Infectious diseases are the worldwide leading cause of morbidity and mortality. Sepsis is the major cause of death in infectious diseases. It is one of the most serious and also one of the most difficult treatable conditions of contemporary medicine. Sepsis is the main cause of death in intensive care units. Causal therapy of sepsis does not yet exist. With a far better understanding of patho/physiological mechanism of sepsis, it is possible to model new preclinical experiments to verify the efficiency and security of new therapeutic procedures. Large animal experiments in progressive sepsis, with the use of domestic porcine, play a vital role. Long-standing experience with this model and similarity to human facilitate the realization of more complex experiments with potential for the relevant translation of results into the subsequent clinical studies on human subjects. The objective of this doctoral dissertation was to assess on the clinically relevant model: 1) the efficiency and security of extracorporeal membrane oxygenation in the event of vasoplegic septic shock; and 2) the benefit of two innovative therapeutic approaches to treatment of sepsis: a) the intravenous administration of mesenchymal stem cells; and b) the activation of the neuro-inflammatory reflex through the vagus nerve stimulation.

Background: Interactions between the neuroendocrine and immune system play an importatnt role in maintaining homeostasis. This communication is mediated by cytokines, neurotransmiters and hormones through endocrine, paracrine and autocrine signaling. Prolactin (PRL), hormone of anterior pituitary, is produced by a number of other tissues and cells of immune system. On periphery, PRL is cytokine. Sepsis is an inflamatory response of the organism to severe infection, Th1 immune response is activated and PRL could participate in it. Toll-like receptors (TLR) play a key role in a recognition of bacteial components and mediate a systemic response (with PRL secretion) during infection. It is supposed that activated immune system leads to increasing of PRL, TLR2 and TLR4 gene expression. We detected PRL, TLR2 a TLR4 mRNA levels in monocytes from patiens with system inflammation. We studied influence of single nucleotide polymorphism (SNP -1149 G/T) in PRL gene promotor, it supposed that G allele increases PRL expression. Materials and Methods: For the pilot study 30 patients diagnose with severe infectious event. Collectoin of patiens blood samples was performed consequently three times. Control group comprised 40 healthy individuals. One blood sample was taken from each healthy subject. For testing of...

Sepsis (blodförgiftning) är ett allvarligt tillstånd och behöver en snabb diagnos som fastställs genom blodprov. Blodprov från hela Stockholms Län skickas till Karolinska Universitetslaboratoriet (KUL) för analys, där biomedicinska analytiker och läkare fastställer arten på infektionen och beslutar vilken antibiotikabehandling är optimal. Målet med arbetet var att identifiera eventuella problem som skapar flaskhalsar hos Klinisk Mikrobiologi i Solna som är en del av KUL. Därefter skulle lösningsförslag framföras och möjligheten att införa blodprover i ett nytt automatiskt system, BD Kiestra Total Lab Automation undersökas. Verksamhetskedjan för laboratoriet analyserades genom observationer och läsning av metodbeskrivningar. Utifrån verksamhetsanalysen identifierades sedan problem. En utbildning med BD Kiestra TLA gav en översikt av hur systemet fungerar samt fördelarna med att använda det. Genom litteraturstudier samt öppna intervjuer kunde förslag på lösningar och förbättringar framföras. Resultaten behandlar de lösningsförslagen för de identifierade problemen, och sammanfattas i en tabell. BD Kiestra TLA behandlades separat med en systembeskrivning, följt av de problem som finns för införandet av blodprov och hur dessa problem kan lösas. / Sepsis (blood poisoning) is a serious affliction that requires a fast diagnosis that can be confirmed through blood samples. Blood samples from all of Stockholm County are sent to Karolinska University Laboratory (KUL) for analysis, where biomedical scientists and medical doctors identify the species of the infection, and decide on the optimum antibiotic treatment. The goal of this assignment was to identify possible problems resulting in bottlenecks at the department of Clinical Microbiology in Solna, which is a part of KUL. Afterwards possible solutions were presented and possibilities of introducing blood samples into a new automated system, BD Kiestra Total Lab Automation, were investigated. The activity line for the blood sample and the laboratory’s operations were analysed by observation and reading of work instructions. The problems were identified through the operational analysis. A course for the BD Kiestra TLA was attended to give an overview of the system and what benefits it gives. The proposed solutions and improvements were formed through literary studies and open interviews. The results present the proposed solutions to the problems, and are summarized in a table. The BD Kiestra TLA was discussed separately with a description of the system, followed by the problems preventing the introduction of blood samples and how these problems can be solved.

