependent on other factors, such as the presence of T cells within an individual’s repertoire that can be activated with the β-lactam hapten and/or an imbalance in immune regulation.

The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2((-/-))) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2((-/-)) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR <1%). One hundred sixty-one were significantly different (P<0.05) between WT and Nrf2((-/-)) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2((-/-)) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway. The Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2((-/-)) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers.

Microclots have been associated with various conditions, including postacute sequelae of severe acute respiratory syndrome coronavirus 2 infection. They have been postulated to be amyloid-fibrin(ogen) aggregates, but their role as a prognostic biomarker remains unclear. To examine their possible clinical utility, blood samples were collected for the first 96 hours from critically ill patients (n = 104) admitted to the intensive care unit (ICU). Detection was by staining platelet-poor plasma samples with thioflavin T and visualized by fluorescent microscopy. Image J software was trained to identify and quantify microclots, which were detected in 44 patients (42.3%) on ICU admission but not in the remaining 60 (57.7%) or the 20 healthy controls (0.0%). Microclots on admission to ICU were associated with a primary diagnosis of sepsis (microclots present in sepsis, 23/44 [52.3%] vs microclots absent in sepsis, 19/60 [31.7%]; P = .044). Multicolor immunofluorescence demonstrated that microclots consisted of amyloid-fibrinogen aggregates, which was supported by proteomic analysis. Patients with either a high number or larger-sized microclots had a higher likelihood of developing disseminated intravascular coagulation (odds ratio [OR], 51.4; 95% confidence interval [CI], 6.3-6721.1; P < .001) and had an increased probability of 28-day mortality (OR, 5.3; 95% CI, 2.0-15.6; P < .001). This study concludes that microclots, as defined by amyloid-fibrin(ogen) aggregates, are potentially useful in identifying sepsis and predicting adverse coagulopathic and clinical outcomes.