Deciphering TCR signaling in metabolically activated T-cells in NASH and liver cancer development

Due to the consumption of high caloric food combined with an increased sedentary lifestyle, the incidence of overweight and obesity is growing rapidly in western cultures, like the USA, Europe and notably also in developing countries (e.g. India, China) - as a consequence of adaptation to the western lifestyle. Thus, obesity-related pathologies like metabolic syndrome have become a major issue in modern medicine, and thus far therapeutic options are limited. The liver - the major metabolic organ of the body - is particularly affected by constant high caloric food intake. Consequently, the liver undergoes dramatic changes, including the development of fatty liver disease, termed non-alcoholic fatty liver disease (NAFLD). In 25% of all NAFLD cases, progression to a more severe pathology termed non-alcoholic steatohepatitis (NASH) can be observed. Immune cell activation in NASH leads to liver fibrosis and subsequently to hepatocellular carcinoma (HCC). Today, more than 90 million people in the USA and 30 million people in Europe are affected by NAFLD. Although chronic viral infections with Hepatitis B or C are the leading etiology causing HCC, it has become clear that NASH is an increasingly important factor for HCC development, a notion supported by the fact that HCC is currently the fastest rising cancer in the USA, with a similar trend in Europe. At the same time, knowledge about the key mechanisms causing NASH and NASH-triggered HCC are scarce and therefore efficient therapies to treat this diseases are lacking. The group of Professor Heikenwälder generated a mouse model of NASH and NASH-driven HCC in the context of a chronic metabolic syndrome6. Mice fed a long-term choline-deficient high fat diet (CD-HFD) develop obesity, steatosis, fibrosis, NASH and NASH-triggered HCC, recapitulating most of the key features found in human patients. In this model, activated CD8+ and NKT-cells drive NASH and HCC through cytokine-mediated crosstalk with hepatocytes. Remarkably, a similar T-cell activation, cytokine and immune cell pattern was found in NASH patients - establishing the clinical relevance of the CD-HFD mouse model. Aim 1: Deciphering TCR signaling in metabolically activated T-cells in NASH and liver cancer development In this scientific context, I investigated the role of T-cells in greater detail and T-cell receptor (TCR) dependent signaling in NASH development and NASH-induced HCC. By utilizing different dietary (high fat diet (HFD), CD-HFD and western-style diet with trans-fat (WD-HTF), genetic mouse models (C57Bl6, TCRβδ-/-, OT-1, Prf1-/-, Jα18-/-, CD1d-/-) and interventional antibody (α-CD8, α-PD-1, α-NK1.1) -based strategies in dietary mouse models, I shed new light on the key mechanisms of NASH pathology and its progression towards HCC. The data of this PhD thesis indicates that from early time points onwards, activation of subsets of CD8+ T-cells correlate with NASH pathology of different severity. Further, with progression of NASH towards advanced stages, CD8+ T-cells of animals fed NASH-inducing diets (CD-HFD or WD-HTF) expressed increasing amounts of early activation marker CD69 and activation/exhaustion marker PD-1. Also, PD-1+ CD8+ T-cells expressed different TCR variable β chains (TCR vβ), indicating that distinct exhausted repertoires develop over the course of NASH. Next, I showed that NASH pathology is dependent on a functional TCRαβ repertoire with intact TCRαβ effector function, but less on natural killer T-cell (NKT) cell-dependent mechanisms. Consistent with TCRαβ dependency in NASH development, I demonstrated a differential role of CD8+ T-cells depending on the progression state of NASH pathology, with a protective function of CD8+ T-cells in early states of NASH. However, in advanced NASH CD8+ T-cells drive hepatic immune-related adverse effects (irAEs), resulting in liver damage and tumor formation potentially involving a TNF-a-mediated mechanism. Aim 2: Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer After deciphering the role of T-cells in NASH and NASH-induced hepatocarcinogenesis, I investigated alternative ways to target hepatic inflammation in NASH without targeting potentially inflammation-driving immune cell populations directly. Thus, I investigated the key mechanisms of cell-immune cell interactions driving early NASH pathogenesis and identified platelets and platelet activation as major contributors to NASH pathology and subsequent HCC development. Further, I deciphered that platelets could be targeted by antiplatelet therapy (therapeutic Ticagrelor), thereby ameliorating NASH pathology and potentially subsequent hepatocarcinogenesis. I could show that platelet interaction with Kupffer cells and CD44-hyaluronan are key mechanisms of NASH progression and that GPIbα is an interventional target for NASH therapy. I published these results as a co-first author in Nature Medicine in April 2019 (https://doi.org/10.1038/s41591-019-0379-5)

