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- Fn14 (2)
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- Roux-en-Y gastric bypass surgery (2)
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- Klinik und Poliklinik für Allgemein-, Viszeral-, Gefäß- und Kinderchirurgie (Chirurgische Klinik I) (27)
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Sonstige beteiligte Institutionen
Die Transplantation ist die einzige effektive Therapie für irreversibel geschädigte Organe. Um jedoch das Transplantat vor der Zerstörung zu schützen, wird die Abwehrfunktion des Immunsystems gezielt geschwächt. Diese notwendige dauerhafte Schwächung mit so genannten Immunsuppressiva haben jedoch in ihrer Langzeitanwendung erhebliche Nebenwirkungen für die Patienten. Sie sind einer größeren Gefahr als die Normalbevölkerung ausgesetzt, an schwerwiegenden Infektionen und Tumore zu erkranken. Zwei antigenspezifische Strategien als Grundlage für zukünftige therapeutische Konzepte, die nicht die Immunabwehr behindern, werden unter dem Begriff "ideale Therapie" erläutert.
Background: Esophageal adenocarcinomas (EACs) arise due to gastroesophageal reflux, with Barrett’s esophagus (BE) regarded as precancerous lesion. Matrix metalloproteinases (MMPs) might play a role during the multistep carcinogenetic process. Methods: Expression of MMP-1 and -13 was analyzed in esophageal cancer (n = 41 EAC with BE, n = 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC), furthermore in BE without intraepithelial neoplasia (IN) (n = 18), and the cell line OE-33. MMP-1 was co-labelled with Ki-67 (proliferation), Cdx-2 (marker for intestinal metaplasia, BE) and analyzed on mRNA level. MMP-1 staining results were correlated with clinicopatholocical parameters. Results: On protein level, MMP-1 expression was found in 39 of 41 (95%) EAC with BE, in 19 of 19 (100%) EAC without BE, in 6 of 10 (60%) ESCC, and in 10 of 18 (56%) BE without IN. No expression of MMP-13 was found in these specimens. Quantification showed 48% MMP-1 positive cells in EAC with BE, compared to 35% in adjacent BE (p < 0.05), 44% in EAC without BE, 32% in ESCC, and 4% in BE without IN. Immunofluorescence double staining experiments revealed increased MMP-1 expressing in proliferating cells (MMP-1+/Ki-67+) (r = 0.943 for BE and r = 0.811 for EAC). On mRNA-level, expression of MMP-1 was significantly higher in EAC compared to BE (p = 0.01) and confirmed immunohistochemical staining results. High MMP-1 levels were associated with lymph node metastases but not with poorer survival (p = 0.307). Conclusions: Our findings suggest that MMP-1 plays a role as preinvasive factor in BE-associated EAC. Expression of MMP-1 in proliferating BE and EAC cells suggest malignant proliferation following the clonal expansion model.
Background: Investigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barrett’s Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis. Materials and methods: Expression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates. Results: LgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNAlevel, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis. Conclusion: The stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.
Background
Investigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barrett's Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis.
Materials and methods
Expression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates.
Results
LgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNA-level, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis.
Conclusion
The stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.
Background: Ascorbic acid demonstrates a cytotoxic effect by generating hydrogen peroxide, a reactive oxygen species (ROS) involved in oxidative cell stress. A panel of eleven human cancer cell lines, glioblastoma and carcinoma, were exposed to serial dilutions of ascorbic acid (5-100 mmol/L). The purpose of this study was to analyse the impact of catalase, an important hydrogen peroxide-detoxifying enzyme, on the resistance of cancer cells to ascorbic acid mediated oxidative stress. Methods: Effective concentration (EC50) values, which indicate the concentration of ascorbic acid that reduced the number of viable cells by 50%, were detected with the crystal violet assay. The level of intracellular catalase protein and enzyme activity was determined. Expression of catalase was silenced by catalase-specific short hairpin RNA (sh-RNA) in BT-20 breast carcinoma cells. Oxidative cell stress induced apoptosis was measured by a caspase luminescent assay. Results: The tested human cancer cell lines demonstrated obvious differences in their resistance to ascorbic acid mediated oxidative cell stress. Forty-five percent of the cell lines had an EC50>20 mmol/L and fifty-five percent had an EC50<20 mmol/L. With an EC50 of 2.6–5.5 mmol/L, glioblastoma cells were the most susceptible cancer cell lines analysed in this study. A correlation between catalase activity and the susceptibility to ascorbic acid was observed. To study the possible protective role of catalase on the resistance of cancer cells to oxidative cell stress, the expression of catalase in the breast carcinoma cell line BT-20, which cells were highly resistant to the exposure to ascorbic acid (EC50: 94,9 mmol/L), was silenced with specific sh-RNA. The effect was that catalase-silenced BT-20 cells (BT-20 KD-CAT) became more susceptible to high concentrations of ascorbic acid (50 and 100 mmol/L). Conclusions: Fifty-five percent of the human cancer cell lines tested were unable to protect themselves against oxidative stress mediated by ascorbic acid induced hydrogen peroxide production. The antioxidative enzyme catalase is important to protect cancer cells against cytotoxic hydrogen peroxide. Silenced catalase expression increased the susceptibility of the formerly resistant cancer cell line BT-20 to oxidative stress.
