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International journal of molecular sciences 23(23), 15126 (2022). doi:10.3390/ijms232315126 special issue: "Special Issue "Neutrophil Extracellular Traps (NETs) in Immunity and Diseases" / Special Issue Editor: Małgorzata Wachowska, Guest Editor", Published by Molecular Diversity Preservation International, Basel

Purpose Obstructive Meibomian gland dysfunction (MGD) is one of the leading causes of evaporative dry eye disease. Meibomian glands at the eyelid secrete lipids that prevent evaporation of the aqueous tear film. The pathogenesis of obstructive MGD is incompletely understood to date. Herein, we aim to investigate the pathogenesis of obstructive MGD using murine and human samples with various forms of ocular surface inflammation. Method The presence of Neutrophil extracellular Traps (NETs) was detected with immunofluorescence analysis of ocular surface discharge and biopsy samples from patients with blepharitis. Tear fluid from patients with MGD and blepharitis were evaluated for the presence of inflammatory mediators using bead based immunoassay. Murine model of allergic eye disease (AED) was performed to investigate the role of NETs in MG occlusion. Results we show that the ocular discharge from patients with blepharitis contains aggregated neutrophil extracellular traps (aggNETs). Furthermore, the ducts of human Meibomian glands affected by blepharitis were largely congested by aggNETs. Tear fluid from patients with MGD showed elevated neutrophil chemoattractants (C5a, IL6, IL8 and IL18). C5a and IL8 correlated with the degree of deficiency of tear fluid. In the murine model of allergic eye disease (AED), aggNETs accumulated in the MG leading to occlusion of their ducts and the retrograde pent-up of the fluid followed by acinar atrophy. Constraining aggNET formation by genetic or pharmacological inhibition of peptidyl arginine deiminase type 4 (PADI4) effectively reduced MG damage. Conclusion We conclude that aggNETs occlude MG causing MGD after ocular surface inflammation.

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland and often results in sialadenitis with the need for surgical intervention. The mechanism behind the formation of sialoliths has been elusive. Here we show that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in their dense aggregation, and the subsequent mineralization creates alternating layers of dense mineral, predominantly calcium salt deposits and DNA. Further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis in which an immune system mediated response essentially participates in the physico-chemical process of concrement formation and growth.

Background Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are incompletely understood. Methods Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry. Patient findings Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage. Interpretation These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage. Funding Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung

The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.

This paper reports on the kinetic and thermodynamic parameters describing the interaction of selected digitalis derivatives with hog and guinea-pig cardiac ( Na + + K + )- ATPase (Na + /K + -transporting ATPase EC 3.6.1.37). 32 digitalis derivatives were characterized as to the values of the ΔG °′ , Δ G ≠ , and Δ G ≠ quantities in their interaction with ( Na + + K + )- ATPase from hog cardiac muscle in the presence of ATP, Mg 2+ , Na + and K + . Nine derivatives were additionally characterized as to the values of the ΔH °′ , ΔS °′ , Δ H ≠ , Δ S ≠ , Δ H ≠ , Δ S ≠ , and Δ S ≠ quantities in their interaction with the hog enzyme promoted by ATP, Mg 2+ and Na + in the presence or absence of K + . The formation of the inhibitory complexes is in any case an endothermic, entropically driven process. The Gibbs energy barriers in the formation and dissociation of the complexes, Δ G ≠ and Δ G ≠ , are imposed by large, unfavourable ΔH ≠ values. K + decreases the ΔG°′ value by increasing the Δ G ≠ value more than the Δ G ≠ value. In comparison with hog ( Na + + K + )- ATPase , the interaction of three derivatives with guinea-pig cardiac enzyme in the presence of ATP, Mg 2+ , Na + and K + is characterized by lower ΔG °′ values caused by lower favourable ΔS °′ values, and is accompanied by lower Δ G ≠ values. The magnitude of the kinetic parameters and the characteristic of the thermodynamic quantities describing the interaction between various digitalis derivatives and ( Na + + K + )- ATPase , indicate the induction of substantial conformational changes in the enzyme protein. A large entropy gain in the enzyme protein, observed irrespective of enzyme origin and ligation, appears to be the common denominator of the inhibitory action of all digitalis derivatives studied, suggesting that the digitalis-elicited relaxation of high conformational energy (negentropy strain) of the enzyme protein is the thermodynamic essence of the reversible inactivation of ( Na + + K + )- ATPase .

