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The site of confluence of the artery and the portal vein in the liver still appears to be controversial. Anatomical studies suggested a presinusoidal or an intrasinusoidal confluence in the first, second or even final third of the sinusoids. The objective of this investigation was to study the problem with functional biochemical techniques. Rat livers were perfused through the hepatic artery and simultaneously either in the orthograde direction from the portal vein to the hepatic vein or in the retrograde direction from the hepatic vein to the portal vein. Arterial how was linearly dependent on arterial pressure between 70 cm H2O and 120 cm H2O at a constant portal or hepatovenous pressure of 18 cm H2O. An arterial pressure of 100 cm H2O was required for the maintenance of a homogeneous orthograde perfusion of the whole parenchyma and of a physiologic ratio of arterial to portal how of about 1:3. Glucagon was infused either through the artery or the portal vein and hepatic vein, respectively, to a submaximally effective ''calculated'' sinusoidal concentration after mixing of 0.1 nmol/L. During orthograde perfusions, arterial and portal glucagon caused the same increases in glucose output. Yet during retrograde perfusions, hepatovenous glucagon elicited metabolic alterations equal to those in orthograde perfusions, whereas arterial glucagon effected changes strongly reduced to between 10% and 50%. Arterially infused trypan blue was distributed homogeneously in the parenchyma during orthograde perfusions, whereas it reached clearly smaller areas of parenchyma during retrograde perfusions. Finally, arterially applied acridine orange was taken up by all periportal hepatocytes in the proximal half of the acinus during orthograde perfusions but only by a much smaller portion of periportal cells in the proximal third of the acinus during retrograde perfusions. These findings suggest that in rat liver, the hepatic artery and the portal vein mix before and within the first third of the sinusoids, rather than in the middle or even last third.
Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation and in the pathogenesis of inflammation. The mouse genome involves seven functional Alox genes and the encoded enzymes share a high degree of amino acid conservation with their human orthologs. There are, however, functional differences between mouse and human ALOX orthologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative (15S-HpETE), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis for this functional difference has been explored and in vitro mutagenesis humanized the reaction specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the reaction specificity of mouse Alox15b in vivo, we created Alox15b knock-in mice expressing the arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain significantly less body weight than outbred wildtype controls, but this effect was not observed for female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here again, these differences were not observed in female individuals. These data suggest that humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest.
In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations.
Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples.
Aging is associated with bone loss, which can lead to osteoporosis and high fracture risk. This coincides with the enhanced formation of bone marrow adipose tissue (BMAT), suggesting a negative effect of bone marrow adipocytes on skeletal health. Increased BMAT formation is also observed in pathologies such as obesity, type 2 diabetes and osteoporosis. However, a subset of bone marrow adipocytes forming the constitutive BMAT (cBMAT), arise early in life in the distal skeleton, contain high levels of unsaturated fatty acids and are thought to provide a physiological function. Regulated BMAT (rBMAT) forms during aging and obesity in proximal regions of the bone and contain a large proportion of saturated fatty acids. Paradoxically, BMAT accumulation is also enhanced during caloric restriction (CR), a life-span extending dietary intervention. This indicates, that different types of BMAT can form in response to opposing nutritional stimuli with potentially different functions.
To this end, two types of nutritional interventions, CR and high fat diet (HFD), that are both described to induce BMAT accumulation were carried out. CR markedly increased BMAT formation in the proximal tibia and led to a higher proportion of unsaturated fatty acids, making it similar to the physiological cBMAT. Additionally, proximal and diaphyseal tibia regions displayed higher adiponectin expression. In aged mice, CR was associated with an improved trabecular bone structure. Taken together, these findings demonstrate, that the type of BMAT that forms during CR might provide beneficial effects for local bone stem/progenitor cells and metabolic health. The HFD intervention performed in this thesis showed no effect on BMAT accumulation and bone microstructure. RNA Seq analysis revealed alterations in the composition of the collagen-containing extracellular matrix (ECM).