Im klinischen Alltag stellt die Diagnosestellung der Sepsis bei Schwerbrandverletzten eine Herausforderung dar. Ursächlich sind pathophysiologische Besonderheiten der Verbrennungskrankheit, die in ihrer klinischen Präsentation dem Bild einer Sepsis ähnelt und somit die differentialdiagnostische Abgrenzung erschwert. Es existieren aktuell keine Parameter, Scores, oder Sepsiskriterien, die mit ausreichender Kompetenz eine Sepsis bei Brandverletzten feststellen können. Traditionelle Infektionsparameter (WBC, CRP, Fieber) gelten als zu unspezifisch. Das in der allgemeinen Intensivmedizin weithin etablierte Procalcitonin wird bei Brandverletzten ebenfalls kontrovers diskutiert. Für die Sepsis-3-Definition unter Verwendung des SOFA-Scores konnten bislang keine ausreichende Sensitivität und Spezifität nachgewiesen werden. IL-6 ist ein etablierter Marker der Inflammation mit sehr rascher Induktion bei einer Sepsis. Während das Ergebnis von Blutkulturen häufig erst nach 24-48 Stunden vorliegt, ist die Bestimmung des IL-6 innerhalb weniger Stunden möglich. Der diagnostische Wert des IL6 in der Frühphase der Sepsis bei Brandverletzten ist bislang ungeklärt. Das Ziel unserer Studie war es, die mögliche Beziehung zwischen positiven Blutkulturen und Il- 6-Serumspiegel bei schwerbrandverletzten Patienten mit klinischem Verdacht auf eine Sepsis zu überprüfen und den prädiktiven Wert des IL-6 mit dem anderer Parameter (PCT, WBC, Tmax und SOFA Score) zu vergleichen. In einer retrospektiven Studie wurden über einen 7-Jahreszeitraum alle Patienten eingeschlossen, bei denen bei klinischem Verdacht auf eine Sepsis Blutkulturen abgenommen sowie IL6, PCT und WBC bestimmt wurden. Die Patienten mit vollständigen Daten wurden gemäß Blutkultur in zwei Gruppen eingeteilt: Blutkultur-positive (BSI) und Blutkultur-negative Gruppe (Non-BSI). Die Gruppen wurden für alle gemessenen Biomarker, SOFA-Score und die maximale Körpertemperatur (T max) verglichen. Insgesamt wurden 101 Patienten eingeschlossen (BSI= 39, non-BSI= 62). Beide Gruppen waren hinsichtlich demographischer Daten und der Verletzungsschwere vergleichbar. IL-6 war bei den Patienten der BSI-Gruppe signifikant höher [1047 (339,9; 9000,5) vs. 198,5 (112,43; 702,52) ng/ l; p = 0,001]. In einer ROC-Analyse ergab sich eine AUC von 0,7 (59; 80,8 %). Somit ist IL-6 ein Biomarker mit moderater Aussagekraft. Als optimaler Cut-Off Wert erwies sich ein IL-6-Serumspiegel von 312,8 ng/ l (Sensitivität 79,5 %, Spezifität 56,5 %). Hinsichtlich der PCT, WBC und der Tmax zeigten die Gruppen keine signifikanten Unterschiede. Der SOFA-Score in der BSI-Gruppe war signifikant höher, allerdings wurde ein Anstieg um mindestens 2 Punkte im Vergleich zum Vortag nicht