Evaluation of novel Epstein-Barr virus-derived antigen formulations for monitoring virus-specific T cells in pediatric patients with infectious mononucleosis.

BACKGROUND: Infection with the Epstein-Barr virus (EBV) elicits a complex T-cell response against a broad range of viral proteins. Hence, identifying potential differences in the cellular immune response of patients with different EBV-associated diseases or different courses of the same disorder requires interrogation of a maximum number of EBV antigens. Here, we tested three novel EBV-derived antigen formulations for their ability to reactivate virus-specific T cells ex vivo in patients with EBV-associated infectious mononucleosis (IM). METHODS: We comparatively analyzed EBV-specific CD4+ and CD8+ T-cell responses to three EBV-derived antigen formulations in 20 pediatric patients during the early phase of IM: T-activated EBV proteins (BZLF1, EBNA3A) and EBV-like particles (EB-VLP), both able to induce CD4+ and CD8+ T-cell responses ex vivo, as well as an EBV-derived peptide pool (PP) covering 94 well-characterized CD8+ T-cell epitopes. We assessed the specificity, magnitude, kinetics, and functional characteristics of EBV-specific immune responses at two sequential time points (v1 and v2) within the first six weeks after IM symptom onset (Tonset). RESULTS: All three tested EBV-derived antigen formulations enabled the detection of EBV-reactive T cells during the early phase of IM without prior T-cell expansion in vitro. EBV-reactive CD4+ and CD8+ T cells were mainly mono-functional (CD4+: mean 64.92%, range 56.15-71.71%; CD8+: mean 58.55%, range 11.79-85.22%) within the first two weeks after symptom onset (v1) with IFN-γ and TNF-secreting cells representing the majority of mono-functional EBV-reactive T cells. By contrast, PP-reactive CD8+ T cells were primarily bi-functional (>60% at v1 and v2), produced IFN-γ and TNF and had more tri-functional than mono-functional components. We observed a moderate correlation between viral load and EBNA3A, EB-VLP, and PP-reactive CD8+ T cells (rs = 0.345, 0.418, and 0.356, respectively) within the first two weeks after Tonset, but no correlation with the number of detectable EBV-reactive CD4+ T cells. CONCLUSIONS: All three EBV-derived antigen formulations represent innovative and generic recall antigens suitable for monitoring EBV-specific T-cell responses ex vivo. Their combined use facilitates a thorough analysis of EBV-specific T-cell immunity and allows the identification of functional T-cell signatures linked to disease development and severity.

Role of Epigenetics for the Efficacy of Cisplatin.

The clinical utility of the chemotherapeutic agent cisplatin is restricted by cancer drug resistance, which is either intrinsic to the tumor or acquired during therapy. Epigenetics is increasingly recognized as a factor contributing to cisplatin resistance and hence influences drug efficacy and clinical outcomes. In particular, epigenetics regulates gene expression without changing the DNA sequence. Common types of epigenetic modifications linked to chemoresistance are DNA methylation, histone modification, and non-coding RNAs. This review provides an overview of the current findings of various epigenetic modifications related to cisplatin efficacy in cell lines in vitro and in clinical tumor samples. Furthermore, it discusses whether epigenetic alterations might be used as predictors of the platinum agent response in order to prevent avoidable side effects in patients with resistant malignancies. In addition, epigenetic targeting therapies are described as a possible strategy to render cancer cells more susceptible to platinum drugs.

Chromate Affects Gene Expression and DNA Methylation in Long-Term In Vitro Experiments in A549 Cells.

