Long-term outcomes of open and endovascular treatment of recurrent carotid artery stenosis - a 16-year retrospective single centre case series.

Background: The aim of this study is to evaluate perioperative as well as long-term outcomes in patients operated with carotid endarterectomy (CEA) or stenting (CAS) due to symptomatic or asymptomatic high-grade restenosis of the internal carotid artery (ICA). Patients and methods: In a retrospective analysis of our electronic database including 2980 patients who underwent carotid endarterectomy or stenting due to a symptomatic or asymptomatic high-grade stenosis of the ICA, between 2000 and 2016, we enrolled 111 patients with recurrent ICA stenosis. Results: An ipsilateral 2nd time restenosis (> 80 % in the asymptomatic and > 50 % in the symptomatic patients according to NASCET criteria) of ICA was detected in 13 patients (12 %); 3 of them were symptomatic. These patients were managed with either CEA (n = 5/38 %) or CAS (n = 8/62 %) with no perioperative stroke or death. The stroke-free survival rates at 2 and 8 years for CEA were 98 % and 98 % versus 100 % and 100 % for CAS respectively (P = .271). The type of the initial procedure (patch, CAS or interposition) did not play any significant role for the development of a 2nd time restenosis (P = .841). Conclusions: Redo-CEA/CAS seem to have similar results as primary procedures (as reported in the literature) with favorable periprocedural and long-term outcomes.

Reduction of DNA damage in peripheral lymphocytes of obese patients after bariatric surgery-mediated weight loss.

Obesity is associated with several detrimental health consequences, among them an increased risk for development of cancer, and an overall elevated mortality. Multiple factors like hyperinsulinemia, chronic microinflammation and oxidative stress may be involved. The comet assay has been proven to be very sensitive for detection of DNA damage and has been used to explore the relationship between overweight/obesity and DNA damage, but results are controversial. Very few investigations have been performed to correlate weight loss of obese individuals and possible reduction of DNA damage and these studies have not provided clear results. As currently, only surgical interventions (metabolic/bariatric surgery) enable substantial and sustained weight loss in the vast majority of morbidly obese patients, we analyzed whole blood samples of 56 subsequent patients prior, 6 and 12 months after bariatric surgery. No reduction of DNA damage was observed in comet assay analysis after 6 months despite efficient weight loss, but a significant reduction was observed 12 months after surgery. Concurrently, the ferric-reducing antioxidant power assay showed a significant reduction after 6 and 12 months. The level of oxidised glutathione and lipid peroxidation products were increased at 6 months but normalised at 12 months after surgery. As conclusion, a significant weight reduction in obese patients may help to diminish existing DNA damage besides improving many other health aspects in these patients.

Impairments of postural control, functional performance and strength in morbidly obese patients awaiting bariatric surgery in comparison to healthy individuals.

[Purpose] There is a lack of information evaluating specific markers of performance in patients awaiting bariatric surgery. We aimed to assess the postural control, functional performance, strength and endurance performance for morbidly obese patients awaiting bariatric surgery compared to lean controls. [Subjects and Methods] All parameters were assessed by modified Y-balance test, timed-up-and-go-test, maximum strength testing on resistance exercise equipment and cardio-pulmonary exercise testing on a cycle ergometer in 10 morbidly obese patients awaiting bariatric surgery and 10 age- and sex-matched lean controls. [Results] It was found that significant differences existed for overall modified Y-balance test in morbidly obese patients awaiting bariatric surgery versus lean controls (0.37 ± 0.03 vs. 0.47 ± 0.02 cm.cm-1), timed-up-and-go-test (9.33 ± 1.23 vs. 7.85 ± 1.73 sec) and several variables of cardio-pulmonary exercise testing. Overall absolute strength expressed in kilogram was similar, yet when relativized to body weight strength differences were notable (0.4 ± 0.17 vs. 0.83 ± 0.32 kg.kg-1). [Conclusion] The results of this study demonstrate the need for comprehensive functional assessment prior to surgery with an identified demand for subsequent tailored physical training prescription that should begin before surgery.