The environmental relevance of freshwater consumption in global power production

Purpose: Freshwater use and consumption is of high environmental concern. While research has primarily focused on agricultural water use, industrial water use has recently become more prominent. Because most industries employ relatively low amounts of water, our study focuses on electricity production, which is involved in almost all economic activities and has a considerable share of the global water consumption. Materials and methods: Water consumption data for different power production technologies was calculated from literature. Due to the global importance of hydropower and the high variability of its specific water consumption, a climate-dependent estimation scheme for water consumption in hydroelectric generation was derived. Applying national power production mixes, we analyzed water consumption and related environmental damage of the average power production for all countries. For the European and North American countries, we further modeled electricity trade to assess the electricity market mix and the power-consumption related environmental damages. Using the Eco-indicator 99 single-score and compatible freshwater consumption damage assessments, the contribution of water consumption to the total environmental impact was quantified. Results and discussion: Water consumption dominates the environmental damage of hydropower, but is generally negligible for fossil thermal, nuclear, and alternative power production. However, as the impact of water consumption has high regional variation, it can be relevant for many power technologies in water-scarce areas. The variability among country production mixes is substantial, both from a water consumption and overall environmental impact perspective. The difference between electricity production and market mixes is negligible for most countries, especially for big countries such as the USA. In Europe, where intensive international electricity trade exists, the difference is more significant. When contrasted with the relatively high uncertainties in water consumption figures particularly for hydropower, the additional error from using production mixes instead of market mixes is rather small. Conclusions: Power production is one of the major global water consumers and involved in life cycles of almost any human activity. Covering the water-consumption-related environmental damage of power generation closes one important gap in life cycle assessment and also improves data availability for the emerging field of water footprint

Khaos: Dynamically Optimizing Checkpointing for Dependable Distributed Stream Processing

Distributed Stream Processing systems are becoming an increasingly essential part of Big Data processing platforms as users grow ever more reliant on their ability to provide fast access to new results. As such, making timely decisions based on these results is dependent on a system's ability to tolerate failure. Typically, these systems achieve fault tolerance and the ability to recover automatically from partial failures by implementing checkpoint and rollback recovery. However, owing to the statistical probability of partial failures occurring in these distributed environments and the variability of workloads upon which jobs are expected to operate, static configurations will often not meet Quality of Service constraints with low overhead. In this paper we present Khaos, a new approach which utilizes the parallel processing capabilities of virtual cloud automation technologies for the automatic runtime optimization of fault tolerance configurations in Distributed Stream Processing jobs. Our approach employs three subsequent phases which borrows from the principles of Chaos Engineering: establish the steady-state processing conditions, conduct experiments to better understand how the system performs under failure, and use this knowledge to continuously minimize Quality of Service violations. We implemented Khaos prototypically together with Apache Flink and demonstrate its usefulness experimentally

Influence of a novel, versatile bifunctional chelator on theranostic properties of a minigastrin analogue