erreicht. Unsere Ergebnisse deuten darauf hin, daß IL-6 bei klinischem Verdacht auf eine Sepsis bereits frühzeitig eine positive Blutkultur vorhersagen kann. In diesem Kontext sind andere getestete Parameter (WBC, T max, PCT, SOFA-Score) weniger geeignet. Der frühe Anstieg des IL-6 könnte im klinischen Alltag hilfreich sein, da die Ergebnisse der IL-6-Bestimmung im Gegensatz zur Kultivierung von Blutkulturen innerhalb weniger Stunden vorliegen. Insbesondere in unklaren Fällen und Situationen, bei denen ein Zuwarten bezüglich der Einleitung einer Antibiotikatherapie möglich erscheint (z.B. fehlende Organdysfunktion) kann IL-6 einen Beitrag bei der Diagnosestellung leisten und einen Hinweis geben, ob differentialdiagnostische Überlegungen weiter verfolgt werden müssen. Die Bestimmung des IL-6 könnte somit zur Vermeidung eines zu liberalen, unkritischen Antibiotikaeinsatzes und den damit assoziierten Folgen beitragen. Das retrospektive Design und die Fallzahl sind relevante Limitationen unserer Studie. Eine Aussage zu möglichen Unterschieden zwischen grampositiven und gramnegativen Keimen kann daher nicht sicher getroffen werden. Weitere prospektive Untersuchungen mit einer höheren Patientenzahl sind daher unbedingt zu empfehlen.:Inhaltsverzeichnis 1. Einleitung…………………………………………………………………………..2 1.1 Epidemiologie der Schwerbrandverletzungen in Deutschland…...……..2 1.2 Pathophysiologie………………..……..…………………………….……….2 1.2.1 Verbrennungskrankheit………………………………….....…………….3 1.2.2 Schockphase……………....………………………………………..…….3 1.2.3 Phase der Ödemrückresorption…………………….………………..….4 1.2.4 Phase der Inflammation/ Infektion und des Hypermetabolismus…....4 1.2.5 Spätphase………………………………………………………......…..…4 1.3 Sepsis bei schwerbrandverletzten Patienten…………...…………………5 1.4 Stellenwert von Infektionsparametern und Scores in der Sepsis bei schwerbrandverletzten brandverletzten Patienten………………………..….6 1.5 Interleukin 6…………………………………..……………………………….7 1.5.1 Zelluläre Mechanismen und die Rolle von Interleukin 6 in der Verbrennungskrankheit……………..……………………………...………….7 1.5.2 Interleukin 6 in der Sepsis……………………...……………………….8 1.6 Material und Methoden………………...…………………...………………..9 1.7 Fragestellung und Zielsetzung des Forschungsvorhabens…………..…12 2. Publikation…………………………………………………………… ………….13 3. Zusammenfassung der Arbeit..…………………………..……………………..22 4. Literaturverzeichnis…………...……………….…………………………………25 5. Anlagen……………………………………………….…………………………...29 5.1 Erklärung über den wissenschaftlichen Beitrag des Promovenden zur Publikation………………………………………..……………………………….29 5.2 Erklärung über die eigenständige Abfassung der Arbeit…….……………….30 5.3 Lebenslauf…………………………….......………………………………...……31 5.4 Danksagung………………………………………......………………………….32

