Monoclonal antibodies and ligands targeting CD40 exhibit a wide range of agonistic activities and antitumor responses. Studies have shown that the flexibility and affinity of antibodies play a crucial role in their immunostimulatory activity. However, a systematic comparison with the natural ligand is yet missing and a detailed investigation with respect to molecular rigidity, binding kinetics, and bond lifetime has not been undertaken to date. Here, we study the dynamic binding features of clinically relevant anti-hCD40 antibody subclasses, ChiLob 7/4, and the trimeric human CD40L to hCD40 at the single-molecule level. We visualize resembling of hCD40 receptors into dimers and higher-order oligomers that are dynamically captured and released by both ChiLob 7/4 and hCD40L with their multiple binding sites. Thereby, ChiLob 7/4 acts as a nanomechanical calliper and rotates its Fab arms in a highly dynamic fashion to screen for hCD40 binding, while hCD40L undergoes significantly less conformational changes. Despite its minor molecular flexibility, hCD40L performs association, dissociation, and re-association of hCD40 ten times faster when compared to ChiLob 7/4. We uncover a distinct binding mechanism that may explain the enhanced cluster formation potential and agonistic activity of the natural ligand and will inspire the design of novel ligand formats.

Adipose tissue enlargement in obesity leads to hypoxia, which may promote premature aging. This study aimed to understand the hypoxic response in 3D cultures of SGBS cells, a model for brown-like adipose tissue expressing uncoupling protein 1 (UCP1). Single-nucleus RNA sequencing of SGBS organoids revealed a heterogeneous composition and sub-population-specific responses to hypoxia. The analysis identified a cluster of transcriptional repression, indicating dying cells, and implied a role of ferroptosis in this model. Further experiments with SGBS cells and white adipose tissue-derived stem/progenitor cells showed that Acyl-CoA synthetase long-chain family member 4 (ACSL4), a key enzyme in ferroptosis, is expressed only in the presence of browning factors. Hypoxia downregulated ACSL4 protein in SGBS organoids but induced an inflammaging phenotype. Analysis of brown-like epicardial adipose tissue from cardiac surgery patients revealed a significant positive correlation of ACSL4 mRNA with UCP1 and hypoxia-inducible pro-inflammatory markers, while ACSL4 protein appeared to be inversely correlated. In conclusion, this study demonstrates that adipocytes’ capability to undergo ACSL4-mediated ferroptosis is linked to brown-like adipogenesis, suggesting an opportunity to modulate ferroptotic signaling in adipose tissue. The dual role of hypoxia by inhibiting ACSL4 but promoting inflammaging indicates a relationship between ferroptosis and aging that warrants further investigation.

Aluminum is added to many food colors to change their solubility. This study compares the aluminum-containing food color carmine with its aluminum-free version carminic acid (both E 120), hypothesizing that the addition of aluminum does not only change the color's solubility, but also its effects on human cells. We could show that carmine, but not carminic acid, is taken up by gastrointestinal Caco-2 and umbilical vein endothelial cells (HUVEC). Clear differences between gene expression profiles of Caco-2 cells exposed to carmine, carminic acid or control were shown. KEGG analysis revealed that carmine-specific genes suppress oxidative phosphorylation, and showed that this suppression is associated with neurodegenerative diseases such as Alzheimer and Parkinson disease. Furthermore, carmine, but not carminic acid, increased proliferation of Caco-2 cells. Our findings show that a food color containing aluminum induces different cellular effects compared to its aluminum-free form, which is currently not considered in EU legislation.

