Insights to HIV-1 coreceptor usage by estimating HLA adaptation with Bayesian generalized linear mixed models.

The mechanisms triggering the human immunodeficiency virus type I (HIV-1) to switch the coreceptor usage from CCR5 to CXCR4 during the course of infection are not entirely understood. While low CD4+ T cell counts are associated with CXCR4 usage, a predominance of CXCR4 usage with still high CD4+ T cell counts remains puzzling. Here, we explore the hypothesis that viral adaptation to the human leukocyte antigen (HLA) complex, especially to the HLA class II alleles, contributes to the coreceptor switch. To this end, we sequence the viral gag and env protein with corresponding HLA class I and II alleles of a new cohort of 312 treatment-naive, subtype C, chronically-infected HIV-1 patients from South Africa. To estimate HLA adaptation, we develop a novel computational approach using Bayesian generalized linear mixed models (GLMMs). Our model allows to consider the entire HLA repertoire without restricting the model to pre-learned HLA-polymorphisms. In addition, we correct for phylogenetic relatedness of the viruses within the model itself to account for founder effects. Using our model, we observe that CXCR4-using variants are more adapted than CCR5-using variants (p-value = 1.34e-2). Additionally, adapted CCR5-using variants have a significantly lower predicted false positive rate (FPR) by the geno2pheno[coreceptor] tool compared to the non-adapted CCR5-using variants (p-value = 2.21e-2), where a low FPR is associated with CXCR4 usage. Consequently, estimating HLA adaptation can be an asset in predicting not only coreceptor usage, but also an approaching coreceptor switch in CCR5-using variants. We propose the usage of Bayesian GLMMs for modeling virus-host adaptation in general.

A rabbit model for outer retinal atrophy caused by surgical RPE removal.

PURPOSE: We aimed to establish a rabbit model with retinal atrophy induced by an iatrogenic retinal pigment epithelium (RPE) removal, for future testing of the efficacy and safety of cell therapy strategies. METHODS: A localized detachment of the retina from the RPE/choroid layer was created in 18 pigmented rabbits. The RPE was removed by scraping with a custom-made extendable loop instrument. The resulting RPE wound was observed over a time course of 12 weeks with optical coherence tomography and angiography. After 4 days (group 1) and 12 weeks (group 2), histology was done and staining with hematoxylin and eosin, as well as immunofluorescence performed to further investigate the effects of debridement on the RPE and the overlying retina. RESULTS: Already after 4 days, we observed a closure of the RPE wound by proliferating RPE and microglia/macrophage cells forming a multilayered clump. This pattern continued over the observation time course of 12 weeks, whereby the inner and outer nuclear layer of the retina became atrophic. No neovascularization was observed in the angiograms or histology. The observed changes were limited to the site of the former RPE wound. CONCLUSIONS: Localized surgical RPE removal induced an adjacent progressive retinal atrophy. Altering the natural course of this model may serve as a basis to test RPE cell therapeutics.

Validation of human microRNA target pathways enables evaluation of target prediction tools.

MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting.

Long-term monitoring of mercury in young German adults: Time trend analyses from the German Environmental Specimen Bank, 1995-2018.

As highlighted in the Minamata Convention, Mercury (Hg) in its various forms poses a substantial risk to human health and the environment. The health relevance of Hg is also recognized by the European Human Biomonitoring Initiative (HBM4EU), which classifies Hg as a priority substance, since considerable knowledge and data gaps on Hg exposure levels and their changes over time still exist in Europe. The German Environmental Specimen Bank (German ESB) provides valuable policy relevant data and long-term trends of substance exposure on a national level for international comparison and evaluation. In this study we analysed data of the German ESB on Hg exposure of young adults aged 20 to 29 including data on urinary Hg levels from 1995 to 2018 and whole blood Hg levels from 2001 to 2010. Results show a clear decrease in both, about 86% in urine total daily Hg excretion from 1995 (0.76 µg/L) to 2018 (0.11 µg/L) (n = 10,069) and about 57% in blood concentrations of Hg from 2001 (1.76 µg/L) to 2010 (0.77 µg/L) (n = 4085). Over the investigated timeframe only a few values exceeded the toxicologically derived health based guidance value HBM I for blood and urine, with these exceedances decreasing over time in line with the general trend. The factors mostly influencing Hg excretion identified in this study are dental amalgam as well as fish and seafood consumption. Besides other factors (e.g. age and sex), also airborne Hg exposure appears to be a low but evident influencing factor in Germany. Although a considerable decrease in internal Hg exposure is recognized in the last decades, the current low-level exposure may cause adverse health effects especially to vulnerable groups such as pregnant women and children. To further elucidate and evaluate current exposure sources and to reduce human exposure to Hg, continuous environmental and human biomonitoring is needed.