Vessel wall morphology is equivalent for different artery types and localizations of advanced human aneurysms.

Aneurysm formation occurs most frequently as abdominal aortic aneurysm (AAA), but is also seen in other localizations like thoracic or peripheral aneurysm. While initial mechanisms for aneurysm induction remain elusive, observations from AAA samples show transmural inflammation with proteolytic imbalance and repair mechanisms triggered by the innate immune system. However, limited knowledge exists about aneurysm pathology, especially for others than AAA. We compared 42 AAA, 15 popliteal, 3 ascending aortic, five iliac, two femoral, two brachial, one visceral and two secondary aneurysms to non-aneurysmatic controls by histologic analysis, immunohistochemistry and cytokine expression. Muscular and elastic type arteries show a uniform way of aneurysm formation. All samples show similar morphology. The changes compared to controls are distinct and include matrix remodeling with smooth muscle cell phenotype switch and angiogenesis, adventitial lymphoid cell accumulation and M1 macrophage homing together with neutrophil inflammation. Inflammatory cytokines are up-regulated accordingly. Comparative analysis of different disease entities can identify characteristic pathomechanisms. The phenotype of human advanced aneurysm disease is observed for elastic and muscular type arteries, does not differ between disease localizations and might, thus, be a unique response of the vasculature to the still unknown trigger of aneurysm formation.

Boost and loss of immune responses against tumor-associated antigens in the course of pregnancy as a model for allogeneic immunotherapy.

Donor-derived immunity against tumor-associated antigens (TAAs) may exert selective antileukemic activity reprieving the allogeneic recipient from graft-versus-host disease. As TAAs are highly expressed in placental tissues we hypothesized that pregnancy could drive respective immunity in healthy individuals. Thus, we investigated the frequency and level of immune responses against clinically relevant TAAs in 114 blood donors and 44 women during their first pregnancy. Quantitative reverse-transcription polymerase chain reaction was employed to detect low levels of interferon-γ after primary peptide stimulation of CD8(+) T lymphocytes. In blood donors, primary immune responses of low and/or high avidity were found against WT1 (15%), MUC1 (14%), PRAME (7%), and HER2/neu (5%) and exerted killing functions against leukemic cells. Men had higher responses than women, likely due to gonadal cancer-testis-antigen expression. Interestingly, a history of prior delivery was not associated with increased responses, whereas the strongest responses during pregnancy were found in early trimesters to disappear after delivery. This boost and loss of TAA-specific immunity suggests that virtually every donor harbors the potential to mount antileukemic immune responses in a recipient. However, in the absence of the driving target and a permissive environment, they are short-lived and thus require supplemental strategies such as vaccination or immunomodulation to facilitate their persistence.

Heterogeneous histomorphology, yet homogeneous vascular smooth muscle cell dedifferentiation, characterize human aneurysm disease.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a frequent, potentially life-threatening, disease that can only be treated by surgical means such as open surgery or endovascular repair. No alternative treatment is currently available, and despite expanding knowledge about the pathomechanism, clinical trials on medical aneurysm abrogation have led to inconclusive results. The heterogeneity of human AAA based on histologic examination is thereby generally neglected. In this study we aimed to further elucidate the role of these differences in aneurysm disease. METHODS: Tissue samples from AAA and popliteal artery aneurysm patients were examined by histomorphologic analysis, immunohistochemistry, Western blot, and polymerase chain reaction. The results were correlated with clinical data such as aneurysm diameter and laboratory results. RESULTS: The morphology of human AAA vessel wall probes varies tremendously based on the grade of inflammation. This correlates with increasing intima/media thickness and upregulation of the vascular endothelial growth factor cascade but not with any clinical parameter or the aneurysm diameter. The phenotypic switch of vascular smooth muscle cells occurred regardless of the inflammatory state and expressional changes of the transcription factors Kruppel-like factor-4 and transforming growth factor-ß lead to differential protein localization in aneurysmal compared with control arteries. These changes were in similar manner also observed in samples from popliteal artery aneurysms, which, however, showed a more homogenous phenotype. CONCLUSIONS: Heterogeneity of AAA vessel walls based on inflammatory morphology does not correlate with AAA diameter yet harbors specific implications for basic research and possible aneurysm detection.

