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Institute
BTU
Paraneoplastic neurological syndromes (PNS) are caused by an immune response against neuronal proteins upon their ectopical expression in tumor cells. Ma2 belongs to the protein family of paraneoplastic Ma antigens (PNMA). Detection of Ma2-specific autoantibodies is relevant for diagnostics of anti-Ma2 PNS and an underlying tumor such as germ testicular cancer, small cell lung cancer or breast cancer. Thus, early tumor treatment should improve the outcome for PNS therapy either.
Dot blot immunoassay based on recombinantly expressed and purified autoantigens could offer a sensitive method for identification of paraneoplastic autoantibodies from sera of PNS patients. Here we present purification with IMAC and FPLC of human Ma2 autoantigen upon its recombinant expression in E.coli. Furthermore, we provide evidence that dot blot immunoassays with purified Ma2 autoantigen can be used for detection of Ma2-specific autoantibodies from sera of PNS patients.
Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3‘-yl)-2‘-desoxyguanosine (E3′N2dG) and N6-(isoestragole-3‘-yl)-desoxyadenosine (E3′N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3′N6dA at 10 μM, E3′N2dG at 1μM estragole). E3′N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 μM after 24 h and < 0.5 μM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 μM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 μM estragole led to a 3.2 fold and 300 μM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a ‘practical threshold’ dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.
1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration–response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration–response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.
Atrial fibrillation (AF) is regularly accompanied by cardiac fibrosis and concomitant heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge about the underlying mechanisms is limited, which prevents effective pharmacotherapy. A deeper understanding of cardiac fibroblasts is essential to meet this need. We previously described phenotypic and functional differences between atrial fibroblasts from patients in sinus rhythm and with AF. Herein, we established and characterized a novel human atrial fibroblast line, which displays typical fibroblast morphology and function comparable to primary cells but with improved proliferation capacity and low spontaneous myofibroblast differentiation. These traits make our model suitable for the study of fibrosis mechanisms and for drug screening aimed at developing effective antifibrotic pharmacotherapy.
COVID-19 and the endothelium
(2020)
There is growing evidence that COVID-19 not only affects the lungs but beyond that the endothelial system. Recent studies showed that this can lead to microcirculatory impairments and in consequence to functional disorders of all inner organs. The combination of endothelial dysfunction with a generalized inflammatory state and complement elements may together contribute to the overall pro-coagulative state described in COVID-19 patients leading to venular as well as to arteriolar occlusions.
Some months ago, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan, China, and spread rapidly around the world. Some states, such as the Netherlands, Germany, Great Britain, Sweden and the USA initially focused on keeping the restrictions for economy and society as low as possible. The responsible authorities were of the opinion - and still are e.g. in Sweden - that it is sufficient enough to protect particularly vulnerable persons such as the elderly or people with pre-existing conditions. The idea behind this is that as soon as 60 to 70 percent of the population is infected with a pathogen, a so-called “herd immunity” has developed. However, the increasing numbers of deaths and modelling studies showed the expected overload of the hospitals. Therefore, most countries decided for a temporary lockdown with the exception of Sweden.
Based on the number of the total population, three times more people died from COVID-19 in Sweden (2679 deaths per 10 million inhabitants) compared to Germany (6848 deaths per 80 million inhabitants). The comparison Sweden versus Taiwan is even worse because 1072 times more people died in Sweden based on the number of the population (6 deaths per 24 million inhabitants).
Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO–induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.
Endothelialization of cardiovascular implants is regarded as a promising strategy for long-term compatibility. While umbilical vein endothelial cells are typically applied in research, human arterial endothelial cells (HAEC) from elderly donors would be the obvious source for autologous cellularization strategies.
In our approach, HAEC from 16 donors of varying age (16–63 years) were divided into two groups (<30 years and >30 years) and analyzed regarding morphology, viability, proliferation, function and senescence status.
No age-related differences were found regarding morphology, viability, density, prostacyclin and nitrite secretion or collagen and laminin production. However, the metabolic activity was slightly decreased (p = 0.0374) and the membrane integrity marginally impaired (p = 0.0404) in cells from older donors. Two out of three senescence assays detected more senescence markers in cells from older donors.