Chromate has been shown to dysregulate epigenetic mechanisms such as DNA methylation, leading to changes in gene expression and genomic instability. However, most in vitro studies are limited to short incubation periods, although chronic exposure may be more relevant for both environmental and occupational exposure. In this study, human adenocarcinoma A549 cells were treated with 1, 2 or 5 µM chromate for 24 h and compared with incubations with 0.2, 0.5 or 1 µM chromate for 1 to 5 weeks. Chromium accumulated in a pronounced time- and concentration-dependent manner after short-term treatment, whereas a plateau of intracellular chromium content was observed after long-term treatment. While short-term treatment induced a G2 arrest of the cell cycle, this effect was not observed after long-term treatment at lower concentrations. The opposite was observed for global DNA methylation: while short-term treatment showed no effect of chromate, significant dose-dependent hypomethylation was observed in the long-term experiments. Time-dependent effects were also observed in a high-throughput RT-qPCR gene expression analysis, particularly in genes related to the inflammatory response and DNA damage response. Taken together, the results suggest specific differences in toxicity profiles when comparing short-term and long-term exposure to chromate in A549 cells.

Effect of Long-Term Low-Dose Arsenic Exposure on DNA Methylation and Gene Expression in Human Liver Cells.

Millions of people around the world are exposed to elevated levels of arsenic through food or drinking water. Epidemiological studies have linked chronic arsenic exposure to an increased risk of several cancers, cardiovascular disease, central nervous system neuropathies, and genotoxic as well as immunotoxic effects. In addition to the induction of oxidative stress and inhibition of DNA repair processes, epigenetic effects, including altered DNA methylation patterns resulting in aberrant gene expression, may contribute to carcinogenicity. However, the underlying mechanisms by which chronic micromolar concentrations of arsenite affect the methylation status of DNA are not fully understood. In this study, human HepG2 hepatocarcinoma cells were treated with 0.5-10 µM sodium arsenite for 24 h, 10, or 20 days. During these periods, the effects on global DNA methylation, cell cycle phase distribution, and gene expression were investigated. While no impact on DNA methylation was seen after short-term exposure, global hypomethylation was observed at both long-term exposure periods, with concomitant induction of the DNA methyltransferase genes DNMT1 and DNMT3B, while DNMT3A was slightly down-regulated. Pronounced time- and concentration-dependent effects were also seen in the case of genes involved in DNA damage response and repair, inflammation, oxidative stress response, and metal homeostasis. These results suggest that chronic low-dose arsenite exposure can lead to global hypomethylation. As an underlying mechanism, the consistent down-regulation of DNA methyltransferase genes could be excluded; alternatively, interactions at the protein level could play an important role.

[The Interdisciplinary Family Consultation in Pediatrics - An Integrative Approach to Complex Burdened Families of Chronically ill Children]. / Die Interdisziplinäre Familienmedizinische Sprechstunde in der Kinderheilkunde ­ Ein integrativer Ansatz für komplex belastete Familien mit einem chronisch kranken Kind.

For families of chronically ill children with a high level of psychosocial stress, it is necessary to involve child and adolescent psychiatric or psychosomatic specialists directly in the pediatric treatment process. For this purpose, a family consultation was set up in the Heidelberg University Pediatrics as part of a model project, which deals with these families in an interdisciplinary and systemic way. It shows that the implementation of the consultation works despite a high organizational effort and is regularly used. The first evaluation results confirm the already described high levels of psychosocial stress in the affected families. They indicate a positive assessment of the consultation and a desire for a permanent offer. First follow-up results indicate a significant decrease in treatment-related concerns, uncertainty and a reduction in treatment-related problems. The lack of a viable funding model poses a challenge for long-term implementation.

Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures.

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

[Psychosomatic-psychotherapeutic support for Covid-19 patients in domestic quarantine: a content analysis of supportive telephone calls]. / Psychosomatisch-psychotherapeutische Unterstützung von Covid-19-Patient:innen in häuslicher Quarantäne: Eine Inhaltsanalyse supportiver Telefongespräche.