Rapidly diagnosing the precise drug resistance present in sepsis-inducing bacteria is a continuing need to maintain the efficacy of our medical systems. Diagnostics currently being developed for such scenarios are either sensitive or rapid, but not both. Sequence-specific single DNA molecule analysis could fill this gap if it could be adapted to work on smaller targets, similar to those produced by classical biological methods. In this work, I demonstrate that immobilized ssDNA in the appropriate hybridization buffer can rapidly pull its complementary sequence out of solution. I also demonstrate that such systems in a microfluidic chip can be used to capture bacterial plasmids as a step toward subsequent multiplexed analysis. Finally, I demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be modified with multiple fluorophores and used to label captured targets in a sequence-specific manner. This system shows that it is possible to label bacterial plasmids in a manner that can bridge the technological gap between single molecule counting and small oligonucleotide targets. Such a system can achieve lower limits of detection for clinically relevant samples while maintaining rapid processing times.

The spleen is an important site for central regulation of immune function by noradrenergic sympathetic nerves, but little is known about this major region of neuroimmune communication in humans. Experimental studies using animal models have established that sympathetic innervation of the spleen is essential for cholinergic anti-inflammatory responses evoked by vagal nerve stimulation, and clinical studies are evaluating this approach for treating inflammatory diseases. Most data on sympathetic nerves in spleen derive from rodent studies, and this work has established that remodeling of sympathetic innervation can occur during inflammation. However, little is known about the effects of sepsis on spleen innervation. Our primary goals were to (i) localize noradrenergic nerves in human spleen by immunohistochemistry for tyrosine hydroxylase (TH), a specific noradrenergic marker, (ii) determine if nerves occur in close apposition to leukocytes, and (iii) determine if splenic sympathetic innervation is altered in patients who died from end stage sepsis. Staining for vesicular acetylcholine transporter (VAChT) was done to screen for cholinergic nerves. Archived paraffin tissue blocks were used. Control samples were obtained from trauma patients or patients who died after hemorrhagic stroke. TH + nerves were associated with arteries and arterioles in all control spleens, occurring in bundles or as nerve fibers. Individual TH + nerve fibers entered the perivascular region where some appeared in close apposition to leukocytes. In marked contrast, spleens from half of the septic patients lacked TH + nerves fibers and the average abundance of TH + nerves for the septic group was only 16% of that for the control group (control: 0.272 ± 0.060% area, n = 6; sepsis: 0.043 ± 0.026% area, n = 8; P < 0.005). All spleens lacked cholinergic innervation. Our results provide definitive evidence for the distribution of noradrenergic nerves in normal human spleen and the first evidence for direct sympathetic innervation of leukocytes in human spleen. We also provide the first evidence for marked loss of noradrenergic nerves in patients who died from sepsis. Such nerve loss could impair neuroimmunomodulation and may not be limited to the spleen.

Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.

Heat shock protein A12B (HSPA12B) is predominately expressed in endothelial cells (ECs) and has been reported to protect against cardiac dysfunction from endotoxemia or myocardial infarction. This study investigated the mechanisms by which endothelial HSPA12B protects polymicrobial sepsis–induced cardiomyopathy. Wild-type (WT) and endothelial HSPA12B knockout (HSPA12B–/–) mice were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP). Cecal ligation and puncture sepsis accelerated mortality and caused severe cardiac dysfunction in HSPA12B–/– mice compared with WT septic mice. The levels of adhesion molecules and the infiltrated immune cells in the myocardium of HSPA12B–/– septic mice were markedly greater than in WT septic mice. The levels of microRNA-126 (miR-126), which targets adhesion molecules, in serum exosomes from HSPA12B–/– septic mice were significantly lower than in WT septic mice. Transfection of ECs with adenovirus expressing HSPA12B significantly increased miR-126 levels. Increased miR-126 levels in ECs prevented LPS-stimulated expression of adhesion molecules. In vivo delivery of miR-126 carried by exosomes into the myocardium of HSPA12B–/– mice significantly attenuated CLP sepsis increased levels of adhesion molecules, and improved CLP sepsis–induced cardiac dysfunction. The data suggest that HSPA12B protects against sepsis-induced severe cardiomyopathy via regulating miR-126 expression which targets adhesion molecules, thus decreasing the accumulation of immune cells in the myocardium.

Cardiac dysfunction is correlated with detrimental prognosis of sepsis and contributes to a high risk of mortality. After an initial hyperinflammatory reaction, most patients enter a protracted state of immunosuppression (late sepsis) that alters both innate and adaptive immunity. The changes of cardiac function in late sepsis are not yet known. MicroRNA-155 (miR-155) is previously found to play important roles in both regulations of immune activation and cardiac function. In this study, C57BL/6 mice were operated to develop into early and late sepsis phases, and miR-155 mimic was injected through the tail vein 48 h after cecal ligation and puncture (CLP). The effect of miR-155 on CLP-induced cardiac dysfunction was explored in late sepsis. We found that increased expression of miR-155 in the myocardium protected against cardiac dysfunction in late sepsis evidenced by attenuating sepsis-reduced cardiac output and enhancing left ventricular systolic function. We also observed that miR-155 markedly reduced the infiltration of macrophages and neutrophils into the myocardium and attenuated the inflammatory response via suppression of JNK signaling pathway. Moreover, overexpression of β-arrestin 2 (Arrb2) exacerbated the mice mortality and immunosuppression in late sepsis. Furthermore, transfection of miR-155 mimic reduced Arrb2 expression, and then restored immunocompetence and improved survival in late septic mice. We conclude that increased miR-155 expression through systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling and Arrb2 mediated immunosuppression.

Sepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. β-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.