Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.

Celiac disease (CD) is a chronic inflammatory condition caused by the ingestion of gliadin-containing food in genetically susceptible individuals. Undigested peptides of gliadin exert various effects, including increased intestinal permeability and inflammation in the small intestine. Although many therapeutic approaches are in development, a gluten-free diet is the only effective treatment for CD. Affecting at least 1% of the population in industrialized countries, it is important to generate therapeutic options against CD. Here, we describe the establishment of a high-throughput screening (HTS) platform based on AlphaLISA and electrical cell-substrate impedance sensing (ECIS) technology for the identification of anti-inflammatory and barrier-protective compounds in human enterocytes after pepsin-trypsin-digested gliadin (PT-gliadin) treatment. Our results show that the combination of these HTS technologies enables fast, reliable, simple, and label-free screening of IgY antibodies against PT-gliadin. Using this platform, we have identified a new chicken anti-PT-gliadin IgY antibody as a potential anti-CD agent.

Melanoma is the most aggressive type of skin cancer and one of the most frequent tumours in young adults. Identification of primary tumours prone to develop metastasis is of paramount importance for further patient stratification. However, till today, no markers exist that are routinely used to predict melanoma progression. To ameliorate this problem, we generated antiserum directed against metastatic melanoma tissue lysate and applied a novel approach to purify the obtained serum via consecutive affinity chromatography steps. The established antibody, termed MHA-3, showed high reactivity against metastatic melanoma cell lines both in vitro and in vivo. We also tested MHA-3 on 227 melanoma patient samples and compared staining with the melanoma marker S100b. Importantly, MHA-3 was able to differentiate between metastatic and non-metastatic melanoma samples. By proteome analysis we identified 18 distinct antigens bound by MHA-3. Combined expression profiling of all identified proteins revealed a significant survival difference in melanoma patients. In conclusion, we developed a polyclonal antibody, which is able to detect metastatic melanoma on paraffin embedded sections. Hence, we propose that this antibody will represent a valuable additional tool for precise melanoma diagnosis.

Modern society is heavily dependent on critical raw materials, such as rare earth elements (REEs), for use in electronic devices. The increasing demand for these materials has led to the need for environmentally friendly methods of processing non-recycled materials from e-waste and wastewater, as well as waste streams from cleaning and manufacturing facilities. Modern society’s dependence on such materials is growing by the day, and with it, the need for environmentally friendly processing of non-recycled materials from e-waste and wastewater in the form of “end-of-life” products, as well as waste streams from cleaning and manufacturing facilities, also increases. As these are problematic indications for modern isolation methods in the industry, these sources may be more suitable for new techniques as they have low concentration and high throughput for bioaccumulation. Chemical methods using nanomaterials are already being tested for their possibilities but still depend on acids and harsh chemicals. Microorganisms, on the other hand, can adsorb/absorb REEs in a more ecological way. Previous studies could already show that it is possible to accumulate REEs in the precipitates of bacterial cultures spiked with REEs to a value of over 50%. However, the question arose whether rare earths were spun into the pellets by centrifugation, adsorbed, or really incorporated in the cells. Therefore, we established a new easy-to-use experimental design in which the microorganisms were spiked with an REE standard and washed to minimize the falsification of measurements by peripheral binding of ions before being analyzed for REE contents by ICP-OES. The bioaccumulation of rare earths in microorganisms was monitored, yielding an uptake rate of up to 53.12% of the overall present ionic REE concentration. In this manuscript, we present the different concentration measurements that were taken during the process, before and after washing of the cells, to create a full picture of the localization, binding, incorporation, and occurrence of the ions of interest. The setup also showed a correlation between the introduction method of rare earths and the uptake of certain elements that might be correlated with the differentiation between light and heavy rare earth elements, while Y and Sc often seem to represent outliers.