Background: Thoracic aortic dissections (TAD) are life-threatening events mostly requiring immediate surgical treatment. Although dissections mainly occur independently of thoracic aortic aneurysms (TAA), both share a high comorbidity. There are several indications for an involvement of the immune system in the development of TAD, just as in TAA. Nevertheless, specific disease-relevant genes, biomolecular processes, and immune-specific phenotypes remain unknown. Methods: RNA from isolated aortic smooth muscle cells from TAD (n = 4), TAA (n = 3), and control patients were analyzed using microarray-based technologies. Additionally, three publicly available bulk RNA-seq studies of TAD (n = 23) and controls (n = 17) and one single-cell RNA-seq study of TAA (n = 8) and controls (n = 3) were analyzed. Differentially expressed genes were identified and used to identify affected pathways in TAD. Five selected genes were validated by quantitative real-time polymerase chain reaction (PCR). Results: We identified 37 genes that were significantly dysregulated in at least three TAD studies—24 of them were not shown to be associated with TAD, yet. Gene ontology analysis showed that immune response was significantly affected. Five of the genes (CCL2, RNASE2, HAVCR2, CXCL8, and IL6R) were revealed as core genes that affect immune response in TAD. We compared the gene expression of those genes to TAA and found that CXCL8, IL6R, and potentially also CCL2 were upregulated in TAD. Conclusions: The identified immune-related genes showed TAD-specificity, independent of possible pre-existing comorbidities like TAA. So, these genes represent potential biomarkers and therapeutic targets linked to the immune response in acute TAD. Additionally, we identified a set of differentially expressed genes that represents a resource for further studies.

Aluminum (Al) is the most abundant metal in the earth's crust, and humans are exposed to Al through sources like food, cosmetics, and medication. So far, no comprehensive data on the Al distribution between and within human tissues were reported. We measured Al concentrations in 24 different tissue types of 8 autopsied patients using ICP-MS/MS (inductively coupled plasma-tandem mass spectrometry) under cleanroom conditions and found surprisingly high concentrations in both the upper and inferior lobes of the lung and hilar lymph nodes. Al/Si ratios in lung and hilar lymph node samples of 12 additional patients were similar to the ratios reported in urban fine dust. Histological analyses using lumogallion staining showed Al in lung erythrocytes and macrophages, indicating the uptake of airborne Al in the bloodstream. Furthermore, Al was continuously found in PM2.5 and PM10 fine dust particles over 7 years in Upper Austria, Austria. According to our findings, air pollution needs to be reconsidered as a major Al source for humans and the environment.

Essential trace elements play an important role in human physiology and are associated with various functions regulating cellular metabolism. Non-essential trace elements, on the other hand, often have well-documented toxicities that are dangerous for the initiation and development of diseases due to their widespread occurrence in the environment and their accumulation in living organisms. Non-essential trace elements are therefore regarded as serious environmental hazards that are harmful to health even in low concentrations. Many representatives of these elements are present as pollutants in our environment, and many people may be exposed to significant amounts of these substances over the course of their lives. Among the most common non-essential trace elements are heavy metals, which are also associated with acute poisoning in humans. When these elements accumulate in the body over years of chronic exposure, they often cause severe health damage in a variety of tissues and organs. In this review article, the role of selected essential and non-essential trace elements and their role in the development of exemplary pathophysiological processes in the cardiovascular system will be examined in more detail.

Pathogenic mechanisms in degenerative thoracic aortic aneurysms (TAA) are still unclear. There is an ongoing debate about whether TAAs are caused by uniform or distinct processes, which would obviously have a major impact on future treatment strategies. Clearly, the ultimate outcome of TAA subgroups associated with a tricuspid aortic valve (TAV) or a bicuspid aortic valve (BAV) is the same, namely a TAA. Based on results from our own and others' studies, we decided to compare the different TAAs (TAV and BAV) and controls using a broad array of analyses, i.e., metabolomic analyses, gene expression profiling, protein expression analyses, histological characterization, and matrix-assisted laser desorption ionization imaging. Central findings of the present study are the presence of noncanonical atherosclerosis, pathological accumulation of macrophages, and disturbances of lipid metabolism in the aortic media. Moreover, we have also found that lipid metabolism is impaired systemically. Importantly, all of the above-described phenotypes are characteristic for TAV-TAA only, and not for BAV-TAA. In summary, our results suggest different modes of pathogenesis in TAV- and BAV-associated aneurysms. Intimal atherosclerotic changes play a more central role in TAV-TAA formation than previously thought, particularly as the observed alterations do not follow classical patterns. Atherosclerotic alterations are not limited to the intima but also affect and alter the TAV-TAA media. Further studies are needed to i) clarify patho-relevant intima-media interconnections, ii) define the origin of the systemic alteration of lipid metabolism, and iii) to define valid biomarkers for early diagnosis, disease progression, and successful treatments in TAV-TAAs.

