Antimicrobial peptides (AMPs) have been identified as important therapeutic agents that can be developed as new multifunctional antibiotic compounds, which may address antibiotic resistance. AMPs have a wide range of bioactivities, including antimicrobial, antioxidant, anti-inflammatory and anticancer properties. Os and Os-C (a derivative of Os, lacking cysteine residues) are two synthetic AMPs derived from the tick defensin OsDef2 which have been shown to have antibacterial, antioxidant and anti-inflammatory activity. Differences in bacterial killing times between these peptides indicate differences in the modes of bacterial killing. For the further development of Os and Os-C for therapeutic application, the aim of this study was to establish the mode of bacterial killing, to determine if these peptides are cytotoxic to human erythrocytes and leukocytes. Lastly, to determine if these peptides have additional beneficial cellular effects such as antioxidant activity. Ultrastructural analysis with electron microscopy techniques revealed that both peptides adversely affected the membranes and intracellular structures of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria. Effects included membrane ruffling, cytoplasmic retraction, intracellular granulation and the formation of dense fibres. At the minimum bactericidal concentrations (MBCs) of 0.77 μM for Os and 1.74 μM for Os-C membrane permeabilisation measured with the SYTOX green assay was found not to be the principle mode of action. In stationary phase bacteria, fluorescent triple staining showed that both peptides caused permeabilisation. Studies using fluorescently labelled peptides revealed that the membrane penetrating activities of Os and Os-C were similar to buforin II, a cell-penetrating peptide. Os was able to enter stationary phase E. coli and B. subtilis while Os-C was unable to enter E. coli cells and accumulated on B. subtilis septa. Using plasmid binding and fluorescence displacement assays both peptides could bind DNA, while a dosage effect was only observed for Os. Evaluation of cytotoxicity revealed that Os and Os-C caused no erythrocyte haemolysis or changes to erythrocyte morphology. Only the highest concentration of Os (100 μM), which is 130 fold greater than the MBC for E. coli and B. subtilis, caused cellular damage to peripheral mononuclear (MN) and polymorphonuclear (PMN) cells. In contrast, Os-C caused leukocyte activation identified by associated morphological features and reactive oxygen species (ROS) formation. Chemical and erythrocyte antioxidant assays indicated that both Os and Os-C had antioxidant activity. Both peptides provided extracellular protection of erythrocytes against 2,2'-azobis(2-amidinopropane) dihydrochloride induced oxidative damage. In MN and PMN cells Os showed low levels of antioxidant activity while Os-C had minimal activity. In conclusion, both peptides showed a dual mechanism of bacterial killing, targeting both the membrane and intracellular elements. Os had a predominant membrane effect while Os-C targeted the septa of B. subtilis and had a higher affinity for DNA. Cytotoxicity in erythrocytes and leukocytes was minimal. In addition, Os exhibited antioxidant properties while Os-C caused leukocyte activation. Both peptides have been identified as promising therapeutic agents although activity in plasma and the effect on coagulation must still be determined. / Thesis (PhD)--University of Pretoria, 2017. / Anatomy / PhD / Unrestricted

Many complications associated with heart surgery are due to the negative effects of extracorporeal circulation (ECC). Some of these complications may be attributed to ECC-induced activation of inflammation and coagulation pathways. The inflammatory reaction may be caused by the interaction of blood components with air and the artificial surfaces of the ECC, from substances produced due to ischaemia-reperfusion injury of the heart and lungs, and from increased release of endotoxin from ischemic intestines. Staphylococcus aureus (S. aureus) infections are the leading cause of respiratory, skin and soft tissue, and bloodstream infections. Nitric oxide (NO) is a gaseous signaling molecule involved in many physiological and pathological processes. The role of NO in infection and inflammation is complex. NO may contribute to morbidity by acting as a vasodilator, myocardial depressant, and cytotoxic mediator. On the other hand, NO may have a salutary role through microvascular, cytoprotective, immunoregulatory, and antimicrobial properties. A simulated extracorporeal circulation (SECC) model is a closed circuit, including a roller pump, an oxygenator, a venous reservoir and polyvinyl chloride (PVC) tubing, where human blood is circulated. The SECC model allows studies of the blood and its components, without any influence from other organ systems. The aim of this work was to investigate NO effects during SECC and in S. aureus infection. Study I. Human blood was circulated through SECC during 3 hours, and leukocyte granule release was studied. Results indicated that NO addition during SECC is pro-inflammatory by stimulating leukocyte activation and granule release, and has no effect on oxygen free radical production and interleukin release. Study II. Investigating the effect of NO on S. aureus growth in whole blood during 180 min SECC, results showed a 6.2 fold growth in the presence of NO. Results indicated that by stimulating the expression of inducible lactate dehydrogenase, specific to S. aureus, NO may improve S. aureus resistance to oxidative stress, giving the pathogen a survival advantage. Study III. In an in vitro system of SECC, we measured glyceryl trinitrate (GTN) induced changes in leukocyte activation in whole blood caused by S. aureus infestation, as well as the effect of GTN on S. aureus growth. Results indicated that GTN does not affect S. aureus growth during SECC and has no effect on SECC-induced leukocyte activation. Study IV. Whole blood concentrations of selected leukocyte adhesion molecules, complement system components and myeloperoxidase  were measured in an in vitro system of SECC. Results indicated that SECC induces the increased expression of some leukocyte markers and that GTN addition significantly reduces the expression of some leukocyte activation markers.