According to the assays applied here, HAEC from young and elderly donors up to the age of 63 years could be judged equally suitable for autologous cellularization strategies. However, this finding should be regarded with caution due to the extremely large variability between individual donors. Further studies comprising a larger sample size are necessary to investigate this issue more thoroughly.
Within the last years a comprehensive number of scientific studies demonstrated beneficial effect of Arthropira platensis (AP) as dietary supplement due to a high content of proteins, minerals and vitamins. Positive effects like promoting the immune system, reducing inflammation and an anti-oxidant capacity are reported. In this study, the effect of an aqueous AP extract on primary human venous endothelial cells (HUVEC) was investigated. In addition, the effect of AP on HUVEC treated with a bacterial toxin (lipopolysaccharide, LPA), inducing an activation of HUVEC and cellular detachment, was analyzed. Depending on the concentration of AP extract a significantly accelerated formation of an endothelial cell monolayer was observed. Furthermore, the detachment of HUVEC after LPA addition was dramatically reduced by AP. In conclusion, the data are promising and indicatory for an application of Arthrospira platensis in the clinical field.
The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.
Prostanoids are bioactive lipid mediators and take part in many physiological and pathophysiological processes in practically every organ, tissue and cell, including the vascular, renal, gastrointestinal and reproductive systems. In this review, we focus on their influence on platelets, which are key elements in thrombosis and hemostasis. The function of platelets is influenced by mediators in the blood and the vascular wall. Activated platelets aggregate and release bioactive substances, thereby activating further neighbored platelets, which finally can lead to the formation of thrombi. Prostanoids regulate the function of blood platelets by both activating or inhibiting and so are involved in hemostasis. Each prostanoid has a unique activity profile and, thus, a specific profile of action. This article reviews the effects of the following prostanoids: prostaglandin-D2 (PGD2), prostaglandin-E1, -E2 and E3 (PGE1, PGE2, PGE3), prostaglandin F2α (PGF2α), prostacyclin (PGI2) and thromboxane-A2 (TXA2) on platelet activation and aggregation via their respective receptors.
There is an urgent need to develop technologies for removing CO2 from the atmosphere to combat climate change. Microalgae and cyanobacteria, such as Arthrospira platensis (AP), have shown promise due to their high photoautotrophic biomass production. Conventional AP culture media are supplemented with high concentrations of NaHCO3 since AP utilizes as a carbon source. These culture conditions result in significant amounts of CO2 escaping into the atmosphere, instead of being sequestered during cultivation. Here, we investigated whether ambient air (0.042% CO2) can be used for growing AP in a culture medium lacking a fossil-based carbon source. AP was cultured in 2 L glass bioreactors containing: (1) Zarrouk medium with 16.8 g/L NaHCO3 and aeration with 0.236 vvm air with 2% CO2 (“NaHCO3/CO2-based”) to compensate carbon loss due to CO2 outgassing, and (2) Zarrouk medium without NaHCO3 and a gas flow with ambient air (0.926 vvm) as the only carbon source (“air-based”). The air-based production resulted in the biofixation of 3.78 gCO2/L during the linear growth phase. With NaHCO3/CO2-based production, a comparable amount of 3.42 gCO2/L was obtained while 659.12 g of CO2 was released into the atmosphere. Total protein, phycocyanin, chlorophyll-a, and carotenoids were present in similar or increased amounts in AP produced by the air-based method. We concluded that cultivation of AP with Zarrouk medium lacking NaHCO3 but using ambient air with atmospheric CO2 as the only carbon source is possible without reducing productivity. These results improve our understanding of how atmospheric CO2 can be reduced by culturing AP.
Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.
The cyanobacterium Arthrospira platensis is a well-known source of bioactive substances. Growth and the generation of bioactive ingredients of Arthrospira platensis depend mainly on the quantity of light in a controlled environment. Photoinhibition is a time, nutrient, and light intensity-dependent decrease in photosynthetic efficiency. However, too strong illumination can induce two harmful effects: (1) photoinhibition as a reduction in photosynthetic rate and (2) photooxidation which can have lethal effects on the cells, and which can lead to total loss of the culture. The results of this microscopical study demonstrate, that under the procedures described and very high photon flux den-sities, not only a decrease in the photosynthetic efficiency but beyond, also destruction of Arthrospira platensis can occur.