Extra- and Intraluminal Elastase Induce Morphologically Distinct Abdominal Aortic Aneurysms in Mice and Thus Represent Specific Subtypes of Human Disease.

Topical application of elastase to induce arterial aneurysm formation is an emerging murine model of vascular disease. In the context of aortic abdominal aneurysm (AAA), angiotensin II infusion and porcine pancreatic elastase perfusion models are commonly used today. This study, therefore, compares matrix remodeling, inflammation, and angiogenesis as distinct features of aneurysms in two models treated with intra-/extraluminal elastase. C57BL/6 mice underwent intra-/extraluminal elastase application via laparotomy and were followed up for 4 weeks. Basic histology and immunohistochemistry were performed at different time points along with transmission electron microscopy, PCR analysis, TUNEL assays, and blood analysis. Both models did not differ in aneurysm growth rate, but they showed distinct features and results depending on the way of elastase application. Extraluminal aneurysm induction preserved endothelial cell function and elastic fibers but showed ongoing acute inflammation, mainly in the adventitia. The destruction of elastic layers followed by chronic inflammation was a characteristic of intraluminal elastase perfusion, as well as medial angiogenesis, a key feature in human AAA. Different animal models harbor different features of human AAA and must, therefore, be chosen wisely. External elastase application mimics an acute inflammatory aneurysm, whereas intraluminal elastase perfusion shows chronic inflammation with angiogenesis and endothelial destruction, thus better mimicking human disease.

Antiproliferative and antimetabolic effects behind the anticancer property of fermented wheat germ extract.

BACKGROUND: Fermented wheat germ extract (FWGE) sold under the trade name Avemar exhibits anticancer activity in vitro and in vivo. Its mechanisms of action are divided into antiproliferative and antimetabolic effects. Its influcence on cancer cell metabolism needs further investigation. One objective of this study, therefore, was to further elucidate the antimetabolic action of FWGE. The anticancer compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ) is the major bioactive compound in FWGE and is probably responsible for its anticancer activity. The second objective of this study was to compare the antiproliferative properties in vitro of FWGE and the DMBQ compound. METHODS: The IC50 values of FWGE were determined for nine human cancer cell lines after 24 h of culture. The DMBQ compound was used at a concentration of 24 µmol/l, which is equal to the molar concentration of DMBQ in FWGE. Cell viability, cell cycle, cellular redox state, glucose consumption, lactic acid production, cellular ATP levels, and the NADH/NAD(+) ratio were measured. RESULTS: The mean IC50 value of FWGE for the nine human cancer cell lines tested was 10 mg/ml. Both FWGE (10 mg/ml) and the DMBQ compound (24 µmol/l) induced massive cell damage within 24 h after starting treatment, with changes in the cellular redox state secondary to formation of intracellular reactive oxygen species. Unlike the DMBQ compound, which was only cytotoxic, FWGE exhibited cytostatic and growth delay effects in addition to cytotoxicity. Both cytostatic and growth delay effects were linked to impaired glucose utilization which influenced the cell cycle, cellular ATP levels, and the NADH/NAD(+) ratio. The growth delay effect in response to FWGE treatment led to induction of autophagy. CONCLUSIONS: FWGE and the DMBQ compound both induced oxidative stress-promoted cytotoxicity. In addition, FWGE exhibited cytostatic and growth delay effects associated with impaired glucose utilization which led to autophagy, a possible previously unknown mechanism behind the influence of FWGE on cancer cell metabolism.

Aneurysm miRNA Signature Differs, Depending on Disease Localization and Morphology.

Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways.

TKTL1 expression in human malign and benign cell lines.