Background: 6-[Bis(carboxymethyl)amino]-1,4-bis(carboxymethyl)-6-methyl-1,4-diazepane (AAZTA ) is a promising chelator with potential advantages over 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radiopharmaceutical applications. Its mesocyclic structure enables fast radiolabelling under mild conditions with trivalent metals including not only 68Ga for positron emission tomography (PET) but also 177Lu and 111In for singlephoton emission computed tomography (SPECT) and radionuclide therapy. Here, we describe the evaluation of a bifunctional AAZTA derivative conjugated to a model minigastrin derivative as a potential theranostic agent. Methods: An AAZTA derivative with an aliphatic C9 chain as linker was coupled to a minigastrin, namely [AAZTA0, D-Glu1, desGlu2\u20136]-minigastrin (AAZTA-MG), and labelled with 68Ga, 177Lu and 111In. The characterisation in vitro included stability studies in different media and determination of logD (octanol/PBS). Affinity determination (IC50) and cell uptake studies were performed in A431-CCK2R cells expressing the human CCK2 receptor. \u3bcPET/CT and ex vivo biodistribution studies were performed in CCK2 tumour xenograft-bearing nude mice and normal mice. Results: AAZTA-MG showed high radiochemical yields for 68Ga (>95 %), 177Lu (>98 %) and 111In (>98 %). The logD value of 123.7 for both [68Ga]- and [177Lu]-AAZTA-MG indicates a highly hydrophilic character. Stability tests showed overall high stability in solution with some degradation in human plasma for [68Ga]- and transchelation towards DTPA for and [177Lu]-AAZTA-MG. An IC50 value of 10.0 nM was determined, which indicates a high affinity for the CCK2 receptor. Specific cell uptake after 60 min was >7.5 % for [68Ga]-AAZTA-MG and >9.5 % for [177Lu]-AAZTA-MG, comparable to other DOTA-MG-analogues. \u3bcPET/CT studies in CCK2 receptor tumour xenografted mice not only revealed high selective accumulation in A431-CCK2R positive tumours of 68Ga-labelled AAZTA-MG (1.5 % ID/g in 1 h post injection) but also higher blood levels as corresponding DOTA-analogues. The 111In-labelled peptide had a tumour uptake of 1.7 % ID/g. Biodistribution in normal mice with the [177Lu]-AAZTA-MG showed a considerable uptake in intestine (7.3 % ID/g) and liver (1.5 % ID/g). Conclusion: Overall, AAZTA showed interesting properties as bifunctional chelator for peptides providing mild radiolabelling conditions for both 68Ga and trivalent metals having advantages over the currently used chelator DOTA. Studies are ongoing to further investigate in vivo targeting properties and stability issues and the influence of spacer length on biodistribution of AAZTA

Influence of a novel, versatile bifunctional chelator on theranostic properties of a minigastrin analogue.

BACKGROUND: 6-[Bis(carboxymethyl)amino]-1,4-bis(carboxymethyl)-6-methyl-1,4-diazepane (AAZTA ) is a promising chelator with potential advantages over 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radiopharmaceutical applications. Its mesocyclic structure enables fast radiolabelling under mild conditions with trivalent metals including not only (68)Ga for positron emission tomography (PET) but also (177)Lu and (111)In for single-photon emission computed tomography (SPECT) and radionuclide therapy. Here, we describe the evaluation of a bifunctional AAZTA derivative conjugated to a model minigastrin derivative as a potential theranostic agent. METHODS: An AAZTA derivative with an aliphatic C9 chain as linker was coupled to a minigastrin, namely [AAZTA(0), D-Glu(1), desGlu(2-6)]-minigastrin (AAZTA-MG), and labelled with (68)Ga, (177)Lu and (111)In. The characterisation in vitro included stability studies in different media and determination of logD (octanol/PBS). Affinity determination (IC50) and cell uptake studies were performed in A431-CCK2R cells expressing the human CCK2 receptor. μPET/CT and ex vivo biodistribution studies were performed in CCK2 tumour xenograft-bearing nude mice and normal mice. RESULTS: AAZTA-MG showed high radiochemical yields for (68)Ga (>95 %), (177)Lu (>98 %) and (111)In (>98 %). The logD value of -3.7 for both [(68)Ga]- and [(177)Lu]-AAZTA-MG indicates a highly hydrophilic character. Stability tests showed overall high stability in solution with some degradation in human plasma for [(68)Ga]- and transchelation towards DTPA for and [(177)Lu]-AAZTA-MG. An IC50 value of 10.0 nM was determined, which indicates a high affinity for the CCK2 receptor. Specific cell uptake after 60 min was >7.5 % for [(68)Ga]-AAZTA-MG and >9.5 % for [(177)Lu]-AAZTA-MG, comparable to other DOTA-MG-analogues. μPET/CT studies in CCK2 receptor tumour xenografted mice not only revealed high selective accumulation in A431-CCK2R positive tumours of (68)Ga-labelled AAZTA-MG (1.5 % ID/g in 1 h post injection) but also higher blood levels as corresponding DOTA-analogues. The (111)In-labelled peptide had a tumour uptake of 1.7 % ID/g. Biodistribution in normal mice with the [(177)Lu]-AAZTA-MG showed a considerable uptake in intestine (7.3 % ID/g) and liver (1.5 % ID/g). CONCLUSION: Overall, AAZTA showed interesting properties as bifunctional chelator for peptides providing mild radiolabelling conditions for both (68)Ga and trivalent metals having advantages over the currently used chelator DOTA. Studies are ongoing to further investigate in vivo targeting properties and stability issues and the influence of spacer length on biodistribution of AAZTA