Microfluidic In Vitro Platform for (Nano)Safety and (Nano)Drug Efficiency Screening.

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments.

Non-pooled Human Platelet Lysate: A Potential Serum Alternative for In Vitro Cell Culture.

Serum supplementation is crucial in in vitro cell culture to provide all the essential nutrients needed for cellular processes. Fetal bovine serum (FBS) is considered the 'gold standard', but its production raises serious ethical concerns. Human-derived alternatives to FBS exist in the form of human platelet lysates (hPLs) or human AB serum (ABS). However, these serum products are usually pooled from several donors, in order to have a standardised product without patient-specific deviations. Nevertheless, the use of patient-specific serum in cell culture might be the key to successful transplantation of the cultured cells in medical applications, particularly as it avoids the transmission of infectious components or xenogenic proteins. In addition, the production of non-pooled hPL from single donors is likely to be a cost-effective and time-saving method. The current study used hPL units isolated from single donors and tested their performance as medium supplements for cell culture in comparison with FBS or ABS. This proof-of-concept study aimed to assess the potential of non-pooled hPL for personalised serum supplementation, and thus optimise in vitro models by making them more relevant to human physiology. We showed that A549, HepG2 and Caco-2 human cell lines were generally able to adapt to the new culture conditions and maintain viability, morphology and certain cell-specific characteristics. These results indicate that non-pooled, single patient-derived hPL could be a suitable alternative for in vitro serum supplementation.

Poly(amidoamine)-alginate hydrogels: directing the behavior of mesenchymal stem cells with charged hydrogel surfaces.

The surface charge of a biomaterial represents a promising tool to direct cellular behavior, which is crucial for therapeutic approaches in regenerative medicine. To expand the understanding of how the material surface charge affects protein adsorption and mesenchymal stem cell behavior, differently charged surfaces with zeta potentials spanning from -25 mV to +15 mV were fabricated by the conjugation of poly(amidoamine) to alginate-based hydrogels. We showed that the increase of the biomaterials surface charge resulted in enhanced quantities of biologically available, surface-attached proteins. Since different surface charges were equalized after protein adsorption, mesenchymal stem cells interacted rather with diverse protein compositions instead of different surface features. Besides an enhanced cell attachment to increasingly positively charged surfaces, the cell spreading area and the expression of adhesion-related genes integrin α5 and tensin 1 were found to be increased after adhesion. Moreover, first results indicate a potential impact of the surface charge on mesenchymal stem cell differentiation towards bone and fat cells. The improved understanding of surface charge-related cell behavior has significant impact on the design of biomedical devices and artificial organs.

Ligand-Modified Human Serum Albumin Nanoparticles for Enhanced Gene Delivery.

The development of nonviral gene delivery systems is a great challenge to enable safe gene therapy. In this study, ligand-modified nanoparticles based on human serum albumin (HSA) were developed and optimized for an efficient gene therapy. Different glutaraldehyde cross-linking degrees were investigated to optimize the HSA nanoparticles for gene delivery. The peptide sequence arginine-glycine-aspartate (RGD) and the HIV-1 transactivator of transduction sequence (Tat) are well-known as promising targeting ligands. Plasmid DNA loaded HSA nanoparticles were covalently modified on their surface with these different ligands. The transfection potential of the obtained plasmid DNA loaded RGD- and Tat-modified nanoparticles was investigated in vitro, and optimal incubation conditions for these preparations were studied. It turned out that Tat-modified HSA nanoparticles with the lowest cross-linking degree of 20% showed the highest transfection potential. Taken together, ligand-functionalized HSA nanoparticles represent promising tools for efficient and safe gene therapy.

Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library.

HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIV-neutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development.

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.

The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions.

Experimental Comparison of Primary and hiPS-Based In Vitro Blood-Brain Barrier Models for Pharmacological Research.

In vitro model systems of the blood-brain barrier (BBB) play an essential role in pharmacological research, specifically during the development and preclinical evaluation of new drug candidates. Within the past decade, the trend in research and further development has moved away from models based on primary cells of animal origin towards differentiated models derived from human induced pluripotent stem cells (hiPSs). However, this logical progression towards human model systems from renewable cell sources opens up questions about the transferability of results generated in the primary cell models. In this study, we have evaluated both models with identical experimental parameters and achieved a directly comparable characterisation showing no significant differences in protein expression or permeability even though the achieved transendothelial electrical resistance (TEER) values showed significant differences. In the course of this investigation, we also determined a significant deviation of both model systems from the in vivo BBB circumstances, specifically concerning the presence or absence of serum proteins in the culture media. Thus, we have further evaluated both systems when confronted with an in vivo-like distribution of serum and found a notable improvement in the differential permeability of hydrophilic and lipophilic compounds in the hiPS-derived BBB model. We then transferred this model into a microfluidic setup while maintaining the differential serum distribution and evaluated the permeability coefficients, which showed good comparability with values in the literature. Therefore, we have developed a microfluidic hiPS-based BBB model with characteristics comparable to the established primary cell-based model.