Already at the very beginning of the COVID-19 pandemic, an extensive PCR and antigen testing strategy was considered necessary and subsequently also proved successful in order to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on international and national levels. However, equally important will be the continuous monitoring of the seroprevalence status of populations from defined regions to detect-in a timely manner-any recurrence of infections or an eventual decline in antibody levels of vaccinated individuals, especially in the emerging post-pandemic situation. The aim of this study was to estimate the prevalence of SARS-CoV-2-specific immunoglobulin G antibodies in the federal state of Upper Austria (Austria) during the period of December 2020 until April 2021. To achieve this goal, we have analyzed anonymized data on the immune status of self-referral volunteers that have been determined at local pharmacies through a low-entry-barrier point-of-care analysis approach. The seroprevalence values for immunoglobulin type G antibodies against SARS-CoV-2 antigens obtained by rapid diagnostic testing on peripheral blood from volunteers reflect the current population-based estimates reported in the literature as well as the positivity rates detected by PCR-screening analyses. In conclusion, broad-based monitoring of IgG antibodies by means of a point-of-care testing network represents a valuable tool to assess the current immune situation within regionally defined populations.

Background: There is much debate about the role of schools and children in the SARS-CoV-2 pandemic. We aimed to quantify reliably the prevalence of SARS-CoV-2 infections at schools detected with reverse-transcription polymerase-chain-reaction (RT-PCR). Methods: This nationwide prospective cohort study monitors a representative sample of pupils (grade 1-8) and teachers at Austrian schools throughout the school year 2020/2021. We repeatedly test participants for SARS-CoV-2 infection using a gargling solution and RT-PCR. We herein report on the first two rounds of examinations. We used mixed-effect logistic regression to estimate odds ratios and robust 95% confidence intervals (95% CI). Findings: We analysed data on 10734 participants from 245 schools (9465 pupils, 1269 teachers). Prevalence of RT-PCR-detected SARS-CoV-2 infection increased from 0.39% at round 1 (95% CI 0.28-0.55%, 29 September-22 October 2020) to 1.42% at round 2 (95% CI 1.06-1.90%, 10-16 November). Odds ratios for SARS-CoV-2 infection were 2.29 (95% CI 1.26-4.17, P=0.007) in regions with >500 vs. [≤]500 inhabitants/km2, 1.69 (95% CI 1.42-2.00, P<0.001) for a two-fold higher regional 7-day incidence, and 2.71 (95% CI 1.68-4.39, P<0.001) in pupils at schools with a high/very high vs. low/moderate social deprivation index. Associations of community incidence and social deprivation persisted in multivariable models. There were no differences between age groups, sexes, pupils vs. teachers, or primary (grade 1-4) vs. secondary schools (grade 5-8). Interpretation: This monitoring study in Austrian schools revealed SARS-CoV-2 infection in 0.39%-1.42% of participants and identified associations of regional community incidence and social deprivation with higher prevalence.

This study evaluates the performance of the antigen-based anterior nasal screening programme implemented in all Austrian schools to detect SARS-CoV-2 infections. We combined nationwide antigen-based screening data obtained in March 2021 from 5,370 schools (Grade 1-8) with an RT-qPCR-based prospective cohort study comprising a representative sample of 244 schools. Considering a range of assumptions, only a subset of infected individuals are detected with the programme (low to moderate sensitivity) and non-infected individuals mainly tested negative (very high specificity).