BACKGROUND: Overexpression of transketolase-like 1 protein TKTL1 in cancer cells has been reported to correlate with enhanced glycolysis and lactic acid production. Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed in vitro for TKTL1 expression. METHODS: 17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the "Warburg effect". RESULTS: Considerable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively. CONCLUSION: Using RT-qPCR and three different antibodies we observed only exceptional occurrence of TKTL1 in a panel of malignant human cell lines in vitro. The presence of TKTL1 was unrelated to either the rate of glucose consumption/lactic acid production or resistance against chemo- and radiotherapy.

The impact of pyrvinium pamoate on colon cancer cell viability.

PURPOSE: The in vitro and in vivo effects of pyrvinium pamoate (PP), a newly identified WNT signaling inhibitor, were evaluated against colon cancer cell lines and primary colon cancer samples. EXPERIMENTAL DESIGN: Antiproliferative activity of PP and its effects on protein and RNA levels of WNT targets were evaluated on adenomatous polyposis coli (APC (mut)) and ß-catenin(mut) cell lines, one WNT(wt) colon cancer cell line, as well as six primary colon cancer samples with mutant APC in vitro. In addition, the effect of PP on the growth of liver metastasis was examined. RESULTS: PP blocked colon cancer cell growth in vitro in a dose-dependent manner with great differences in the inhibitory concentration (IC(50)), ranging from 0.6 × 10(-6) to 65 × 10(-6) mol/L for colon cancer cells with mutations in WNT signaling. In addition, PP demonstrated a cytotoxic effect on primary colon cancer samples. A combined cytotoxic effect of PP with 5-fluorouracil (5-FU) was observed for two cell lines. PP decreased messenger RNA (mRNA) and protein levels of known WNT target genes as c-MYC and thereby led to the induction of p21. PP inhibited the migration of HCT116 colon cancer cells in vitro and decreased tumor growth in vivo after intraportal injection of HCT116 cells in nude mice. CONCLUSIONS: PP displays promising anticancer activity against a broad panel of human colon cancer cell lines, as well as primary colon cancer samples. However, our findings do not demonstrate a predominant cytotoxic effect of PP on colon cancer cells with mutations in WNT signaling.

Blocking the angiopoietin-2-dependent integrin ß-1 signaling axis abrogates small cell lung cancer invasion and metastasis.

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin ß-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin ß-1 or blocking Integrin ß-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin ß-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin ß-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.

Duodenal-jejunal bypass improves glycemia and decreases SGLT1-mediated glucose absorption in rats with streptozotocin-induced type 2 diabetes.

OBJECTIVE: To elucidate whether duodenal-jejunal-bypass (DJB), which improves blood glucose control, changes activity of Na-D-glucose cotransporter SGLT1 in small intestine. BACKGROUND: DJB has been shown to improve oral glucose tolerance in normal rats and a genetic diabetic rat model. Because intestinal D-glucose absorption is mediated by SGLT1 localized in the brush border membrane of small intestinal enterocytes, it is unclear whether function of SGLT1 is altered by DJB and whether this contributes to the improvement of glycemic control. METHODS: A high-fat diet and low-dose streptozotocin administration were used to induce a type 2 diabetes in male Lewis rats. The diabetic animals underwent DJB or sham surgery. An oral glucose tolerance test (OGTT) was used to evaluate glucose control 3 weeks after surgery. SGLT1-mediated glucose transport was assessed using everted rings of different small intestinal segments. SGLT1 mRNA expression was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: DJB improved the OGTT significantly (P < 0.001) compared with sham-operated rats while body weight was not different among the surgical groups. DJB induced a 50% reduction of SGLT1-mediated glucose uptake into enterocytes of duodenum and jejunum (P < 0.001). The concentration of D-glucose in the blood following glucose gavage increased more slowly after DJB versus sham. CONCLUSIONS: The data indicate that DJB surgery decreases glucose absorption in the small intestine by downregulation of SGLT1-mediated glucose uptake. We suggest that the downregulation of SGLT1 contributes to the body-weight independent improvement of diabetes type 2.

Noninvasive visualization of tumor growth in a human colorectal liver metastases xenograft model using bioluminescence in vivo imaging.