Objectives: During their domestic quarantine, Covid-19 patients face major physical, psychological and social challenges. The description of support needs and specific topics brought to supportive conversations will be used to add to the body of knowledge about stressors and resources. Methods: A total of 109 telephone conversations with 69 quarantined Corona patients were documented by psychotherapists and physicians at Heidelberg University Hospital from November 2020 to April 2021. Subsequently, clinical documentations were analyzed according to a qualitative content analysis. Results: Most physical complaints related to cardio-respiratory symptoms (29 %), previous illnesses (24 %), and exhaustion or fatigue (16 %). On the psychological level, patients reported mainly anxiety (31 %) and depressive symptoms (16 %). On a social level, patients described stress related to family (56 %), work (20 %), and time in quarantine (16 %). Social support, individual coping strategies, a positive prognosis on the course of the corona disease, psychotherapy, and satisfactory medical care were mentioned as relieving factors. Therapeutic interventions aimed at stabilization and consisted of psychoeducation, relaxation techniques, and general counseling. Conclusions: The study shows that physical complaints, psychological symptoms, and social factors are brought into telephone support conversations. The support offer met a high demand and was well accepted.

Analysis of microplastics in drinking water and other clean water samples with micro-Raman and micro-infrared spectroscopy: minimum requirements and best practice guidelines.

Microplastics are a widespread contaminant found not only in various natural habitats but also in drinking waters. With spectroscopic methods, the polymer type, number, size, and size distribution as well as the shape of microplastic particles in waters can be determined, which is of great relevance to toxicological studies. Methods used in studies so far show a huge diversity regarding experimental setups and often a lack of certain quality assurance aspects. To overcome these problems, this critical review and consensus paper of 12 European analytical laboratories and institutions, dealing with microplastic particle identification and quantification with spectroscopic methods, gives guidance toward harmonized microplastic particle analysis in clean waters. The aims of this paper are to (i) improve the reliability of microplastic analysis, (ii) facilitate and improve the planning of sample preparation and microplastic detection, and (iii) provide a better understanding regarding the evaluation of already existing studies. With these aims, we hope to make an important step toward harmonization of microplastic particle analysis in clean water samples and, thus, allow the comparability of results obtained in different studies by using similar or harmonized methods. Clean water samples, for the purpose of this paper, are considered to comprise all water samples with low matrix content, in particular drinking, tap, and bottled water, but also other water types such as clean freshwater.

Tunable Fluorescence of a Semiconducting Polythiophene Positioned on DNA Origami.

A novel approach for the integration of π-conjugated polymers (CPs) into DNA-based nanostructures is presented. Using the controlled Kumada catalyst-transfer polycondensation, well-defined thiophene-based polymers with controllable molecular weight, specific end groups, and water-soluble oligoethylene glycol-based side chains were synthesized. The end groups were used for the easy but highly efficient click chemistry-based attachment of end-functionalized oligodeoxynucleotides (ODNs) with predesigned sequences. As demonstrated by surface plasmon resonance spectroscopy, the prepared block copolymers (BCPs), P3(EO)3T-b-ODN, comprising different ODN lengths and specific or repetitive sequences, undergo specific hybridization with complementary, thiol-functionalized ODNs immobilized on a gold surface. Furthermore, the site-specific attachment of the BCPs to DNA origami structures is studied. We demonstrate that a nanoscale object, that is, a single BCP with a single ODN handle, can be directed and bound to the DNA origami with reasonable yield, site-specificity, and high spatial density. On the basis of these results, we are able to demonstrate for the first time that optical properties of CP molecules densely immobilized on DNA origami can be locally fine-tuned by controlling the attractive π-π-stacking interactions between the CPs. In particular, we show that the fluorescence of the immobilized CP molecules can be significantly enhanced by surfactant-induced breakup of π-π-stacking interactions between the CP's backbones. Such molecular control over the emission intensity of the CPs can be valuable for the construction of sophisticated switchable nanophotonic devices and nanoscale biosensors.

Mechanochemical Knoevenagel condensation investigated in situ.