Valvulotomy of the great saphenous vein in ex situ non-reversed and in situ setting: a multicenter post-market study to assess the safety and efficacy of the AndraValvulotome™”

Purpose To evaluate the safety and technical success of the AndraValvulotome™ device (Andramed GmbH, Reutlingen, Germany) in patients with peripheral arterial disease (PAD) requiring bypass surgery using the great saphenous vein (GSV) as graft. Methods This was a multicenter, post-market observational study conducted in 2021 in 11 German centers. Safety and efficacy data were prospectively collected and analyzed. Primary endpoints were the absence of device-related serious adverse events until 30 ± 7 days follow-up, the clinical efficacy of valvulotomy, which was defined as pulsatile blood flow in the bypass and the number of insufficiently destroyed vein valves. Secondary endpoints were the number of valvulotomy passages, the primary patency rate of the venous bypass (determined by a color-duplex sonography showing a normal blood flow through the bypass and absence of stenosis or occlusion), and the primary technical success defined as the absence of product-specific (serious) adverse events and clinical efficacy. Results Fifty-nine patients were enrolled. The mean age of the patients was 71 years (46–91), and 74.6% were males. The vein material used for bypass grafting had a median length of 47.5 cm (range 20–70 cm) with a median diameter of 5.0 mm (range 3–6 mm) and 4.0 mm (range 2–6 mm) in the proximal and distal segments, respectively. The technical success rate was 96.6%. The primary patency rate was 89.9% at 30 days follow-up. The clinical efficacy was rated as very good in 81% of patients, fair in 17%, and poor in 2%. Between 1 and 5 (average 2.9) valvulotome passages were performed. One product-related serious adverse event was recorded (bypass vein dissection). Conclusion The AndraValvulotome™ can be considered a safe and effective device to disrupt venous valves during in situ non-reversed bypass surgeries using GSV grafts in patients with PAD

Nitrogen Oxides and Ozones from B-747 Measurements (NOXAR) during POLINAT 2 and SONEX: Overview and Case-Studies on Continental and Marine Convection

In the framework of the project POLINAT 2 (Pollution in the North Atlantic Flight Corridor) we measured NO(x) (NO and NO2) and ozone on 98 flights through the North Atlantic Flight Corridor (NAFC) with a fully automated system permanently installed aboard an in-service Swissair B-747 airliner in the period of August to November 1997. The averaged NO, concentrations both in the NAFC and at the U.S. east coast were similar to that measured in autumn 1995 with the same system. The patchy occurrence of NO(x), enhancements up to 3000 pptv over several hundred kilometers (plumes), predominately found over the U.S. east coast lead to a log-normal NO(x) probability density function. In three case-studies we examine the origins of such plumes by combining back-trajectories with brightness temperature enhanced (IR) satellite imagery, with lightning observations from the U.S. National Lightning Detection Network (NLDN) or with the Optical Transient Detector (OTD) satellite. For frontal activity above the continental U.S., we demonstrate that the location of NO(x) plumes can be well explained with maps of convective influence. For another case we show that the number of lightning flashes in a cluster of marine thunderstorms is proportional to the NO(x) concentrations observed several hundred kilometers downwind of the anvil outflows and suggest that lightning was the dominant source. From the fact that in autumn the NO, maximum was found several hundred kilometers off the U.S. east coast, it can be inferred that thunderstorms triggered over the warm Gulf Stream current are an important source for the regional upper tropospheric NO(x) budget in autumn

An Immune Gene Expression Signature Associated With Development of Human Hepatocellular Carcinoma Identifies Mice That Respond to Chemopreventive Agents

Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis.We analyzed gene expression profiles of non-tumor liver tissues from 392 patients with early-stage HCC (training set, n=167 and validation set, n=225) and liver tissue from patients with cirrhosis without HCC (n=216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of N-nitrosodiethylamine followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then given orally the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, given orally aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet.We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of non-tumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor beta signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% 1.21-4.80). Mice with chemically-induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel downregulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle.We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that associates with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and developed fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis