SARS-CoV-2 vaccine ChAdOx1 nCov-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models and analysis of VITT patient samples we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the EDTA-containing vaccine. Injected vaccine increased vascular leakage in mice leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release NETs in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drive thrombosis in VITT. The data support a two-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

BackgroundThe emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs.ResultsIn an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted.ConclusionThe metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria.

BackgroundSARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that—like autoimmune heparin-induced thrombocytopenia—is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies.MethodsWe investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using dynamic light scattering, 3D-super-resolution microscopy, and electron microscopy. Mass spectrometry was used to analyze vaccine composition. We investigated the mechanism for early post-vaccine inflammatory reactions as potential co-stimulant for anti-PF4 immune response. Finally, we evaluated VITT antibodies for inducing release of procoagulant DNA-containing neutrophil extracellular traps (NETs), and measured DNase activity in VITT patient serum.ResultsBiophysical analyses showed formation of complexes between PF4 and vaccine constituents, including virus proteins that were recognized by VITT antibodies. EDTA, a vaccine constituent, increased microvascular leakage in mice allowing for circulation of virus- and virus-producing cell culture-derived proteins. Antibodies in normal sera cross-reacted with human proteins in the vaccine and likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. Polyphosphates and DNA enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven procoagulant NETs formation, while DNase activity was reduced in VITT sera, with granulocyte-rich cerebral vein thrombosis observed in a VITT patient.ConclusionsChAdOx1 nCoV-19 vaccine constituents (i) form antigenic complexes with PF4, (ii) EDTA increases microvascular permeability, and (iii) vaccine components cause acute inflammatory reactions. Antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. High-titer anti-PF4 antibodies activate platelets and induce neutrophil activation and NETs formation, fueling the VITT prothrombotic response.

ABSTRACT:The in vitro metabolism of flupirtine, ethyl-N-[2-amino-6-(4-fluorophenylmethyl-amino)pyridine-3-yl]carbamate, a centrally acting analgesic with muscle tone-reducing activity, was studied. Two flupirtine metabolites were already known: the N-acetylated analog D13223 and 4-fluorohippuric acid. The structure of flupirtine suggested that redox chemistry may play a role in metabolism, and cyclic voltammetry studies showed that the drug undergoes facile and irreversible redox reactions. Thus, oxidative metabolism was investigated first. With CYP3A1-induced rat liver microsomes an 18% turnover of flupirtine and a 20 to 25% turnover of D13223 took place over 30 min, but less than 5% turnover of flupirtine was observed with all human liver microsomal preparations tested, evidence that cytochrome P450 does not contribute appreciably to the metabolism in humans. Likewise, no involvement of human monoamine oxidase (isoforms A and B) was found for either flupirtine or D13223. In contrast, flupirtine was an excellent substrate for both human myeloperoxidase and horse radish peroxidase (HRP). These enzymes produced detectable amounts of oxidation products. Incubations of flupirtine with HRP produced an oxidation product that could be trapped with glutathione, the resulting glutathione conjugate was characterized by mass spectrometry and NMR. Metabolism of D13223 by both peroxidases was also observed but to a much lesser extent. Porcine liver esterases cleave the carbamate group of flupirtine, and both human N-acetyltransferases 1 and 2 acetylated the hydrolysis product, presumably descarboethoxyflupirtine, with nearly equal efficiencies to yield D13223. Incubations of human liver microsomes with flupirtine or the metabolite D13223 together with UDP-glucuronic acid gave two isomeric N-glucuronides in both cases.Flupirtine maleate (1) (ethyl-N-[2-amino-6-(4-fluoro-phenylmethylamino)pyridin-3-yl]carbamate maleate, Katadolon) (Scheme 1), a centrally acting nonopiate analgesic with muscle tone-reducing activity, does not show the classic side effects of opiates or nonsteroidal anti-inflammatory analgesics (Jakovlev et al., 1985;Szelenyi and Nickel, 1991). The drug has been effectively and safely used in Germany for 20 years. The mode of action is now mostly understood. Modification of prostaglandin formation is of minor importance for the analgesic action of flupirtine (Darius and Schrör, 1985). Evidence has accumulated that flupirtine interacts indirectly with the N-methyl-D-aspartate-responsive subtype of the glutamate receptor, possibly by opening K ϩ channels (Jakob and Krieglstein, 1997; Kornhuber et al., 1999a,b). Clinical interest in the compound is growing as further indications for the drug are found. For example, successful treatment of fibromyalgia has been reported (Stoll, 2000). In particular, the neuroprotective properties attributed to flupirtine are making it a possible candidate for treatment of Parkinson's and Alzheimer's diseases, Creutzfeldt-Jacob disease, and other neurodegenerative ailments (Schuste...