Digitoxin wurde mit Blut oder mit Gewebsschnitten aus der Leber verschiedener Species inkubiert. Mit Hilfe einer mikrochemischen Methode wurde die Geschwindigkeit des Abbaus zu chloroformunloslichen, unwirksamen Metaboliten bestimmt. Eine Biotransformation von Digitoxin durch Blut von Ratte, Mensch oder Meerschweinchen war nicht nachweisbar. In vitro erwiesen sich die Lebern von Krote, Frosch oder Mensch zu einem Abbau des Glykosids als unfahig. Leberschnitte von Taube, Katze, Meerschweinchen, Kaninchen und Hund zeigten in dieser Reihenfolge eine steigende Fahigkeit, Digitoxin in chloroformunlosliche Derivate umzuwandeln. — Die Speciesdifferenzen in der akuten Giftigkeit des Digitoxins stehen mit der jeweiligen Geschwindigkeit der Glykosidentgiftung nicht in Beziehung. Auf Grund der relativen Bedeutungslosigkeit der Digitoxinausscheidung bei Saugern einerseits und der Parallelitat zwischen der Abbaugeschwindigkeit und Kumulationsneigung von Digitoxin bei verschiedenen Tierarten andererseits wird geschlossen, das die Abbaugeschwindigkeit bestimmend fur die Dauer der Digitoxinwirkung bei Saugern ist. Nur bei Froschen und vielleicht auch Kroten ist die Ausscheidung von Digitoxin mit dem Harn der entscheidende Faktor. — Die unterschiedliche, kumulative Wirkung von Digitalisverbindungen verschiedener Struktur wird mit der Reversibilitat ihrer Bindung am Digitalisreceptor beziehungsweise mit der Geschwindigkeit ihrer Ausscheidung oder Entgiftung in Zusammenhang gebracht.

Mit Hilfe einer mikrochemischen Methodik wurde das Stoffwechselschicksal von i.v. injiziertem Digitoxin bei der Krote (B. bufo L.) untersucht. Dabei wurden keine Schwierigkeiten angetroffen, Digitoxin von den chloroform-loslichen Bufadienoliden zu unterscheiden, die im Parotidensekret, in der Haut und im Urin gefunden werden. Digitoxin wird innerhalb 48 Std nach Injektion weder zerstort noch verstoffwechselt. Das Verteilungsmuster von Digitoxin in den Geweben und Organen ist bei der Krote im wesentlichen das gleiche wie bei anderen, digitoxinempfindlicheren Species. Die Krote scheidet Digitoxin weder durch die Parotiden noch durch die Haut aus und auch die Ausscheidung mit dem Kot ist nur von geringer Bedeutung. Die Ausscheidung unveranderten Digitoxins im Harn ist bei der Krote viel groser als bei anderen Species mit Ausnahme des Frosches, jedoch scheint sie unzureichend, um die akute Digitoxintoxizitat zu senken. Die Ergebnisse legen den Schlus nahe, das die Unempfindlichkeit der Krote gegenuber Digitoxin nicht verursacht ist durch schnelle Entgiftung des Glykosids, durch Fehlen der Biotransformation zu einer eigentlichen Wirksubstanz, durch ungewohnlich niedrige Digitoxinaufnahme in den Herzmuskel oder durch umgehende Ausscheidung von Digitoxin. Wie in der Diskussion dargelegt wird, konnten die Speciesunterschiede in der Digitalisempfindlichkeit auf Unterschieden in der Assoziationstendenz von Digitalismolekul und Digitalisreceptor (Na+ + K+ aktivierte Membran-ATPase des Herzmuskels) beruhen.

After incubation of digitoxigenin with slices from livers of man, rabbit or dog, a total of 5 chloroform-insoluble, highly polar metabolites has been isolated by extraction with ether-ethanol. Two of the products were the sulfuric acid esters of digitoxigenin or 3-epi-digitoxigenin, as demonstrated earlier, and two further ones could not be identified. The fifth metabolite appeared to be identical with 3-epi-digitoxigenin glucuronide.