The mechanochemical Knoevenagel condensation of malononitrile with p-nitrobenzaldehyde was studied in situ using a tandem approach. X-ray diffraction and Raman spectroscopy were combined to yield time-resolved information on the milling process. Under solvent-free conditions, the reaction leads to a quantitative conversion to p-nitrobenzylidenemalononitrile within 50 minutes. The in situ data indicate that the process is fast and proceeds under a direct conversion. After stopping the milling process, the reaction continues until complete conversion. The continuous and the stopped milling process both result in crystalline products suitable for single crystal X-ray diffraction.

Quantitative determination of activation energies in mechanochemical reactions.

Mechanochemical reactions often result in 100% yields of single products, making purifying procedures obsolete. Mechanochemistry is also a sustainable and eco-friendly method. The ever increasing interest in this method is contrasted by a lack in mechanistic understanding of the mechanochemical reactivity and selectivity. Recent in situ investigations provided direct insight into formation pathways. However, the currently available theories do not predict temperature T as an influential factor. Here, we report the first determination of an apparent activation energy for a mechanochemical reaction. In a temperature-dependent in situ study the cocrystallisation of ibuprofen and nicotinamide was investigated as a model system. These experiments provide a pivotal step towards a comprehensive understanding of milling reaction mechanisms.

Clinical experience with eplerenone to treat chronic central serous chorioretinopathy.

PURPOSE: Chronic central serous chorioretinopathy (CSC) is a vision-threatening eye disease for which there is still no approved treatment. Recent studies suggest that the corticosteroid pathway in the choroid is implicated in CSC pathogenesis, and that therapy with the aldosterone antagonist eplerenone improves clinical outcomes. However, there is still little clinical data to support this hypothesis. We performed a retrospective chart review to further investigate the clinical value of eplerenone treatment in patients with chronic CSC and to identify possible response predictors. METHODS: Twenty-four patients with chronic CSC resistant to conventional therapy over at least 4 months were included in this retrospective study. Patients were initially treated with 25 mg/day of eplerenone administered orally for 1 week, followed by a sustained daily dose of 50 mg. The primary outcome measure was percentage of eyes achieving complete resolution of subretinal fluid (SRF), recorded by spectral domain optical coherence tomography (SD-OCT). Secondary outcomes included changes in central macular thickness (CMT) and best-corrected visual acuity (BCVA). Baseline SD-OCT images were also evaluated as possible predictors of treatment response. RESULTS: Twenty-nine percent of patients experienced complete resolution of SRF after a median of 106 days of treatment, while 33 % of patients showed a transient initial decrease in SRF, and 25 % failed to respond to treatment. Treatment had to be stopped in 13 % of patients because of adverse effects of the eplerenone treatment. In the study population, CMT decreased from 342 to 275 µm after treatment, which was associated with a modest improvement in mean BCVA from 0.35 to 0.3 logMar. The integrity of the ellipsoid zone and the retinal pigment epithelium (RPE) at baseline were associated with a tendency towards a favourable visual outcome. CONCLUSION: This study confirms the proposed clinical value of eplerenone for treating patients with therapy-resistant CSC. However, patients presenting widespread RPE changes are less likely to benefit from eplerenone treatment, which may argue for an earlier intervention. Larger studies are needed to characterise patient subgroups that may benefit the most from eplerenone treatment.

Mechanochemically Induced Conversion of Crystalline Benzamide Polymorphs by Seeding.

Benzamide has been known for its polymorphism for almost 200 years. Three polymorphic forms are described. To date, it was only possible to crystallize a metastable form in a mixture together with the thermodynamically most stable form I. A complete transformation of form I into the metastable form III by mechanochemical treatment has been achieved. Catalytic amounts of nicotinamide seeds were used to activate the conversion by mechanochemical seeding. NMR experiments indicated that the nicotinamide molecules were incorporated statistically in the crystal lattice of benzamide form III during the conversion. The transformation pathway was evaluated using in situ powder X-ray diffraction.

Survival of the Fittest: Competitive Co-crystal Reactions in the Ball Mill.