Störungen im Bereich der Mikrozirkulation gelten als ursächlich für die Entstehung des Multiorganversagens bei Sepsis, wobei der Darm eine zentrale Rolle einnimmt. Aktivierte Leukozyten setzen u.a. Sauerstoffradikale frei, die entscheidend zur Zerstörung der endothelialen Integrität beitragen. Die Antagonisierung schädigender Mediatoren stellt ein Prinzip der adjunktiven Sepsis-Therapie dar, wobei die antioxidativ wirkenden Substanzen N-Acetylcystein (NAC) und Tirilazad Mesylat (TM) in mehreren Studien positive Effekte gezeigt haben. In einer tierexperimentellen Untersuchung an Ratten wurde der Effekt der kombinierten Gabe von NAC und TM auf die mesenteriale Mikrozirkulation, auf die Freisetzung von TNF-alpha, IL-1beta, IL-6, IL-10 und auf die Leukozytenzahl unter einer kontinuierlichen Lipopolysaccharidbelastung (LPS) von 10 mg/kg KG untersucht. Die Beurteilung der mesenterialen Mikrozirkulation erfolgte mittels Intravitalmikroskopie. Hierbei wurde das Ausmaß der Leukozytenadhärenz am Endothel der mesenterialen Venolen als Maß für die Leukozytenaktivierung und die Plasmaextravasation als Parameter für die Endotheldysfunktion bestimmt. Dabei konnte während zwei Stunden Endotoxinämie die Zunahme der Plasmaextravasation an mesenterialen Venolen durch die kombinierte Gabe von NAC und TM nicht signifikant beeinflusst werden (p>0,05). Eine tendenziell erhöhte Plasmaextravasation unterstützt die Hypothese, dass leukozytenunabhängige Mechanismen für die Plasmaextravasation existieren. Während zwei Stunden Endotoxinämie kam es zu einer signifikanten Reduktion der Anzahl der fest adhärenten Leukozyten in der NAC/TM-Gruppe im Vergleich zur LPS-Gruppe (p=0,001). Durch die kombinierte Gabe von NAC und TM konnte die endotoxininduzierte Freisetzung von TNF-alpha, IL-1beta, IL-6, IL-10 und die endotoxinbedingte Leukopenie nicht signifikant beeinflusst werden. / Disturbances of the microcirculation are causal for the pathophysiology of multiorgan failure related to sepsis in which the gut plays a central part. Activated leukocytes release i.e. oxygen radicals which decisively contribute to the destruction of the endothelial integration. To antagonize the damaging mediators is a principle of the adjunctive sepsis therapy in which the antioxidant agents N-acetylcysteine (NAC) and tirilazad mesylate (TM) showed positive effects in several studies. The effect of the combined administering of NAC and TM under continuous lipopolysaccharide (LPS) exposure of 10 mg/kg BW on the mesenteric microcirculation, on the release of TNF-alpha, IL-1beta, IL-6, IL-10 and on the number of leukocytes was examined in an animal study on rats. The appraisal of the microcirculation was done by intravital microscopy. The degree of leukocyte adherence on the endothelium of mesenteric venules was determined for the degree of leukocyte activation, and the plasma extravasation was the parameter for the endothelial dysfunction. The increase of plasma extravasation on mesenteric venules during 2 hours of endotoxemia could not be affected significantly by the combined administering of NAC and TM. The tendency of increased plasma extravasation supports the hypothesis of the existence of a leukocyte independent mechanism of plasma extravasation. During 2 hours of endotoxemia the NAC/TM group showed a significant decrease in the number of firmly adherent leukocytes in comparison to the LPS group. There was no significant effect on the endotoxin induced release of TNF-alpha, IL-1beta, IL-6, IL-10 and the endotoxin induced leukopenia by the combined administration of NAC and TM.