BACKGROUND: Bioluminescence imaging (BLI) is an ideal tool for noninvasive, quantitative monitoring of tumor progression/regression in animal models. The effectiveness of different treatment strategies is displayed by an altered intensity of bioluminescence, demonstrating a change of the tumor burden. The aim of this study was to establish a reliable, reproducible colorectal hepatic metastases cancer animal model. METHODS: Cells of the human colon carcinoma cell line HCT-116 Luc(pos) expressing the firefly luciferase enzyme gene were used. HCT-116 Luc(pos) cells (2.5 × 10(6)) were injected through the portal vein into the liver of immunoincompetent nude mice. BLI was used to analyze intrahepatic tumor burden and growth kinetic. RESULTS: HCT-116 Luc(pos) cells demonstrated a progressive and reproducible growth in the liver after intraportal injection. Four days after injection, the animals were analyzed for tumor growth by BLI, and mice without or too low bioluminescence signals were excluded (between 10% and 20% animals). HCT-116 Luc(pos) intrahepatic tumors responded successfully to different dosages (5 and 10 mg/kg) of 5-fluorouracil. CONCLUSIONS: BLI is an important tool with many potential advantages for investigators. The measurement of intrahepatic tumor growth by imaging luciferase activity noninvasively provides valuable information on tumor burden and effectiveness of therapy. Thus, the presented intrahepatic metastases model based on the growth of HCT-116 Luc(pos) cells is suitable for in vivo testing of different cancer therapy strategies.

Gastric bypass surgery weight loss-independently induces gut Il-22 release in association with improved glycemic control in obese Zucker fatty rats.

Background: Roux-en-Y gastric bypass surgery (RYGB) improves glycemic control in individuals with severe obesity beyond the effects of weight loss alone. To identify potential underlying mechanisms, we asked how equivalent weight loss from RYGB and from chronic caloric restriction impact gut release of the metabolically beneficial cytokine interleukin-22 (Il-22). Methods: Obese male Zucker fatty rats were randomized into sham-operated (Sham), RYGB, and sham-operated, body weight-matched to RYGB (BWM) groups. Food intake and body weight were measured regularly for 4 weeks. An oral glucose tolerance test (OGTT) was performed on postoperative day 27. Portal vein plasma, systemic plasma, and whole-wall samples from throughout the gut were collected on postoperative day 28. Gut Il-22 mRNA expression was determined by real-time quantitative PCR. Plasma Il-22 levels were determined by enzyme-linked immunosorbant assay (ELISA). Results: RYGB and BWM rats had lower food intake and body weight as well as superior blood glucose clearing capability compared with Sham rats. RYGB rats also had superior blood glucose clearing capability compared with BWM rats despite having similar body weights and higher food intake. Il-22 mRNA expression was approximately 100-fold higher specifically in the upper jejunum in RYGB rats compared with Sham rats. Il-22 protein was only detectable in portal vein (34.1 ± 9.4 pg/mL) and systemic (46.9 ± 10.5 pg/mL) plasma in RYGB rats. Area under the curve of blood glucose during the OGTT, but not food intake or body weight, negatively correlated with portal vein and systemic plasma Il-22 levels in RYGB rats. Conclusions: These results suggest that induction of gut Il-22 release might partly account for the weight loss-independent improvements in glycemic control after RYGB, and further support the use of this cytokine for the treatment of metabolic disease.

Antibody-based soluble and membrane-bound TWEAK mimicking agonists with FcγR-independent activity.