The driving forces triggering the formation of co-crystals under milling conditions were investigated by using a set of multicomponent competitive milling reactions. In these reactions, different active pharmaceutical ingredients were ground together with a further compound acting as coformer. The study was based on new co-crystals including the coformer anthranilic acid. The results of the competitive milling reactions indicate that the formation of co-crystals driven by intermolecular recognition are influenced and inhibited by kinetic aspects including the formation of intermediates and the stability of the reactants.

Direct in†situ investigation of milling reactions using combined X-ray diffraction and Raman spectroscopy.

The combination of two analytical methods including time-resolved in situ X-ray diffraction (XRD) and Raman spectroscopy provides a new opportunity for a detailed analysis of the key mechanisms of milling reactions. To prove the general applicability of our setup, we investigated the mechanochemical synthesis of four archetypical model compounds, ranging from 3D frameworks through layered structures to organic molecular compounds. The reaction mechanism for each model compound could be elucidated. The results clearly show the unique advantage of the combination of XRD and Raman spectroscopy because of the different information content and dynamic range of both individual methods. The specific combination allows to study milling processes comprehensively on the level of the molecular and crystalline structures and thus obtaining reliable data for mechanistic studies.

Interference of mismatch and base excision repair during the processing of adjacent U/G mispairs may play a key role in somatic hypermutation.

In eukaryotic mismatch repair (MMR), degradation of the error-containing strand initiates at nicks or gaps that can be up to a kilobase away from the mispair. These discontinuities may be the ends of Okazaki fragments or the 3'-termini of the leading strands during replication, whereas the termini of invading strands may fulfill this role during recombination. Here we show that, in extracts of human cells, MMR can initiate also at sites of ongoing base excision repair. Although unlikely under normal circumstances, this situation may arise in vivo during somatic hypermutation (SHM) and class switch recombination of Ig genes, where activation-induced cytidine deaminase (AID) generates multiple U/G mismatches in the variable or switch regions. Uracil should normally be excised by base excision repair (BER), but we show here that MMR proteins activated by a nearby mismatch interfere with uracil processing to generate long single-stranded gaps. We postulate that, in a subset of the repair events, filling-in of the MMR-generated gaps might be catalyzed by the error-prone polymerase-eta, rather than by the high-fidelity polymerase-delta. Because polymerase-eta has a propensity to misinsertions opposite adenine residues, the above mechanism would help explain why SHM affects not only C/G, but also A/T base pairs.

Impact of Nano- and Micro-Sized Chromium(III) Particles on Cytotoxicity and Gene Expression Profiles Related to Genomic Stability in Human Keratinocytes and Alveolar Epithelial Cells.

Exposure to Cr(VI) compounds has been consistently associated with genotoxicity and carcinogenicity, whereas Cr(III) is far less toxic, due to its poor cellular uptake. However, contradictory results have been published in relation to particulate Cr2O3. The aim of the present study was to investigate whether Cr(III) particles exerted properties comparable to water soluble Cr(III) or to Cr(VI), including two nano-sized and one micro-sized particles. The morphology and size distribution were determined by TEM, while the oxidation state was analyzed by XPS. Chromium release was quantified via AAS, and colorimetrically differentiated between Cr(VI) and Cr(III). Furthermore, the toxicological fingerprints of the Cr2O3 particles were established using high-throughput RT-qPCR and then compared to water-soluble Cr(VI) and Cr(III) in A549 and HaCaT cells. Regarding the Cr2O3 particles, two out of three exerted only minor or no toxicity, and the gene expression profiles were comparable to Cr(III). However, one particle under investigation released considerable amounts of Cr(VI), and also resembled the toxicity profiles of Cr(VI); this was also evident in the altered gene expression related to DNA damage signaling, oxidative stress response, inflammation, and cell death pathways. Even though the highest toxicity was found in the case of the smallest particle, size did not appear to be the decisive parameter, but rather the purity of the Cr(III) particles with respect to Cr(VI) content.

Agricultural application of microplastic-rich sewage sludge leads to further uncontrolled contamination.

