Stephan Arni

Systematic inference of enzyme activity in human tumors is key to understanding cancer progression and resistance to therapy. However, standard protein or transcript abundances are blind to the activity status of the measured enzymes, regulated, for example, by active-site amino acid mutations or post-translational protein modifications. Current methods for activity-based proteome profiling (ABPP), which combine mass spectrometry (MS) with chemical probes, quantify the fraction of enzymes that are catalytically active. Here, we describe depletion-dependent ABPP (dd-ABPP) combined with automated SWATH/DIA-MS, which simultaneously determines three molecular layers of studied enzymes: i) catalytically active enzyme fractions, ii) enzyme and background protein abundances, and iii) context-dependent enzyme-protein interactions. We demonstrate the utility of the method in advanced lung adenocarcinoma (LUAD) by monitoring nearly 4000 protein groups and 200 serine hydrolases (SHs) in tumor and adjacent tissue sections routinely collected for patient histopathology. The activity profiles of 23 SHs and the abundance of 59 proteins associated with these enzymes retrospectively classified aggressive LUAD. The molecular signature revealed accelerated lipoprotein depalmitoylation via palmitoyl(protein)hydrolase activities, further confirmed by excess palmitate and its metabolites. The approach is universal and applicable to other enzyme families with available chemical probes, providing clinicians with a biochemical rationale for tumor sample classification.

Cytokine adsorption can resolve different complications characteristic of transplantation medicine, such as cytokine storm activation and blood ABO and immune incompatibilities. Cytokine adsorption is also performed for the treatment of various life-threatening conditions, such as endotoxic septic shock, acute respiratory distress syndrome, and cardiogenic shock, all potentially leading to adverse clinical outcomes during transplantation. After surgery, dysmetabolism and stress response limit successful graft survival and can lead to primary or secondary graft dysfunction. In this clinical context, and given that a major problem in transplant medicine is that the demand for organs far exceeds the supply, a technological innovation such as a hemoadsorption system could greatly contribute to increasing the number of usable organ donors. The objectives of this review are to describe the specific advantages and disadvantages of the application of cytokine adsorption in the context of transplantation and examine, before and/or after organ transplantation, the benefits of the addition of a cytokine adsorption therapy protocol.

Cytokine adsorption can resolve different complications characteristic of transplantation medicine, such cytokine storm activation, blood immuno- and AB0- incompatibilities but can also be performed for the treatment of various life-threatening conditions, such as sepsis, acute respiratory distress syndrome (ARDS), and cardiogenic shock, all of which can contribute to adverse clinical outcomes during transplantation. After surgery, dysmetabolism and stress response limit successful graft survival and can lead to primary (PGD) or secondary graft dysfunction. In this clinical context, and given that a major problem in transplant medicine is that the demand for organs far exceeds the supply, a technological innovation such as a hemoadsoption system could greatly contribute to increasing the number of usable organ donors. The objectives of this review are to describe the specific advantages and disadvantages of the application of cytokine adsorption in the context of transplantation and also to examine, before and/or after organ transplantation, the benefits of addition of a complementary cytokine adsorption therapy protocol.

Supernatant from rat and human MPM cells could stimulate Gli1 Ptch1 and Fn1 expression of NIH3T3 cells

Ischemia-reperfusion injury compromises short- and long-term outcomes after lung transplantation. The scarce existing data on NAD⁺ suggest effects on hypoxia-induced vasoconstriction, on reactive oxygen species and on tampering inflammation. We exposed rat lungs to 14 h of cold ischemic storage and perfused them in a rat ex vivo lung perfusion (EVLP) system for 4 h. A control group (n = 6) was compared to groups receiving 100 µM (n = 6) or 200 µM NAD⁺ (n = 6) in the preservation solution and groups receiving 200 µM (n = 4) or 2000 µM (n = 6) NAD⁺ every 30 min in the perfusate, starting at 1 h of EVLP. Compared to the control, significant effects were only achieved in the 2000 µM NAD⁺ group. During the 4 h of EVLP, we monitored higher vascular flow, lower mean pulmonary arterial pressure and increased oxygenation capacity. Tissue inflammation estimated with the myeloperoxidase assay was lower in the 2000 µM NAD⁺ group. We observed higher levels of anti-inflammatory IL-10, higher anti-inflammatory IL-6/IL-10 ratios and lower levels of pro-inflammatory IL-12 and IL-18 as well as a trend of more anti-inflammatory IFNy in the 2000 µM NAD⁺ perfusate. In the bronchoalveolar lavage, the pro-inflammatory levels of IL-1α and IL-1β were lower in the 2000 µM NAD⁺ group. NAD⁺ administered during EVLP is a promising agent with both anti-inflammatory properties and the ability to improve ischemic lung function.

Lung transplantation improves the outcome and quality of life of patients with end-stage pulmonary disease. However, the procedure is still hampered by the lack of suitable donors, the complexity of the surgery, and the risk of developing chronic lung allograft dysfunction. Over the past decades, translational experiments in animal models have led to a better understanding of physiology and immunopathology following the lung transplant procedure. Small animal models (e.g., rats and mice) are mostly used in experiments regarding immunology and pathobiology and are preferred over large animal models due to the ethical aspects, the cost–benefit balance, and the high throughput possibility. In this comprehensive review, we summarize the reported surgical techniques for lung transplantation in rodent models and the management of perioperative complications. Furthermore, we propose a guide to help identify the appropriate species for a given experiment and discuss recent experimental findings in small animal lung transplant models.

Ex vivo lung perfusion (EVLP) has been implemented to increase the number of donor lungs available for transplantation. The use of K(ATP) channel modulators during EVLP experiments may protect against lung ischemia-reperfusion injury and may inhibit the formation of reactive oxygen species. In a rat model of donation after circulatory death with 2 h warm ischemic time, we evaluated rat lungs for a 4-hour time in EVLP containing either mitochondrial-specific or plasma membrane and/or sarcolemmal-specific forms of K(ATP) channel modulators. Lung physiological data were recorded, and metabolic parameters were assessed. When compared to the control group, in the EVLP performed with diazoxide or 5-hydroxydecanoic acid (5-HD) we recorded significantly lower pulmonary vascular resistance and only in the diazoxide group recorded significant lung weight loss. In the perfusate of the 5-HD group, interleukin-1β and interleukin-1α were significantly lower when compared to the control group. Perfusate levels of calcium ions were significantly higher in both 5-HD and cromakalim groups, whereas the levels of calcium, potassium, chlorine and lactate were reduced in the diazoxide group, although not significantly when compared to the control. The use of a diazoxide mitochondrial-specific K(ATP) channel opener during EVLP improved lung physiological and metabolic parameters and reduced edema.