Vascular adhesion protein-1-targeted PET imaging in autoimmune myocarditis.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and primary amine oxidase, and Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetra-acetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([68Ga]Ga-DOTA-Siglec-9) is a positron emission tomography (PET) tracer targeting VAP-1. We evaluated the feasibility of PET imaging with [68Ga]Ga-DOTA-Siglec-9 for the detection of myocardial lesions in rats with autoimmune myocarditis. METHODS: Rats (n = 9) were immunized twice with porcine cardiac myosin in complete Freund's adjuvant. Control rats (n = 6) were injected with Freund's adjuvant alone. On day 21, in vivo PET/computed tomography (CT) imaging with [68Ga]Ga-DOTA-Siglec-9 was performed, followed by ex vivo autoradiography, histology, and immunohistochemistry of tissue sections. In addition, myocardial samples from three patients with cardiac sarcoidosis were studied. RESULTS: [68Ga]Ga-DOTA-Siglec-9 PET/CT images of immunized rats showed higher uptake in myocardial lesions than in myocardium outside lesions (SUVmean, 0.5 ± 0.1 vs 0.3 ± 0.1; P = .003) or control rats (SUVmean, 0.2 ± 0.03; P < .0001), which was confirmed by ex vivo autoradiography of tissue sections. Immunohistochemistry showed VAP-1-positive staining in lesions of rats with myocarditis and in patients with cardiac sarcoidosis. CONCLUSION: VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 PET is a potential novel technique for the detection of myocardial lesions.

Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are ß-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

Nafamostat is a Potent Human Diamine Oxidase Inhibitor Possibly Augmenting Hypersensitivity Reactions during Nafamostat Administration.

Nafamostat is an approved short-acting serine protease inhibitor. However, its administration is also associated with anaphylactic reactions. One mechanism to augment hypersensitivity reactions could be inhibition of diamine oxidase (DAO). The chemical structure of nafamostat is related to the potent DAO inhibitors pentamidine and diminazene. Therefore, we tested whether nafamostat is a human DAO inhibitor. Using different activity assays, nafamostat reversibly inhibited recombinant human DAO with an IC50 of 300-400 nM using 200 µM substrate concentrations. The Ki of nafamostat for the inhibition of putrescine and histamine deamination is 27 nM and 138 nM, respectively For both substrates, nafamostat is a mixed mode inhibitor with P values of <0.01 compared with other inhibition types. Using 80-90% EDTA plasma, the IC50 of nafamostat inhibition was approximately 360 nM using 20 µM cadaverine. In 90% EDTA plasma, the IC50 concentrations were 2-3 µM using 0.9 µM and 0.18 µM histamine as substrate. In silico modeling showed a high overlap compared with published diminazene crystallography data, with a preferred orientation of the guanidine group toward topaquinone. In conclusion, nafamostat is a potent human DAO inhibitor and might increase severity of anaphylactic reaction by interfering with DAO-mediated extracellular histamine degradation. SIGNIFICANCE STATEMENT: Treatment with the short-acting anticoagulant nafamostat during hemodialysis, leukocytapheresis, extracorporeal membrane oxygenator procedures, and disseminated intravascular coagulation is associated with severe anaphylaxis in humans. Histamine is a central mediator in anaphylaxis. Potent inhibition of the only extracellularly histamine-degrading enzyme diamine oxidase could augment anaphylaxis reactions during nafamostat treatment.