The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor that stabilises the c-Myc protein. CIP2A is overexpressed in several tumours, and expression levels are an independent marker for long-term outcome. To determine whether CIP2A expression is elevated in colon cancer and whether it might serve as a prognostic marker for survival, we analysed CIP2A mRNA expression by real-time PCR in 104 colon cancer samples. CIP2A mRNA was overexpressed in colon cancer samples and CIP2A expression levels correlated significantly with tumour stage. We found that CIP2A serves as an independent prognostic marker for disease-free and overall survival. Further, we investigated CIP2A-dependent effects on levels of c-Myc, Akt and on cell proliferation in three colon cancer cell lines by silencing CIP2A using small interfering (si) and short hairpin (sh) RNAs. Depletion of CIP2A substantially inhibited growth of colon cell lines and reduced c-Myc levels without affecting expression or function of the upstream regulatory kinase, Akt. Expression of CIP2A was found to be dependent on MAPK activity, linking elevated c-Myc expression to deregulated signal transduction in colon cancer.
Clinical prognosis of metastasized colorectal carcinoma (CRC) is still not at desired levels and novel drugs are needed. Here, we focused on the multi-tyrosine kinase inhibitor E7080 (Lenvatinib) and assessed its therapeutic efficacy against human CRC cell lines in vitro and human CRC xenografts in vivo. The effect of E7080 on cell viability was examined on 10 humanCRCcell lines and humanendothelial cells (HUVEC). The inhibitory effect of E7080 on VEGF-induced angiogenesis was studied in an ex vivo mouse aortic ring angiogenesis assay. In addition, the efficacy of E7080 against xenografts derived fromCRC cell lines and CRC patient resection specimenswithmutated KRASwas investigated in vivo. Arelatively low cytotoxic effect of E7080 on CRC cell viabilitywas observed in vitro. Endothelial cells (HUVEC)weremore susceptible to the incubation with E7080. This is in line with the observation that E7080 demonstrated an anti-angiogenic effect in a three-dimensional ex vivo mouse aortic ring angiogenesis assay. E7080 effectively disrupted CRC cell-mediated VEGF-stimulated growth of HUVEC in vitro. Daily in vivo treatment with E7080 (5 mg/kg) significantly delayed the growth of KRAS mutated CRC xenografts with decreased density of tumor-associated vessel formations and without tumor regression. This observation is in line with results that E7080 did not significantly reduce the number of Ki67-positive cells in CRC xenografts. The results suggest antiangiogenic activity of E7080 at a dosage thatwas well tolerated by nudemice. E7080 may provide therapeutic benefits in the treatment of CRC with mutated KRAS.
Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells.
The Quality of Acute Stroke Care-an Analysis of Evidence-Based Indicators in 260 000 Patients
(2014)
Background: Stroke patients should be cared for in accordance with evidence-based guidelines. The extent of implementation of guidelines for the acute care of stroke patients in Germany has been unclear to date. Methods: The regional quality assurance projects that cooperate in the framework of the German Stroke Registers Study Group (Arbeitsgemeinschaft Deutscher Schlaganfall-Register, ADSR) collected data on the care of stroke patients in 627 hospitals in 2012. The quality of the acute hospital care of patients with stroke or transient ischemic attack (TIA) was assessed on the basis of 15 standardized, evidence-based quality indicators and compared across the nine participating regional quality assurance projects. Results: Data were obtained on more than 260 000 patients nationwide. Intravenous thrombolysis was performed in 59.7% of eligible ischemic stroke patients patients (range among participating projects, 49.7-63.6%). Dysphagia screening was documented in 86.2% (range, 74.8-93.1%). For the following indicators, the defined targets were not reached for all of Germany: antiaggregation within 48 hours, 93.4% (range, 86.6-96.4%); anticoagulation for atrial fibrillation, 77.6% (range, 72.4-80.1%); standardized dysphagia screening, 86.2% (range, 74.8-93.1%); oral and written information of the patients or their relatives, 86.1% (range, 75.4-91.5%). The rate of patients examined or treated by a speech therapist was in the target range. Conclusion: The defined targets were reached for most of the quality indicators. Some indicators, however, varied widely across regional quality assurance projects. This implies that the standardization of care for stroke patients in Germany has not yet been fully achieved.