In order to investigate the effects of glucose homeostasis on osteogenesis, differentiation capacity of immortalized multipotent mesenchymal stromal cells (MSCs) and osteochondrogenic progenitor cells (OPCs) was analyzed. Insulin improved differentiation in both cell types, however, combination of with a high glucose concentration led to an impaired mineralization of the ECM. In the MSCs, this was accompanied by the formation of adipocytes, indicating negative effects of the adipocytes formed during hyperglycemic conditions on mineralization processes. However, the altered mineralization pattern and structure of the ECM was also observed in OPCs, which did not form any adipocytes, suggesting further negative effects of a hyperglycemic environment on osteogenic differentiation.
In summary, the work provided in this thesis demonstrated that differentiation commitment of bone-resident stem cells can be altered through nutrient availability, specifically glucose. Surprisingly, both high nutrient supply, e.g. the hyperglycemic cell culture conditions, and low nutrient supply, e.g. CR, can induce adipogenic differentiation. However, while CR-induced adipocyte formation was associated with improved trabecular bone structure, adipocyte formation in a hyperglycemic cell-culture environment hampered mineralization. This thesis provides further evidence for the existence of different types of BMAT with specific functions.
Dermal Delivery of the High-Molecular-Weight Drug Tacrolimus by Means of Polyglycerol-Based Nanogels
(2019)
Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions.
Dermal Delivery of the High-Molecular-Weight Drug Tacrolimus by Means of Polyglycerol-Based Nanogels
(2019)
Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions.
Systemic inflammation is a hallmark of cancer cachexia. Among tumor-host interactions, the white adipose tissue (WAT) is an important contributor to inflammation as it suffers morphological reorganization and lipolysis, releasing free fatty acids (FA), bioactive lipid mediators (LM) and pro-inflammatory cytokines, which accentuate the activation of pro-inflammatory signaling pathways and the recruitment of immune cells to the tissue. This project aimed to investigate which inflammatory factors are involved in the local adipose tissue inflammation and what is the influence of such factors upon enzymes involved in FA or LM metabolism in healthy individuals (Control), weight stable gastro-intestinal cancer patients (WSC) and cachectic cancer patients (CC). The results demonstrated that the inflammatory signature of systemic inflammation is different from local adipose tissue inflammation. The systemic inflammation of the cachectic cancer patients was characterized by higher levels of circulating saturated fatty acids (SFA), tumor-necrosis-factor-α (TNF-α), interleukins IL-6, IL-8 and CRP while levels of polyunsaturated fatty acids (PUFAs), especially n3-PUFAs, were lower in CC than in the other groups. In vitro and in adipose tissue explants, pro-inflammatory cytokines and SFAs were shown to increase the chemokines IL-8 and CXCL10 that were found to be augmented in adipose tissue inflammation in CC which was more profound in the visceral adipose tissue (VAT) than in subcutaneous adipose tissue (SAT). Systemic inflammation was negatively associated with the expression of PUFA synthesizing enzymes, though gene and protein expression did hardly differ between groups. The effects of inflammatory factors on enzymes in the whole tissue could have been masked by differentiated modulation of the diverse cell types in the same tissue. In vitro experiments showed that the expression of FA-modifying enzymes such as desaturases and elongases in adipocytes and macrophages was regulated into opposing directions by TNF-α, IL-6, LPS or palmitate. The higher plasma concentration of the pro-resolving LM resolvin D1 in CC cannot compensate the overall inflammatory status and the results indicate that inflammatory cytokines interfere with synthesis pathways of pro-resolving LM. In summary, the data revealed a complex inter-tissue and inter-cellular crosstalk mediated by pro-inflammatory cytokines and lipid compounds enhancing inflammation in cancer cachexia by feed-forward mechanisms.
The complement fragments C3a and C5a were purified from zymosan-activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti-C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver.
In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the α1-blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied.