In recent years, numerous studies have investigated somatic mutations in mitochondrial DNA in various tumours. The observed high mutation rates might reflect mitochondrial deregulation; consequently, mutation analyses could be clinically relevant. The purpose of this study was to determine if mutations in the mitochondrial D-loop region and/or the level of mitochondrial gene expression could influence the clinical course of human ovarian carcinomas.

Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA-protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods.

Hydrogels prepared from gelatin and lysine diisocyanate ethyl ester provide tailorable elastic properties and degradation behavior. Their interaction with human aortic endothelial cells (HAEC) as well as human macrophages (Mɸ) and granulocytes (Gɸ) were explored. The experiments revealed a good biocompatibility, appropriate cell adhesion, and cell infiltration. Direct contact to hydrogels, but not contact to hydrolytic or enzymatic hydrogel degradation products, resulted in enhanced cyclooxygenase-2 (COX-2) expression in all cell types, indicating a weak inflammatory activation in vitro. Only Mɸ altered their cytokine secretion profile after direct hydrogel contact, indicating a comparably pronounced inflammatory activation. On the other hand, in HAEC the expression of tight junction proteins, as well as cytokine and matrix metalloproteinase secretion were not influenced by the hydrogels, suggesting a maintained endothelial cell function. This was in line with the finding that in HAEC increased thrombomodulin synthesis but no thrombomodulin membrane shedding occurred. First in vivo data obtained after subcutaneous implantation of the materials in immunocompetent mice revealed good integration of implants in the surrounding tissue, no progredient fibrous capsule formation, and no inflammatory tissue reaction in vivo. Overall, the study demonstrates the potential of gelatin-based hydrogels for temporal replacement and functional regeneration of damaged soft tissue.

Hydrogels based on gelatin have evolved as promising multifunctional biomaterials. Gelatin is crosslinked with lysine diisocyanate ethyl ester (LDI) and the molar ratio of gelatin and LDI in the starting material mixture determines elastic properties of the resulting hydrogel. In order to investigate the clinical potential of these biopolymers, hydrogels with different ratios of gelatin and diisocyanate (3-fold (G10_LNCO3) and 8-fold (G10_LNCO8) molar excess of isocyanate groups) were subcutaneously implanted in mice (uni- or bilateral implantation). Degradation and biomaterial-tissue-interaction were investigated in vivo (MRI, optical imaging, PET) and ex vivo (autoradiography, histology, serum analysis). Multimodal imaging revealed that the number of covalent net points correlates well with degradation time, which allows for targeted modification of hydrogels based on properties of the tissue to be replaced. Importantly, the degradation time was also dependent on the number of implants per animal. Despite local mechanisms of tissue remodeling no adverse tissue responses could be observed neither locally nor systemically. Finally, this preclinical investigation in immunocompetent mice clearly demonstrated a complete restoration of the original healthy tissue.

Stent thrombosis (ST) is a rare but serious complication following percutaneous coronary intervention. Analysis of thrombus composition from patients undergoing catheter thrombectomy may provide important insights into the pathological processes leading to thrombus formation. We performed a large-scale multicentre study to evaluate thrombus specimens in patients with ST across Europe.

Medical treatment of arterial thrombosis is mainly directed against platelets and coagulation factors, and can lead to bleeding complications. Novel antithrombotic therapies targeting immune cells and neutrophil extracellular traps (NETs) are currently being investigated in animals. We addressed whether immune cell composition of arterial thrombi induced in mouse models of thrombosis resemble those of human patients with acute myocardial infarction (AMI).

