Performance assessment and clinical validation of OR ventilation systems

Microbiological burden of room-air in operating theatres is a known risk factor for surgical site infections. However, it is unclear how to best evaluate efficacy and efficiency under routine clinical conditions. Moreover, there still is a lack of data to assess the impact on infection rates. To date there still is substantial discussion in the scientific community which ventilation system provides the most effective and economical respectively efficient control of microbial risk factors during surgery. This is especially important as most standards do not require a performance assessment of the operating room ventilation, but rather rely on tests “at rest” in empty rooms. This might be an explanation for the conflicting results regarding infection preventive effects of different OR ventilation systems as well as the ambiguous data for infection rates. Since the release of the latest version of DIN 1946-4 in 2018 in Germany [DIN, 2018] a positioning analysis (worst-case scenario with the largest space requirement) for determining the required protected area in class 1a (highest standard) operating rooms is also mandatory. Therefore, another key point of this investigation was to use typical workflow scenarios to assess existing installations regarding the match of the required and the built size of the protected area. Positioning analyses were done together with the onsite staff for various clinical procedures in different hospitals. In all cases, the positioning analysis revealed that required protected areas need to be significantly larger than provided by the existing setup. The size of the protected area that is actually required can only be determined by individual positioning analysis. Most existing installations of unidirectional flow systems (UDF) are likely to be too small. The larger protected areas actually require significantly larger rooms in order to maintain proper thermodynamics. Furthermore, significantly higher volumetric flow rates are required. Finally, the current mismatch between actual and necessary protected area would be a possible explanation for the controversial data situation regarding the infection preventive effects of UDF systems. Thus, we aimed to evaluate how different widely used qualification techniques as well as several operational parameters impact OR ventilation performance assessment. We specifically studied the desired effect of reducing microbiological air burden and infection rates under routine clinical conditions. Therefore, we evaluated the performance of a temperature controlled ventilation system (TcAF) during surgery and its impact on surgical site infections. This was done under routine clinical conditions in 10 clinical installations of the TcAF system Opragon (Avidicare AB, Sweden) during live surgeries according to the Swedish SIS TS 39: 2015 standard. Furthermore, a retrospective analysis of 1,000 consecutive cases of primary total joint arthroplasty (hip, knee) before and 1000 after installation of the TcAF system was performed. Endpoints for clinical outcome were length of stay and infection rates. Our results show that performance testing is essential for a proper assessment of OR ventilation systems. Moreover, we demonstrated that TcAF systems are able to reliably and robustly ensure "ultra-clean" air (<10 CFU/m3) in the entire operating room demonstrating its capability to reduce the risk of airborne microbial transmission during surgery. The retrospective analysis of clinical patient data shows positive impact of TcAF on key clinical outcome parameters in line with previous research by Charnley and Lidwell.publishedVersio