A HET-CAM based vascularized intestine tumor model as a screening platform for nano-formulated photosensitizers.

The development of new tumor models for anticancer drug screening is a challenge for preclinical research. Conventional cell-based in vitro models such as 2D monolayer cell cultures or 3D spheroids allow an initial assessment of the efficacy of drugs but they have a limited prediction to the in vivo effectiveness. In contrast, in vivo animal models capture the complexity of systemic distribution, accumulation, and degradation of drugs, but visualization of the individual steps is challenging and extracting quantitative data is usually very difficult. Furthermore, there are a variety of ethical concerns related to animal tests. In accordance with the 3Rs principles of Replacement, Reduction and Refinement, alternative test systems should therefore be developed and applied in preclinical research. The Hen's egg test on chorioallantoic membrane (HET-CAM) model provides the generation of vascularized tumor spheroids and therefore, is an ideal test platform which can be used as an intermediate step between in vitro analysis and preclinical evaluation in vivo. We developed a HET-CAM based intestine tumor model to investigate the accumulation and efficacy of nano-formulated photosensitizers. Irradiation is necessary to activate the phototoxic effect. Due to the good accessibility of the vascularized tumor on the CAM, we have developed a laser irradiation setup to simulate an in vivo endoscopic irradiation. The study presents quantitative as well as qualitative data on the accumulation and efficacy of the nano-formulated photosensitizers in a vascularized intestine tumor model.

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells.

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Targeted human serum albumin nanoparticles for specific uptake in EGFR-Expressing colon carcinoma cells.

The specific application and transport of drugs into malignant tissue is a critical point during diagnosis and therapy. Nanoparticles are known as excellent drug carrier systems and offer the possibility of surface modification with targeting ligands, leading to a specific accumulation in the targeted tissue. First, the specificity of such a carrier system has to be proven. In this study, cetuximab-modified nanoparticles based on biodegradable human serum albumin (HSA) are investigated regarding their cellular binding and intracellular accumulation. Different EGFR-expressing colon carcinoma cells were used to test possible cytotoxic potential, specific binding and intracellular accumulation. A specific accumulation targeting the EGFR could be shown. These results emphasize that cetuximab-modified HSA-nanoparticles are a promising carrier system for later drug transport. To our knowledge, this is the first study investigating the specific accumulation of HSA nanoparticles into different EGFR-expressing colon carcinoma cells. FROM THE CLINICAL EDITOR: In this study, cetuximab-modified nanoparticles based on human serum albumin (HSA) are investigated regarding their cellular binding and intracellular accumulation. The results suggest that these nanoparticles are a promising carrier system for EGFR overexpressing colon carcinoma cells.

Novel Cost-Efficient Graphene-Based Impedance Biosensor for the Analysis of Viral Cytopathogenicity and the Effect of Antiviral Drugs.

Biosensors become increasingly relevant for medical diagnostics, pharmaceutical industry, and environmental technology, for example, to test new drugs easily and reliably or to detect cell growth in changing environmental conditions. Novel materials like graphene are promising candidates to produce biosensors on an industrial scale by means of printing processes. To reach this aim, methods for the reliable and automated production of electrode structures and their coating are required. We present an impedance biosensor in the format of a microtiter plate, fabricated by highly efficient roll-to-roll printing of graphene-based microstructures on large-area polymer foils. Proof-of-principle experiments show the evidence of the suitability of the printed graphene biosensors for impedance-based monitoring of viral cytopathogenicity and its inhibition in the presence of antiviral drugs. The developed system is a promising approach toward cost-efficient impedimetric biosensors for high-throughput screening in vaccine research and antiviral drug development.

Comparative cytotoxic and antiproliferative profile of methotrexate and fluorouracil on different ocular cells.