The administration of iodinated contrast media (CM) can cause microcirculatory disorder leading to acute renal dysfunction. In a prospective, randomized investigation two CM (Iodixanol vs Iopromide) were compared in 16 pigs. Each animal received 10 intra-aortal injections (5 ml Iodixanol or 4.32 ml Iopromide). Microcirculation was assessed using contrast-enhanced ultrasound (CEUS) directly on the kidney surface using time-to-peak (TTP) and blood-volume-analysis. Macroscopic observations were documented. Post mortem residual CM distribution in the kidneys was detected using X-ray. TTP was significantly prolonged over the descending vasa recta of the Iopromide group. This coincided with a visible marble-like pattern on the kidney surface occurring in 30 out of 80 Iopromide-injections but in 4 out of 80 Iodixanol-injections (p = 0.007). The blood volume over the entire kidney did not change after Iodixanol-application, but decreased by about 6.1% after Iopromide-application. The regional blood volume in the renal cortex showed a tendency to decrease by about 13.5% (p = 0.094) after Iodixanol-application, and clearly decreased by about 31.7% (p = 0.022) after Iopromide-application. The study revealed a consistent influence of repeated injections of two different CM on the kidney perfusion using three different imaging methods (CEUS analysis, macroscopic observation and X-ray analysis).

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.

Traditionally the female sex, compared with the male sex, has been perceived as having greater variability in many physiological traits, including within the immune system. We investigated effects of biological sex and the female reproductive cycle on numbers of circulating leukocytes in C57BL/6J mice. We show that biological sex, but not female reproductive cyclicity, has a significant effect on peripheral blood immune cell prevalence and variability, and that sex differences were not consistent amongst common inbred laboratory mouse strains. We found that male C57BL/6J mice, compared with female mice, have greater variability in peripheral blood immunophenotype, and that this was influenced by body weight. We created summary tables for researchers to facilitate experiment planning and sample size calculations for peripheral immune cells that consider the effects of biological sex.

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.

Lung cancer causes impairment of health-related quality of life (QoL), but little is known about gender aspects in QoL and symptom burden of lung cancer patients. The aim of this study was to investigate gender differences in QoL as assessed by the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 and the updated lung cancer module.

Inflammation contributes to obesity-related hyperinsulinemia and insulin resistance, which often precede type 2 diabetes. Inflammation is one way that obesity can promote insulin resistance. It is not clear if the extent of obesity, hyperinsulinemia, or hyperglycemia, underpins changes in cellular immunity during diet-induced obesity. In particular, the requirement for obesity or directionality in the relationship between insulin resistance and monocyte characteristics is poorly defined. Inflammatory cytokines such as tumor necrosis factor (TNF) can contribute to insulin resistance. It is unclear if TNF alters monocytosis or specific markers of cellular immunity in the context of obesity. We measured bone marrow and blood monocyte characteristics in WT and TNF-/- mice that were fed obesogenic, high fat (HF) diets. We also used hyperglycemic Akita mice and mice implanted with insulin pellets in order to determine if glucose or insulin were sufficient to alter monocyte characteristics. We found that diet-induced obesity in male mice increased the total number of monocytes in blood, but not in bone marrow. Immature, inflammatory (Ly6Chigh ) monocytes decreased within the bone marrow and increased within peripheral blood of HF-fed mice. We found that neither hyperinsulinemia nor hyperglycemia was sufficient to induce the observed changes in circulating monocytes in the absence of diet-induced obesity. In obese HF-fed mice, antibiotic treatment lowered insulin and insulin resistance, but did not alter circulating monocyte characteristics. Fewer Ly6Chigh monocytes were present within the blood of HF-fed TNF-/- mice in comparison to HF-fed wild-type (WT) mice. The prevalence of immature Ly6Chigh monocytes in the blood correlated with serum insulin and insulin resistance irrespective of the magnitude of adipocyte or adipose tissue hypertrophy in obese mice. These data suggest that diet-induced obesity instigates a TNF-dependent increase in circulating inflammatory monocytes, which predicts increased blood insulin and insulin resistance independently from markers of adiposity or adipose tissue expansion.