Bariatric operations in obese patients with type 2 diabetes often improve diabetes before weight loss is observed. In patients mainly Roux-en-Y-gastric bypass with partial stomach resection is performed. Duodenojejunal bypass (DJB) and ileal interposition (IIP) are employed in animal experiments. Due to increased glucose exposition of L-cells located in distal ileum, all bariatric surgery procedures lead to higher secretion of antidiabetic glucagon like peptide-1 (GLP-1) after glucose gavage. After DJB also downregulation of Na\(^{+}\)-D-glucose cotransporter SGLT1 was observed. This suggested a direct contribution of decreased glucose absorption to the antidiabetic effect of bariatric surgery. To investigate whether glucose absorption is also decreased after IIP, we induced diabetes with decreased glucose tolerance and insulin sensitivity in male rats and investigated effects of IIP on diabetes and SGLT1. After IIP, we observed weight-independent improvement of glucose tolerance, increased insulin sensitivity, and increased plasma GLP-1 after glucose gavage. The interposed ileum was increased in diameter and showed increased length of villi, hyperplasia of the epithelial layer, and increased number of L-cells. The amount of SGLT1-mediated glucose uptake in interposed ileum was increased 2-fold reaching the same level as in jejunum. Thus, improvement of glycemic control by bariatric surgery does not require decreased glucose absorption.
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.
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.
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 IC\(_{50}\) 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 IC\(_{50}\) 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.
Background
Currently ketogenic diets (KDs) are hyped as an anti-tumor intervention aimed at exploiting the metabolic abnormalities of cancer cells. However, while data in humans is sparse, translation of murine tumor models to the clinic is further hampered by small sample sizes, heterogeneous settings and mixed results concerning tumor growth retardation. The aim was therefore to synthesize the evidence for a growth inhibiting effect of KDs when used as a monotherapy in mice.
Methods
We conducted a Bayesian random effects meta-analysis on all studies assessing the survival (defined as the time to reach a pre-defined endpoint such as tumor volume) of mice on an unrestricted KD compared to a high carbohydrate standard diet (SD). For 12 studies meeting the inclusion criteria either a mean survival time ratio (MR) or hazard ratio (HR) between the KD and SD groups could be obtained. The posterior estimates for the MR and HR averaged over four priors on the between-study heterogeneity τ\(^{2}\) were MR = 0.85 (95% highest posterior density interval (HPDI) = [0.73, 0.97]) and HR = 0.55 (95% HPDI = [0.26, 0.87]), indicating a significant overall benefit of the KD in terms of prolonged mean survival times and reduced hazard rate. All studies that used a brain tumor model also chose a late starting point for the KD (at least one day after tumor initiation) which accounted for 26% of the heterogeneity. In this subgroup the KD was less effective (MR = 0.89, 95% HPDI = [0.76, 1.04]).
Conclusions
There was an overall tumor growth delaying effect of unrestricted KDs in mice. Future experiments should aim at differentiating the effects of KD timing versus tumor location, since external evidence is currently consistent with an influence of both of these factors.
Background:
Factors influencing access to stroke unit (SU) care and data on quality of SU care in Germany are scarce. We investigated characteristics of patients directly admitted to a SU as well as patient-related and structural factors influencing adherence to predefined indicators of quality of acute stroke care across hospitals providing SU care.
Methods:
Data were derived from the German Stroke Registers Study Group (ADSR), a voluntary network of 9 regional registers for monitoring quality of acute stroke care in Germany. Multivariable logistic regression analyses were performed to investigate characteristics influencing direct admission to SU. Generalized Linear Mixed Models (GLMM) were used to estimate the influence of structural hospital characteristics (percentage of patients admitted to SU, year of SU-certification, and number of stroke and TIA patients treated per year) on adherence to predefined quality indicators.
Results:
In 2012 180,887 patients were treated in 255 hospitals providing certified SU care participating within the ADSR were included in the analysis; of those 82.4% were directly admitted to a SU. Ischemic stroke patients without disturbances of consciousness (p < .0001), an interval onset to admission time ≤3 h (p < .0001), and weekend admission (p < .0001) were more likely to be directly admitted to a SU. A higher proportion of quality indicators within predefined target ranges were achieved in hospitals with a higher proportion of SU admission (p = 0.0002). Quality of stroke care could be maintained even if certification was several years ago.
Conclusions:
Differences in demographical and clinical characteristics regarding the probability of SU admission were observed. The influence of structural characteristics on adherence to evidence-based quality indicators was low.
Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n = 9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n = 5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC\(_{50}\)< 3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents.