Fibroblast growth factor (FGF)-inducible 14 (Fn14) activates the classical and alternative NFκB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) signaling pathway but also enhances tumor necrosis factor (TNF)-induced cell death. Fn14 expression is upregulated in non-hematopoietic cells during tissue injury and is also often highly expressed in solid cancers. In view of the latter, there were and are considerable preclinical efforts to target Fn14 for tumor therapy, either by exploiting Fn14 as a target for antibodies with cytotoxic activity (e.g. antibody-dependent cellular cytotoxicity (ADCC)-inducing IgG variants, antibody drug conjugates) or by blocking antibodies with the aim to interfere with protumoral Fn14 activities. Noteworthy, there are yet no attempts to target Fn14 with agonistic Fc effector function silenced antibodies to unleash the proinflammatory and cell death-enhancing activities of this receptor for tumor therapy. This is certainly not at least due to the fact that anti-Fn14 antibodies only act as effective agonists when they are presented bound to Fcγ receptors (FcγR). Thus, there are so far no antibodies that robustly and selectively engage Fn14 signaling without triggering unwanted FcγR-mediated activities. In this study, we investigated a panel of variants of the anti-Fn14 antibody 18D1 of different valencies and domain architectures with respect to their inherent FcγR-independent ability to trigger Fn14-associated signaling pathways. In contrast to conventional 18D1, the majority of 18D1 antibody variants with four or more Fn14 binding sites displayed a strong ability to trigger the alternative NFκB pathway and to enhance TNF-induced cell death and therefore resemble in their activity soluble (TNF)-like weak inducer of apoptosis (TWEAK), one form of the natural occurring ligand of Fn14. Noteworthy, activation of the classical NFκB pathway, which naturally is predominately triggered by membrane-bound TWEAK but not soluble TWEAK, was preferentially observed with a subset of constructs containing Fn14 binding sites at opposing sites of the IgG scaffold, e.g. IgG1-scFv fusion proteins. A superior ability of IgG1-scFv fusion proteins to trigger classical NFκB signaling was also observed with the anti-Fn14 antibody PDL192 suggesting that we identified generic structures for Fn14 antibody variants mimicking soluble and membrane-bound TWEAK.

Human organoids are superior to cell culture models for intestinal barrier research.

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.

Apolipoprotein E (ApoE) Rescues the Contractile Smooth Muscle Cell Phenotype in Popliteal Artery Aneurysm Disease.

Popliteal artery aneurysm (PAA) is the most frequent peripheral aneurysm, primarily seen in male smokers with a prevalence below 1%. This exploratory study aims to shed light on cellular mechanisms involved in PAA progression. Sixteen human PAA and eight non-aneurysmatic popliteal artery samples, partially from the same patients, were analyzed by immunohistochemistry, fluorescence imaging, Affymetrix mRNA expression profiling, qPCR and OLink proteomics, and compared to atherosclerotic (n = 6) and abdominal aortic aneurysm (AAA) tissue (n = 19). Additionally, primary cell culture of PAA-derived vascular smooth muscle cells (VSMC) was established for modulation and growth analysis. Compared to non-aneurysmatic popliteal arteries, VSMCs lose the contractile phenotype and the cell proliferation rate increases significantly in PAA. Array analysis identified APOE higher expressed in PAA samples, co-localizing with VSMCs. APOE stimulation of primary human PAA VSMCs significantly reduced cell proliferation. Accordingly, contractile VSMC markers were significantly upregulated. A single case of osseous mechanically induced PAA with a non-diseased VSMC profile emphasizes these findings. Carefully concluded, PAA pathogenesis shows similar features to AAA, yet the mechanisms involved might differ. APOE is specifically higher expressed in PAA tissue and could be involved in VSMC phenotype rescue.

Multiorgan recovery in a cadaver body using mild hypothermic ECMO treatment in a murine model.