PURPOSE: To analyse the cytotoxic and antiproliferative effect of methotrexate (MTX) and fluorouracil (5-FU) in vitro on fibroblasts, retinal pigment epithelial (RPE) and photoreceptor cells as an adjunct for reducing the incidence of proliferative vitreoretinopathy (PVR) after rhegmatogenous retinal detachment surgery. METHODS: Methotrexate and 5-FU were dissolved separately in balanced salt solution (BSS) with concentrations ranging from 0-8000 µg/ml and 0-4000 µg/ml, respectively. All solutions were analysed in terms of pH and osmolarity and applied for 1 h to fibroblasts (BJ), RPE (ARPE-19) and photoreceptor (661W) cell lines adherently cultivated in 96-well cell culture plates (10 000 cells/well). 24 h after incubation, the proliferative (BrdU), metabolic (CellTiter-Glo) and apoptotic (Caspase 3/7) activity of the cells were examined in vitro. RESULTS: 5-FU had an antiproliferative effect on BJ and ARPE-19 cells starting from low concentrations (2 µg/ml). However, the viability of 661W cells decreased and apoptosis was induced with increasing 5-FU concentration. In contrast, MTX up to a concentration of 266 µg/ml did neither result in a significant loss of viability nor in increased caspase 3/7 activity of BJ, ARPE-19 and 661W cells and inhibited the proliferation of ARPE-19 already at low concentrations starting from 8 µg/ml. CONCLUSIONS: Methotrexate dissolved in BSS is biocompatible up to a concentration of 266 µg/ml and may act as an intraoperative rinse solution to inhibit RPE proliferation in PVR-diseased eyes. Contrary, the use of 5-FU within the posterior segment of the eye is limited by its cell-damaging effect on photoreceptor cells.

Influence of Physicochemical Characteristics and Stability of Gold and Silver Nanoparticles on Biological Effects and Translocation across an Intestinal Barrier-A Case Study from In Vitro to In Silico.

A better understanding of their interaction with cell-based tissue is a fundamental prerequisite towards the safe production and application of engineered nanomaterials. Quantitative experimental data on the correlation between physicochemical characteristics and the interaction and transport of engineered nanomaterials across biological barriers, in particular, is still scarce, thus hampering the development of effective predictive non-testing strategies. Against this background, the presented study investigated the translocation of gold and silver nanoparticles across the gastrointestinal barrier along with related biological effects using an in vitro 3D-triple co-culture cell model. Standardized in vitro assays and quantitative polymerase chain reaction showed no significant influence of the applied nanoparticles on both cell viability and generation of reactive oxygen species. Transmission electron microscopy indicated an intact cell barrier during the translocation study. Single particle ICP-MS revealed a time-dependent increase of translocated nanoparticles independent of their size, shape, surface charge, and stability in cell culture medium. This quantitative data provided the experimental basis for the successful mathematical description of the nanoparticle transport kinetics using a non-linear mixed effects modeling approach. The results of this study may serve as a basis for the development of predictive tools for improved risk assessment of engineered nanomaterials in the future.

Long-term time trend of lead exposure in young German adults - Evaluation of more than 35 Years of data of the German Environmental Specimen Bank.

Lead is a ubiquitous pollutant with well-known effects on human health. As there is no lower toxicological threshold for lead in blood and since data gaps on lead exposure still exist in many European countries, HBM data on lead is of high importance. To address this, the European Human Biomonitoring Initiative HBM4EU classified lead as a priority substance. The German Environmental Specimen Bank (German ESB) has monitored lead exposure since more than 35 years. Using data from the early 1980s to 2019 we reveal and discuss long-term trends in blood lead levels (BLLs) and current internal exposure of young adults in Germany. BLLs in young adults decreased substantially in the investigated period. As results from the ESB sampling site Muenster demonstrate, the geometric mean of BLLs of young adults decreased from 1981 (78,7 µg/L) to 2019 (10.4 µg/L) by about 87%. Trends in human exposure closely correlate with air lead levels (ALLs) provided by the European Monitoring and Evaluation Programme (EMEP). Hence, the decrease of BLLs largely reflects the drop in air lead pollution. Known associations of sex, smoking, alcohol consumption, and housing situation with BLLs are confirmed with data of the German ESB. Although internal lead exposure in Germany decreased substantially, the situation might be different in other European countries. Since 2010, BLLs of young adults in Germany levelled out at approximately 10 µg/L. The toxicity of lead even at low levels is known to cause adverse health effects especially in children following exposure of the child or the mother during pregnancy. To identify current exposure sources and to minimize future lead exposure, continuous monitoring of lead intake and exposure levels is needed.

Adsorption of obidoxime onto human serum albumin nanoparticles: drug loading, particle size and drug release.

The standard treatment of poisoning by organophosphorous compounds such as paraoxon includes the administration of oximes. Due to their inability to rapidly cross the blood-brain barrier (BBB) in therapeutically relevant concentrations, these drugs possess insufficient activity in the central nervous system. Since human serum albumin (HSA) nanoparticles enable the delivery of a variety of drugs across the BBB into the brain, in the present study the antidote obidoxime was bound to these particles by adsorption. The resulting sorption isotherms showed a best fit to Langmuir isotherms indicating that obidoxime adsorbs to HSA nanoparticles forming a monolayer. A maximum drug loading of 93.5 microg obidoxime/mg of nanoparticles at pH 8.3 was calculated. At higher concentrations the particle diameter increased significantly with obidoxime concentration leading to instable particle systems. The in vitro release of obidoxime from HSA nanoparticles showed a rapid release of the drug from the nanoparticles within 3 h.