Neue Mechanismen der angeborenen Immunantwort

Over decades, one of the central dogmas of immunity has been that immune cells can be classified as parts of either the adaptive or innate immune system. In recent years, however, the lines between these two systems have become increasingly blurred. For example, subsets of innate natural killer (NK) cells have been demonstrated to display memory phenotypes, while recently identified mucosal-associated invariant CD8 T cells (MAIT cells) realize innate cytotoxicity. MAIT and NK cells both constitute predominant populations in human blood and other organs and share a high expression of CD161. NK cells detect virus-infected and abnormally transformed cells, and MAIT cells are able to identify and directly lyse bacterial-infected cells. Although NK cell activation is well defined, the underlying signaling networks are still obscure. In the first part of this thesis, the role of CaMKII and PKD kinases in NK cell activity was investigated. It could be shown that chemical inhibition leads to a significant decrease of NK cell cytotoxicity and cytokine release, underscoring the importance of CaMKII and PKDs in NK cell activation. Notably, these experiments also indicate significant donor-variations, inhibitory pathways and further involved cell types, which concertedly bias drug-modulated NK responses. In the second part, new mechanisms of innate immunity were investigated for the first time in primary human MAIT cells. Currently, the exact role of MAIT cells in health and disease remains widely undefined, and proteomic data about this innate-like T cell subset is missing. Primary human MAIT were proteomically compared with innate NK cells and conventional CD8 T cells by mass spectrometry. In total, 5500 proteins could be identified from five representative human donors. These data reveal a functional inventory of MAIT cells that integrates phenotypes from both NK and CD8 cells. Donor-dependent analyses then defined a MAIT-specific profile of effector molecules, and regulators of exocytosis and proliferation, respectively. Furthermore, this thesis provides candidates that can now be considered as novel MAIT markers and/or likely contribute to their anti-bacterial unique phenotype. In this context, the first observation of the immunological synapse (IS) of a MAIT cell interacting with a bacterial infected target cell is presented and recruitment of candidate protein S100A4 to the site of anti-microbial activity is confirmed.Eins der zentralen Dogmen der Immunologie ist die Zuordnung der Bestandteile des Immunsystems zur angeborenen oder erworbenen Immunantwort. Aktuelle Forschungsergebnisse haben in den letzten Jahren zu einem Aufweichen dieses Grundsatzes geführt. So konnten bei bestimmten Natürlichen Killerzellen (NK-Zellen) eine Funktionalität ähnlich von Gedächtniszellen nachgewiesen werden. Die erst kürzlich entdeckten Mukosal-Assoziierten Invarianten T-Zellen (MAIT) hingegen zeigen im Gegensatz zu konventionellen T-Zellen einen Phänotyp, der typisch für Zellen der angeborenen Immunantwort ist. Beide dieser Zelltypen sind in größeren Mengen im human Blut präsent, und exprimieren größere Mengen von CD161. Während NK-Zellen virusinfizierte Zellen und Krebszellen abtöten, bekämpfen MAIT-Zellen bakterielle Infektionen. Obwohl ausführlich beschrieben ist, auf welche Arten die Aktivierung von NK-Zellen erfolgen kann, sind die darunter liegenden Signalnetzwerke unerforscht. Im ersten Teil dieser Arbeit wurde daher die Rolle verschiedener Kinasen während der NK-Zell-Aktivierung untersucht. Es konnte gezeigt werden, dass sowohl die Inhibition von CaMKII, als auch die von PKD-Kinasen die Reaktion von NK-Zellen auf aktivierende Stimuli vermindert. Dies deutet auf eine wichtige Rolle dieser Kinasen bei der Kontrolle von NK-Effektorfunktionen hin. Im zweiten Teil dieser Arbeit wurden neue Mechanismen der angeborenen Immunantwort erstmals in MAIT-Zellen untersucht. Momentan ist die genaue Rolle von MAIT-Zellen im Kontext von Krankheiten weitestgehend unbekannt, und eine proteomische Charakterisierung wurde bisher nicht durchgeführt. Daher wurde das Proteom von primären humanen menschlichen MAIT-Zellen quantitativ und spenderabhängig mit dem von NK-Zellen und konventionellen CD8 T-Zellen verglichen. Insgesamt wurden dabei 5500 Proteine aus fünf humanen Spendern nachgewiesen. In den dabei generierten Daten konnte ein MAIT-Zell-spezifisches Arsenal aus Effektorproteinen identifiziert werden, sowie ein individuelles Muster aus Regulatoren für Exozytose und Proliferation. Des Weiteren wurden Kandidaten nachgewiesen, die als Marker für MAIT-Zellen fungieren können und/oder zu ihrem anti-bakteriellen Phänotyp beitragen. In diesem Kontext konnte ebenfalls die immunologische Synapse von MAIT-Zellen erstmalig gezeigt werden

Effects of handling on animals welfare during transport and marketing

Animals can be transported either by trekking or by vehicle, during which they are subjected to different types and levels of stressor. Some key factors affecting animal welfare during handling and transport are mixing of unfamiliar animals, handling procedures, driving methods, stocking density, journey length, vehicle design, animal standing orientation, loading and unloading facilities and transport by walking. Much important research has been done on animal transport and welfare, but many questions remained to be addressed, particularly regarding the effects of transport time and length, vibration, climate conditions and handling during transport and marketing. This thesis investigated the effects of handling on animal welfare during transport and marketing. The main methods employed were comprehensive field measurements to collect data, observations, video filming for behavioural studies, interviews with animal handlers and heart rate modelling. The results showed that during loading for transport, animal heart rate rose exponentially from its mean resting value to a peak value and declined during a recovery period. Driving speed, road conditions and the standing orientation of animals had an effect on levels of vibration. The three most common resonance frequencies identified were 1.3, 5.1 and 12.6 Hz, with a second peak at 23 Hz in the vertical direction on a tarmac road at a driving speed of 85 km/h. In pig and cattle transport, blood cortisol level was elevated during short transport time. Concentrations of lactate and Creatine kinase and animal behaviour were positively correlated with transport time. During transport of animals by trekking from farm to feeder market and on to regional market in Ethiopia, the number of animals that died, were injured and were stolen was 7.6%, 6.9% and 2.8%, respectively. The overall conclusion from the thesis, based on transport conditions, vibration levels, animal behaviour, stress hormones and pH24 values, was that handling and transport had a negative effect on animal welfare