In der vorliegenden Arbeit sind Untersuchungen vorgestellt, die sich mit Wirkungen und Interaktionen von zwei ubiquitär im menschlichen Körper vorkommenden Gasen befassen, i.e. Sauerstoff und Stickstoffmonoxid. Im Falle beider Substanzen ermöglicht die geringe Größe der Moleküle eine freie Diffusion über Membranen hinweg, eine Eigenschaft, die für die Funktion der Signaltransduktion geradezu prädestiniert. Aus den vorgelegten Untersuchungen lassen sich die folgenden Folgerungen ableiten: * Stickstoffmonoxid wirkt in-vitro selektiv bakteriostatisch auf Bakterien, die üblicherweise Früh- und Neugeborene besiedeln. Dabei hängt die Selektivität von den jeweiligen bakteriellen Verteidigungsmechanismen ab, die bakteriostatische Wirkung liegt in einem Konzentrationsbereich, der außerhalb desjenigen liegt, der derzeit klinisch angewendet wird. * Hyperoxie führt im Ganztiermodell der unreifen Ratte zu einer zerebralen Hochregulation von iNOS und damit zur Synthese von NO. Soweit dies anhand der Synthese von Peroxynitrit als definitivem Schädigungsmechanismus beurteilbar ist, wird trotz entsprechender iNOS-Expression wenig bis gar kein Peroxynitrit gebildet. Da das Zusammentreffen von NO und Sauerstoff sonst regelhaft zur Entstehung von Peroxynitrit führt, müssen im Gehirn der unreifen Ratte ausreichende antioxidative Schutzmechanismen präsent sein, die diese Reaktion verhindern. * Im in-vitro-Modell der Gasäquilibrierung von Nabelschnur-PMN zeigte sich unter Hyperoxie das ausgeprägteste Aktivierungsmuster aller verglichenen Sauerstoffkonzentrationen. Dies stand im Gegensatz zur Exposition adulter Zellen, hier fand sich eine größere Hyperoxietoleranz bei gleichzeitig stärkster Aktivierung unter Hypoxiebedingungen. Welche Bedeutung diesen Ergebnissen im klinischen Umgang mit Neugeborenen zukommt muß derzeit noch offen bleiben. Allerdings häufen sich Hinweise aus experimentellen Studien, die darauf hindeuten, daß ein restriktiver Umgang mit hohen Sauerstoffkonzentrationen auch im klinischen Umfeld gerechtfertigt sein könnte. / The present investigations deal with the effects and interactions of gases, which are ubiquitous in the human body i.e. oxygen and nitric oxide. Both substances are small enough to freely diffuse across biological membranes. This ability predestines both molecules for the function of signal transduction. The results of our investigations lead to conclusions as follows: * Nitric oxide has selective bacteriostatic effects in-vitro on some bacterial strains typically isolated from preterm and term newborn infants. Selectivity depends on the presence of bacterial defense mechanisms. The bacteriostatic effect takes place at concentrations above those currently used in clinical practice. * Hyperoxia leads to upregulation of iNOS and subsequent NO production in an animal model of the immature rat. Despite this upregulation of iNOS synthesis there is no increased production of peroxynitrite which is known to cause cellular and DNA damage. Since the combination of NO and high concentrations of oxygen lead to peroxynitrite formation on a regular basis, effective antioxidant mechanisms appear to prevent peroxynitrite formation in the brain of the immature rat. * The most pronounced activation of cord blood polymorphonuclear cells (PMN) during conditions of hyperoxia, normoxia, and hypoxia was found for exposure towards high oxygen concentrations in an in-vitro model of gas equilibration. As opposed to that, hypoxia was the most potent trigger for adult PMN. It remains to be determined which clinical implications must be derived from these results. However, increasing experimental evidence indicates that exposure towards high oxygen concentrations should be restricted also in clinical practice and not only in preterm infants, but also in term newborns.