Long-term slash-and-burn experiments, when compared with intensive tillage without manuring, resulted in a huge data set relating to potential crop yields, depending on soil quality, crop type, and agricultural measures. Cultivation without manuring or fallow phases did not produce satisfying yields, and mono-season cropping on freshly cleared and burned plots resulted in rather high yields, comparable to those produced during modern industrial agriculture - at least ten-fold the ones estimated for the medieval period. Continuous cultivation on the same plot, using imported wood from adjacent areas as fuel, causes decreasing yields over several years. The high yield of the first harvest of a slash-and-burn agriculture is caused by nutrient input through the ash produced and mobilization from the organic matter of the topsoil, due to high soil temperatures during the burning process and higher topsoil temperatures due to the soil’s black surface. The harvested crops are pure, without contamination of any weeds. Considering the amount of work required to fight weeds without burning, the slash-and-burn technique yields much better results than any other tested agricultural approach. Therefore, in dense woodland, without optimal soils and climate, slash-and-burn agriculture seems to be the best, if not the only, feasible method to start agriculture, for example, during the Late Neolithic, when agriculture expanded from the loess belt into landscapes less suitable for agriculture. Extensive and cultivation with manuring is more practical in an already-open landscape and with a denser population, but its efficiency in terms of the ratio of the manpower input to food output, is worse. Slash-and-burn agriculture is not only a phenomenon of temperate European agriculture during the Neolithic, but played a major role in land-use in forested regions worldwide, creating anthromes on a huge spatial scale.
Background:
Ketogenic diets (KDs) or short-term fasting are popular trends amongst supportive approaches for cancer patients. Beta-hydroxybutyrate (3-OHB) is the main physiological ketone body, whose concentration can reach plasma levels of 2–6 mM during KDs or fasting. The impact of 3-OHB on the biology of tumor cells described so far is contradictory. Therefore, we investigated the effect of a physiological concentration of 3 mM 3-OHB on metabolism, proliferation, and viability of breast cancer (BC) cells in vitro.
Methods:
Seven different human BC cell lines (BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D) were cultured in medium with 5 mM glucose in the presence of 3 mM 3-OHB at mild hypoxia (5% oxygen) or normoxia (21% oxygen). Metabolic profiling was performed by quantification of the turnover of glucose, lactate, and 3-OHB and by Seahorse metabolic flux analysis. Expression of key enzymes of ketolysis as well as the main monocarboxylic acid transporter MCT2 and the glucose-transporter GLUT1 was analyzed by RT-qPCR and Western blotting. The effect of 3-OHB on short- and long-term cell proliferation as well as chemo- and radiosensitivity were also analyzed.
Results:
3-OHB significantly changed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BT20 cells resulting in a more oxidative energetic phenotype. MCF-7 and MDA-MB 468 cells had increased ECAR only in response to 3-OHB, while the other three cell types remained uninfluenced. All cells expressed MCT2 and GLUT1, thus being able to uptake the metabolites. The consumption of 3-OHB was not strongly linked to mRNA overexpression of key enzymes of ketolysis and did not correlate with lactate production and glucose consumption. Neither 3-OHB nor acetoacetate did interfere with proliferation. Further, 3-OHB incubation did not modify the response of the tested BC cell lines to chemotherapy or radiation.
Conclusions:
We found that a physiological level of 3-OHB can change the energetic profile of some BC cell lines. However, 3-OHB failed to influence different biologic processes in these cells, e.g., cell proliferation and the response to common breast cancer chemotherapy and radiotherapy. Thus, we have no evidence that 3-OHB generally influences the biology of breast cancer cells in vitro.
In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine.
TNF-like weak inducer of apoptosis (TWEAK) and inhibition of protein synthesis with cycloheximide (CHX) sensitize for poly(I:C)-induced cell death. Notably, although CHX preferentially enhanced poly(I:C)-induced apoptosis, TWEAK enhanced primarily poly(I:C)-induced necroptosis. Both sensitizers of poly(I:C)-induced cell death, however, showed no major effect on proinflammatory poly(I:C) signaling. Analysis of a panel of HeLa-RIPK3 variants lacking TRADD, RIPK1, FADD, or caspase-8 expression revealed furthermore similarities and differences in the way how poly(I:C)/TWEAK, TNF, and TRAIL utilize these molecules for signaling. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for this response in TNF and TRAIL signaling. TRADD-RIPK1-double deficiency differentially affected poly(I:C)-triggered gene induction but abrogated gene induction by TNF completely. FADD deficiency abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis, whereas TRADD elicited protective activity against all three death inducers. A general protective activity against poly(I:C)-, TRAIL-, and TNF-induced cell death was also observed in FLIPL and FLIPS transfectrants.