BACKGROUND: Transplant candidates on the waiting list are increasingly challenged by the lack of organs. Most of the organs can only be kept viable within very limited timeframes (e.g., mere 4-6 h for heart and lungs exposed to refrigeration temperatures ex vivo). Donation after circulatory death (DCD) using extracorporeal membrane oxygenation (ECMO) can significantly enlarge the donor pool, organ yield per donor, and shelf life. Nevertheless, clinical attempts to recover organs for transplantation after uncontrolled DCD are extremely complex and hardly reproducible. Therefore, as a preliminary strategy to fulfill this task, experimental protocols using feasible animal models are highly warranted. The primary aim of the study was to develop a model of ECMO-based cadaver organ recovery in mice. Our model mimics uncontrolled organ donation after an "out-of-hospital" sudden unexpected death with subsequent "in-hospital" cadaver management post-mortem. The secondary aim was to assess blood gas parameters, cardiac activity as well as overall organ state. The study protocol included post-mortem heparin-streptokinase administration 10 min after confirmed death induced by cervical dislocation under full anesthesia. After cannulation, veno-arterial ECMO (V-A ECMO) was started 1 h after death and continued for 2 h under mild hypothermic conditions followed by organ harvest. Pressure- and flow-controlled oxygenated blood-based reperfusion of a cadaver body was accompanied by blood gas analysis (BGA), electrocardiography, and histological evaluation of ischemia-reperfusion injury. For the first time, we designed and implemented, a not yet reported, miniaturized murine hemodialysis circuit for the treatment of severe hyperkalemia and metabolic acidosis post-mortem. RESULTS: BGA parameters confirmed profound ischemia typical for cadavers and incompatible with normal physiology, including extremely low blood pH, profound negative base excess, and enormously high levels of lactate. Two hours after ECMO implantation, blood pH values of a cadaver body restored from < 6.5 to 7.3 ± 0.05, pCO2 was lowered from > 130 to 41.7 ± 10.5 mmHg, sO2, base excess, and HCO3 were all elevated from below detection thresholds to 99.5 ± 0.6%, - 4 ± 6.2 and 22.0 ± 6.0 mmol/L, respectively (Student T test, p < 0.05). A substantial decrease in hyperlactatemia (from > 20 to 10.5 ± 1.7 mmol/L) and hyperkalemia (from > 9 to 6.9 ± 1.0 mmol/L) was observed when hemodialysis was implemented. On balance, the first signs of regained heart activity appeared on average 10 min after ECMO initiation without cardioplegia or any inotropic and vasopressor support. This was followed by restoration of myocardial contractility with a heart rate of up to 200 beats per minute (bpm) as detected by an electrocardiogram (ECG). Histological examinations revealed no evidence of heart injury 3 h post-mortem, whereas shock-specific morphological changes relevant to acute death and consequent cardiac/circulatory arrest were observed in the lungs, liver, and kidney of both control and ECMO-treated cadaver mice. CONCLUSIONS: Thus, our model represents a promising approach to facilitate studying perspectives of cadaveric multiorgan recovery for transplantation. Moreover, it opens new possibilities for cadaver organ treatment to extend and potentiate donation and, hence, contribute to solving the organ shortage dilemma.

Randomized comparison of reduced fat and reduced carbohydrate hypocaloric diets on intrahepatic fat in overweight and obese human subjects.

UNLABELLED: Obesity-related hepatic steatosis is a major risk factor for metabolic and cardiovascular disease. Fat reduced hypocaloric diets are able to relieve the liver from ectopically stored lipids. We hypothesized that the widely used low carbohydrate hypocaloric diets are similarly effective in this regard. A total of 170 overweight and obese, otherwise healthy subjects were randomized to either reduced carbohydrate (n = 84) or reduced fat (n = 86), total energy restricted diet (-30% of energy intake before diet) for 6 months. Body composition was estimated by bioimpedance analyses and abdominal fat distribution by magnetic resonance tomography. Subjects were also submitted to fat spectroscopy of liver and oral glucose tolerance testing. In all, 102 subjects completed the diet intervention with measurements of intrahepatic lipid content. Both hypocaloric diets decreased body weight, total body fat, visceral fat, and intrahepatic lipid content. Subjects with high baseline intrahepatic lipids (>5.56%) lost ≈7-fold more intrahepatic lipids compared with those with low baseline values (<5.56%) irrespective of diet composition. In contrast, changes in visceral fat mass and insulin sensitivity were similar between subgroups, with low and high baseline intrahepatic lipids. CONCLUSION: A prolonged hypocaloric diet low in carbohydrates and high in fat has the same beneficial effects on intrahepatic lipid accumulation as the traditional low-fat hypocaloric diet. The decrease in intrahepatic lipids appears to be independent of visceral fat loss and is not tightly coupled with changes in whole body insulin sensitivity during 6 months of an energy restricted diet.