Background: Most non-small cell lung cancers occur in elderly and frequently comorbid patients. Therefore, it is necessary to evaluate the efficacy of biomodulatory active therapy regimen, concertedly interfering with tumor-associated homeostatic pathways to achieve tumor control paralleled by modest toxicity profiles. Patients and Methods: The ModuLung trial is a national, multicentre, prospective, open-label, randomized phase II trial in patients with histologically confirmed stage IIIB/IV squamous (n = 11) and non-squamous non-small cell (n = 26) lung cancer who failed first-line platinum-based chemotherapy. Patients were randomly assigned on a 1:1 ratio to the biomodulatory or control group, treated with nivolumab. Patients randomized to the biomodulatory group received an all-oral therapy consisting of treosulfan 250 mg twice daily, pioglitazone 45 mg once daily, clarithromycin 250 mg twice daily, until disease progression or unacceptable toxicity. Results: The study had to be closed pre-maturely due to approval of immune checkpoint inhibitors (ICi) in first-line treatment. Thirty-seven patients, available for analysis, were treated in second to forth-line. Progression-free survival (PFS) was significantly inferior for biomodulation (N = 20) vs. nivolumab (N = 17) with a median PFS (95% confidence interval) of 1.4 (1.2-2.0) months vs. 1.6 (1.4-6.2), respectively; with a hazard ratio (95% confidence interval) of 1.908 [0.962; 3.788]; p = 0.0483. Objective response rate was 11.8% with nivolumab vs. 5% with biomodulation, median follow-up 8.25 months. The frequency of grade 3-5 treatment related adverse events was 29% with nivolumab and 10% with biomodulation. Overall survival (OS), the secondary endpoint, was comparable in both treatment arms; biomodulation with a median OS (95% confidence interval) of 9.4 (6.0-33.0) months vs. nivolumab 6.9 (4.6-24.0), respectively; hazard ratio (95% confidence interval) of 0.733 [0.334; 1.610]; p = 0.4368. Seventy-five percent of patients in the biomodulation arm received rescue therapy with checkpoint inhibitors. Conclusions: This trial shows that the biomodulatory therapy was inferior to nivolumab on PFS. However, the fact that OS was similar between groups gives rise to the hypothesis that the well-tolerable biomodulatory therapy may prime tumor tissues for efficacious checkpoint inhibitor therapy, even in very advanced treatment lines where poor response to ICi might be expected with increasing line of therapy.

Gadoxetic acid uptake on hepatobiliary phase MRI has been shown to correlate with ß-catenin mutation in patients with HCC, which is associated with resistance to certain therapies. This study aimed to evaluate the prognostic value of gadoxetic acid uptake on hepatobiliary phase MRI in patients with advanced HCC receiving sorafenib.

Overall survival is the ultimate criterion for the therapy of lung cancer, but psychosocial care, which helps the patient to cope with the disease, becomes a more and more important issue in the treatment of this life-threatening disease.

The adaption of gut microbiota (GM) throughout human life is a key factor in maintaining health. Interventions to restore a healthy GM composition may have the potential to improve health and disease outcomes in the elderly. We performed a comprehensive characterization of changes in the luminal and mucosa-associated microbiota composition in elderly compared with younger healthy individuals. Samples from saliva and feces, and biopsies from the upper and lower gastrointestinal tract (UGIT, LGIT), were collected from 59 asymptomatic individuals grouped by age: 40-55, 56-70, and 71-85 years). All underwent anthropometric, geriatric, and nutritional assessment. RNA was extracted and reverse-transcribed into complementary DNA; the V1-V2 regions of 16S ribosomal RNA genes were amplified and sequenced. Abundances of the taxa in all taxonomic ranks in each sample type were used to construct sample-similarity matrices by the Bray-Curtis algorithm. Significant differences between defined groups were assessed by analysis of similarity. The bacterial community showed strong interindividual variations and a clear distinction between samples from UGIT, LGIT, and feces. While in saliva some taxa were affected by aging, this number was considerably greater in UGIT and was subsequently higher in LGIT. Unexpectedly, aging scarcely influenced the bacterial community of feces over the age range of 40-85 years. The development of interventions to preserve and restore human health with increased age by establishing a healthy gut microbiome should not rely solely on data from fecal analysis, as the intestinal mucosa is affected by more significant changes, which differ from those observed in fecal analyses.

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.

To examine to what extent the high frame rate contrast-enhanced ultrasound (HiFR) diagnostic enables the conclusive diagnosis of liver changes with suspected malignancy.

Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production.