Stinging nettle is appreciated for its antioxidant and anti-inflammatory properties, which renders the plant a popular ingredient in a healthy diet in form of salads or smoothies. The most common use, presumably, is of dried leaves as ingredient in tea mixtures. The plant's health benefits are attributed primarily to phenolic phytochemicals. Here we describe the characterization and quantification of a phylloxanthobilin (PxB), a yellow chlorophyll catabolite, in nettle tea. Despite their abundance in the plant kingdom, chlorophyll catabolites have been overlooked as phytochemicals and as part of human nutrition. Our investigations of tea reveal that one cup of nettle tea contains about 50 mu g of PxB with large variations depending on the supplier. When investigating the bioactivities of PxB, our observations show that PxB has antioxidative and anti-inflammatory activities comparable to known bioactive small molecules found in nettle, indicating the phylloxanthobilin to be an overlooked ingredient of nettle tea.

Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastatic patients. In vitro, miR-486-5p was down-regulated in CSC models and it induced an inhibitory effect on stem factors and oncogenes in the main pathways of CSCs. Our results provide a step forward in understanding the role of mir-486-5p in CRC and CSC, and suggest that further studies are needed to investigate its diagnostic and prognostic power, possibly in combination with other biomarkers.

All-trans-retinoic acid (atRA) is the essential derivative of vitamin A and is of interest due to its various biological key functions. As shown in the recent literature, atRA also plays a role in the failing heart during myocardial infarction, the leading cause of death globally. To date insufficient mechanistic information has been available on related hypoxia-induced cell damage and reperfusion injuries. However, it has been demonstrated that a reduction in cellular atRA uptake abrogates hypoxia-mediated cell and tissue damage, which may offer a new route for intervention. Consequently, in this study, the effect of the novel cardio-protective compound 5-methoxyleoligin (5ML) on cellular atRA uptake was tested in human umbilical-vein endothelial cells (HUVECs). For this purpose, a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to assess intra-cellular levels of the active substance and corresponding levels of vitamin A and its derivatives, including potential cis/trans isomers. This work also focused on light-induced isomerization and the stability of biological sample material to ensure sample integrity and avoid biased conclusions. This study provides evidence of the inhibitory effect of 5ML on cellular atRA uptake, a promising step toward a novel therapy for myocardial infarction.

Background: Serological assays for the determination of the immune status of patients that have tested positive for infection with SARS-CoV-2 by RT-PCR are required for, e.g., contact tracing and epidemiological studies. However, data concerning the performance parameters of commercially available high-throughput ELISA tests are still not available on a large scale. Study design: In our study, we have evaluated an in-house developed ELISA for the detection of the immunoglobulin classes A, G and M directed against the full-length spike glycopmtein from SARS-CoV-2. For this analysis, we have included 110 sera from patients presenting with COVID-19 symptoms or blood donors without symptoms collected at the Austrian Red Cross, Blood Transfusion Service for Upper Austria, Linz. In addition, we have selected four commercially available IgG-based ELISAs as well as one IgA/IgG-based ELISA for the detection of SARS-CoV-2 antigens as well as a multiplexed IgG-based micro-ELISA assay developed for rapid Point of Care testing applications. Conclusions: All assays evaluated in the course of this study demonstrated suitable sensitivity and specificity values for the identification of patients that have experienced a past infection with SARS-CoV-2. However, testing for the presence of additional immunoglobulins (IgA and IgM) as well as using combinations of different viral antigens is highly advised to improve the predictive values of serological assays.

Adipose and muscle tissue – so far underestimated – contribute to the total Cd burden. Cd distribution in testis and kidney correlates with the histologic blood-testis-barrier and renal excretion, respectively.

BACKGROUND:Tylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions.HYPOTHESIS/PURPOSE:Our goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways.STUDY DESIGN AND METHODS:TYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis.RESULTS:TYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia.CONCLUSION:This unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.