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Intracranial hemorrhage results in devastating forms of cerebral damage. Frequently, these results also present with cardiac dysfunction ranging from ECG changes to Takotsubo syndrome (TTS). This suggests that intracranial bleeding due to subarachnoid hemorrhage (SAH) disrupts the neuro–cardiac axis leading to neurogenic stress cardiomyopathy (NSC) of different degrees. Following this notion, SAH and secondary TTS could be directly linked, thus contributing to poor outcomes. We set out to test if blood circulation is the driver of the brain–heart axis by investigating serum samples of TTS patients. We present a novel in vitro model combining SAH and secondary TTS to mimic the effects of blood or serum, respectively, on blood–brain barrier (BBB) integrity using in vitro monolayers of an established murine model. We consistently demonstrated decreased monolayer integrity and confirmed reduced Claudin-5 and Occludin levels by RT-qPCR and Western blot and morphological reorganization of actin filaments in endothelial cells. Both tight junction proteins show a time-dependent reduction. Our findings highlight a faster and more prominent disintegration of BBB in the presence of TTS and support the importance of the bloodstream as a causal link between intracerebral bleeding and cardiac dysfunction. This may represent potential targets for future therapeutic inventions in SAH and TTS.
Neisseria gonorrhoeae is a human-specific pathogen that causes gonorrhea, the second most common sexually transmitted infection worldwide. Disease progression, drug discovery, and basic host-pathogen interactions are studied using different approaches, which rely on models ranging from 2D cell culture to complex 3D tissues and animals. In this review, we discuss the models used in N. gonorrhoeae research. We address both in vivo (animal) and in vitro cell culture models, discussing the pros and cons of each and outlining the recent advancements in the field of three-dimensional tissue models. From simple 2D monoculture to complex advanced 3D tissue models, we provide an overview of the relevant methodology and its application. Finally, we discuss future directions in the exciting field of 3D tissue models and how they can be applied for studying the interaction of N. gonorrhoeae with host cells under conditions closely resembling those found at the native sites of infection.
In vitro rearing of honeybee larvae is an established method that enables exact control and monitoring of developmental factors and allows controlled application of pesticides or pathogens. However, only a few studies have investigated how the rearing method itself affects the behavior of the resulting adult honeybees. We raised honeybees in vitro according to a standardized protocol: marking the emerging honeybees individually and inserting them into established colonies. Subsequently, we investigated the behavioral performance of nurse bees and foragers and quantified the physiological factors underlying the social organization. Adult honeybees raised in vitro differed from naturally reared honeybees in their probability of performing social tasks. Further, in vitro-reared bees foraged for a shorter duration in their life and performed fewer foraging trips. Nursing behavior appeared to be unaffected by rearing condition. Weight was also unaffected by rearing condition. Interestingly, juvenile hormone titers, which normally increase strongly around the time when a honeybee becomes a forager, were significantly lower in three- and four-week-old in vitro bees. The effects of the rearing environment on individual sucrose responsiveness and lipid levels were rather minor. These data suggest that larval rearing conditions can affect the task performance and physiology of adult bees despite equal weight, pointing to an important role of the colony environment for these factors. Our observations of behavior and metabolic pathways offer important novel insight into how the rearing environment affects adult honeybees.
Objective
As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors.
Design
Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX)9,transforming growth factor beta (TGFB) 1or bone morphogenetic protein (BMP) 2cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy.
Results
Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenesSOX9,TGFB1andBMP2as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9).
Conclusions
Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factorsSOX9,TGFB1andBMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.
The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.
Surgical implantation of a biomaterial triggers foreign-body-induced fibrous encapsulation. Two major mechanisms of this complex physiological process are (I) chemotaxis of fibroblasts from surrounding tissue to the implant region, followed by (II) tissue remodeling. As an alternative to animal studies, we here propose a process-aligned \({in}\) \({vitro}\) test platform to investigate the material dependency of fibroblast chemotaxis and tissue remodeling mediated by material-resident macrophages.
Embedded in a biomimetic three-dimensional collagen hydrogel, chemotaxis of fibroblasts in the direction of macrophage-material-conditioned cell culture supernatant was analyzed by live cell imaging. A combination of statistical analysis with a complementary parameterized random walk model allowed quantitative and qualitative characterization of the cellular walk process. We thereby identified an increasing macrophage-mediated chemotactic potential ranking of biomaterials from glass over polytetrafluorethylene to titanium. To address long-term effects of biomaterial-resident macrophages on fibroblasts in a three-dimensional microenvironment, we further studied tissue remodeling by applying macrophage-material-conditioned medium on fibrous \({in}\) \({vitro}\) tissue models. A high correlation of the \({in}\) \({vitro}\) tissue model to state of the art \({in}\) \({vivo}\) study data was found. Titanium exhibited a significantly lower tissue remodeling capacity compared to polytetrafluorethylene. With this approach, we identified a material dependency of both chemotaxis and tissue remodeling processes, strengthening knowledge on their specific contribution to the foreign body reaction.
Polyphenols exert beneficial effects in type 2 diabetes mellitus (T2DM). However, their mechanism of action remains largely unknown. Endothelial Akt-kinase plays a key role in the pathogenesis of cardiovascular complications in T2DM and therefore the modulation of its activity is of interest. This work aimed to characterize effects of structurally different polyphenols on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926) and to describe structure-activity features. A comprehensive screening via ELISA quantified the effects of 44 polyphenols (10 µM) on pAkt Ser473. The most pronounced inhibitors were luteolin (44 ± 18%), quercetin (36 ± 8%), urolithin A (35 ± 12%), apigenin, fisetin, and resveratrol; (p < 0.01). The results were confirmed by Western blotting and complemented with corresponding experiments in HUVEC cells. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested polyphenols on both Akt-residues Ser473 and Thr308 (r = 0.9478, p = 0.0003) was determined by immunoblotting. Interestingly, the structural characteristics favoring pAkt inhibition partially differed from structural features enhancing the compounds’ antioxidant activity. The present study is the first to quantitatively compare the influence of polyphenols from nine different structural subclasses on pAkt in endothelial cells. These effects might be advantageous in certain T2DM-complications involving over-activation of the Akt-pathway. The suggested molecular mode of action of polyphenols involving Akt-inhibition contributes to understanding their effects on the cellular level.
Despite growing effort to advance materials towards a low fibrotic progression, all implants elicit adverse tissue responses. Pre-clinical biomaterial assessment relies on animals testing, which can be complemented by in vitro tests to address the Russell and Burch’s 3R aspect of reducing animal burden. However, a poor correlation between in vitro and in vivo biomaterial assessments confirms a need for suitable in vitro biomaterial tests. The aim of the study was to identify a test setting, which is predictive and might be time- and cost-efficient. We demonstrated how sensitive in vitro biomaterial assessment based on human primary macrophages depends on test conditions. Moreover, possible clinical scenarios such as lipopolysaccharide contamination, contact to autologous blood plasma, and presence of IL-4 in an immune niche influence the outcome of a biomaterial ranking. Nevertheless, by using glass, titanium, polytetrafluorethylene, silicone, and polyethylene representing a specific material-induced fibrotic response and by comparison to literature data, we were able to identify a test condition that provides a high correlation to state-of-the-art in vivo studies. Most important, biomaterial ranking obtained under native plasma test conditions showed a high predictive accuracy compared to in vivo assessments, strengthening a biomimetic three-dimensional in vitro test platform.
Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.
Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.