Municipal sewage sludge has been shown to be high in microplastics (MP) and is applied to agricultural land as fertiliser in many countries. The authors recently proposed in a viewpoint article that MP applied to land in this way may well contaminate other areas in an uncontrolled way. This study examined experimental plots with known history of application of sewage sludge. Results showed that 44% of the MP load found on sludge-applied land was found on nearby land never directly applied with sludge. Examination of polymer type compositions demonstrated marked similarity between the two fields indicating the sludge-applied field was a source of contamination for surrounding areas. Furthermore, MP was detected at a depth of 60-90 cm in the sludge-applied soil indicating that MP may also penetrate deep enough to reach agricultural drainage systems, although this effect is slight (1.6% of surface load). These results show that application of municipal sewage sludge on agricultural land can lead to further uncontrolled contamination, paving the way for future research to improve understanding of the extents of such effects on real farms to better inform future agricultural policy.

Airborne microplastic concentrations and deposition across the Weser River catchment.

Microplastic (MP) appears to be omnipresent in the atmosphere, raising concerns about dispersion across environmental compartments, ecological consequences and human health risks by inhalation. To date, data on the sources of atmospheric MP and deposition to river catchment areas are still sparse. We, therefore, took aerosol and total atmospheric deposition samples in the catchment area of the large German river Weser to estimate microplastic deposition fluxes (DFs) at six specific sites and airborne MP concentrations. Sampling in rural, suburban, and urban environments and wastewater treatment plants (WWTPs) was performed, aiming at a variation in airborne MP pollution and elucidating potential MP source areas. Aerosol samples were taken twice in April and October while monthly total deposition samples were collected over a period from March to October. Microplastics were detected in all analysed aerosol samples by Raman spectroscopy down to 4 µm, and in all 32 total deposition samples by µFT-IR down to 11 µm. Average MP number concentrations of 91 ± 47 m-3 were found in aerosol samples. The measured total MP number DFs ranged between 10 and 367 N m-2 day-1 (99 ± 85 mean ± SD) corresponding to total deposition of 0.05 ± 0.1 kg ha-1 per year and to an estimated 232 metric tons of plastic being deposited in the Weser River catchment annually. MP number DFs were higher in urban than rural sites. An effect of WWTPs on the MP abundance in air was not observed. Polypropylene, polyethylene, polyethylene terephthalate, polyvinyl chloride, polystyrene, and silicone fragments were found as the predominant polymer types in total deposition samples, while polyethylene particles dominated in aerosol samples. The results suggest that proximity to sources, especially to cities, increase the numbers of MP found in the atmosphere. It further indicates that atmospheric MP considerably contributes to the contamination of both aquatic and terrestrial habitats.

Activation of retinal microglia rather than microglial cell density correlates with retinal neovascularization in the mouse model of oxygen-induced retinopathy.

BACKGROUND: Retinal neovascularization has been intensively investigated in the mouse model of oxygen-induced retinopathy (OIR). Here, we studied the contribution of microglial cells to vascular regression during the hyperoxic phase and to retinal neovascularization during the hypoxic phase. METHODS: Mice expressing green fluorescent protein (GFP) under the Cx3cr1 promoter labeling microglial cells were kept in 75% oxygen from postnatal day 7 (P7) to P12. Microglial cell density was quantified at different time points and at different retinal positions in retinal flat mounts. Microglial activation was determined by the switch from ramified to amoeboid cell morphology which correlated with the switch from lectin negative to lectin positive staining of GFP positive cells. RESULTS: Microglial cell density was constant in the peripheral region of the retina. In the deep vascular layer of the central region, however, it declined 14 fold from P12 to P14 and recovered afterwards. Activated microglial cells were found in the superficial layer of the central avascular zone from P8 to P12 and from P16 to P18. In addition, hyalocytes were found in the vitreal layer in the central region and their cell density decreased over time. CONCLUSION: Density of microglial cells does not correlate with vascular obliteration or revascularization. But the time course of the activation of microglia indicates that they may be involved in retinal neovascularization during the hypoxic phase.