The paper describes the dissociation parameters of the complexes between [3H]-digitoxin and Na,K-ATPase (Na+ + K+-activated, Mg2+-dependent ATP phosphohydrolase, E.C. 3.6.1.3) from pig cardiac muscle and brain cortex formed and dissociated in the presence of different combinations and concentrations of the enzyme effectors ATP, Mg2+, Na+ and K+. Systematic variation of effector-ligation of Na,K-ATPase allowed production of glycoside complexes with two enzyme conformers only, which showed either rapid or slow dissociation kinetics. Appropriate changes of enzyme ligation allowed the interconversion of the two conformer types. Biphasic, rapid and slow glycoside release was not bound with the presence of two Na,K-ATPase isozymes, but caused by the enzyme ligation-determined coexistence of the two conformers of Na,K-ATPase. The rate constants for the rapid and slow glycoside release were within the complexes of each dissociation type much alike indicating uniform isomerization kinetics of the two conformers even when differently liganded. Taken together, the observations indicated the effector-controlled isomerizations of two conformers of Na,K-ATPase possessing different geometries of the glycoside binding domain. Present findings and relevant literature data were integrated in a circular, consecutive and simultaneous model for induced conformation changes that accounted for the regulation of the interaction of cardiac glycosides and Na,K-ATPase through an effector-controlled equilibrium between two limit enzyme conformers.

In der vorliegenden Arbeit wird die Biotransformation von insgesamt zehn Cardenolid- und Bufadienolidgeninen (Steroidlactonen) durch Gewebsschnitte aus der Leber von zehn Tierarten einschlieslich des Menschen beschrieben. Die Geschwindigkeit der Verstoffwechselung ist am geringsten bei Krote, Taube und Frosch und am hochsten bei der Katze und den Nagern. Zusatzliche Hydroxylgruppen in den Positionen 12β oder 16β vermindern die Geschwindigkeit der Biotransformation der entsprechenden Steroidlactone. Eine reversible Reduktion der Doppelbindung in der Butenolidgruppe oder eine Umlagerung des Lactonrings aus 17αH- in 17βH-Konfiguration scheint nicht vorzukommen. Der gewohnliche Weg der Biotransformation der Genine mit Ausnahme des Strophanthidins ist eine Umlagerung der Hydroxylgruppe am Kohlenstoff 3 aus β- in α-Stellung. Die Epimerisierung verlauft uber eine Dehydrogenierung des 3β-Hydroxyls zu einer 3-Ketogruppe und deren anschliesende Reduktion zu einer 3α-Hydroxylgruppe. Die Lebern der Species, welche die Steroidlactone mit hoher Geschwindigkeit verstoffwechseln, bilden auch grose Mengen an 3-Epi-Derivaten. Die Epimerisierung ist danach offenbar von hervorragender Bedeutung fur die Biotransformation der Genine. Da die 3-Epi-Verbindungen bekanntlich biologisch relativ unwirksam sind, kann die Epimerisierung der Steroidlactone im tierischen Organismus als ein Entgiftungsprozes betrachtet werden, welcher die Wirkungsdauer der Genine begrenzt. Die Inkubation von Digitoxigenin, Dihydro-digitoxigenin, Uzarigenin, Digoxigenin oder Gitoxigenin mit Leberschnitten verschiedener Species liefert daruber hinaus einige hoher polare, aber noch chloroformlosliche Metaboliten, welche offenbar aus der Hydroxylierung des Steroidkerns hervorgehen. Die Lebern aller untersuchten Species hydroxylieren Digitoxigenin in einer bislang nicht genau identifizierten Position, wobei es sich moglicherweise um C-6 handelt. Das Vorkommen der schon bekannten 12β-Hydroxylierung kann nur fur die Rattenleber gesichert werden.

In der vorliegenden Arbeit wird die Konjugation von Cardenolidgeninen mit Schwefelsaure oder Glucuronsaure unter in vitro-Bedingungen beschrieben. — Die Synthese der Schwefelsaureester von Digitoxigenin, 3-Epi-digitoxigenin, Uzarigenin, 3-Epi-uzarigenin, Sarmentogenin, Digoxigenin und Gitoxigenin wird erreicht durch Verwendung eines enzymatischen Systems, das von anderen Autoren fur die Bereitung der Sulfate von Steroidhormonen beschrieben wurde. Das System funktioniert zumindest mit den loslichen Fraktionen der Lebern von den untersuchten Species (Meerschweinchen, Mensch und menschlicher Fetus).

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. The mechanism behind the formation of sialoliths has been elusive. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland, and often results in sialadenitis with the need for surgical intervention. Here, we show by the use of immunohistochemistry, immunofluorescence, computed tomography (CT) scans and reconstructions, special dye techniques, bacterial genotyping, and enzyme activity analyses that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths in humans. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in the dense aggregation of the latter, and the subsequent mineralization creates alternating layers of dense mineral, which are predominantly calcium salt deposits and DNA. The further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis, in which an immune system-mediated response essentially participates in the physicochemical process of concrement formation and growth.