Simultaneous Incubation Research Articles

Amino Acid Adsorption Onto Magnetic Nanoparticles Reveals Correlations With Physicochemical Parameters

AbstractWe analyze the adsorption of the proteinogenic amino acids (AAs) glutamine, glutamic acid, lysine, tyrosine, proline, and valine onto bare iron oxide nanoparticles (approx. 10 nm). Aiming to identify the governing principles of low molecular weight coronae, which remain underinvestigated, our study covers broad concentration ranges up to the solubility limit of the AAs. Isothermal experiments reveal that the highly soluble AAs valine, proline, and lysine form extensive multilayers on the nanoparticle surface, and infrared measurements indicate intermolecular interactions, particularly with valine and lysine, for higher AA contents. Conversely, the low solubility of tyrosine and glutamic acid restricts their adsorption capacity, despite their higher partitioning on the solid surface. Parameters derived from fitting a classic saturation model seem to align with well‐documented physicochemical properties such as the hydrophobicity and the complexity indices – a promising first step towards formulating design principles. Scaling these parameters by the AA solubility reveals a clear correlation with the adsorption behavior. In adsorption experiments with AA model mixtures, sequential incubation increases the adsorption capacity for valine and proline, whereas simultaneous incubation with these AAs reduces tyrosine's capacity. Future studies should seek to elucidate adsorption patterns to advance our understanding of corona growth and evolution mechanisms.

Apelin Counteracts the Effects of Fusobacterium nucleatum on the Migration of Periodontal Ligament Cells In Vitro

To better understand the link between periodontitis and metabolic diseases, our in vitro study aimed to assess the influence of the adipokine apelin and/or the periodontal pathogen Fusobacterium nucleatum on periodontal cells. Periodontal ligament (PDL) cells were exposed to F. nucleatum in the presence and absence of apelin. Scratch assays were used to analyze the in vitro wound healing and velocity of cell migration. To investigate if F. nucleatum and/or apelin have a regulatory effect on cell proliferation and apoptosis, proliferation and viability assays were performed as well as an analysis of caspase 9 expression. Both the in vitro wound closure and the cell migration rate were significantly reduced by F. nucleatum. Simultaneous incubation with apelin counteracted the adverse effects of F. nucleatum. The proliferation assay demonstrated that neither apelin nor F. nucleatum significantly affected PDL cell proliferation. Furthermore, neither apelin nor F. nucleatum was cytotoxic or affected apoptosis after 48 h. Apelin could play a modulatory role in the pathogenesis of periodontitis, as it was able to compensate for the inhibitory effects of the periodontal pathogen F. nucleatum on PDL cell migration in vitro.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Microfluidics-Based Drying-Wetting Cycles to Investigate Phase Transitions of Small Molecules Solutions.

Drying-wetting cycles play a crucial role in the investigation of the origin of life as processes that both concentrate and induce the supramolecular assembly and polymerization of biomolecular building blocks, such as nucleotides and amino acids. Here, we test different microfluidic devices to study the dehydration-hydration cycles of the aqueous solutions of small molecules, and to observe, by optical microscopy, the insurgence of phase transitions driven by self-assembly, exploiting water pervaporation through polydimethylsiloxane (PDMS). As a testbed, we investigate solutions of the chromonic dye Sunset Yellow (SSY), which self-assembles into face-to-face columnar aggregates and produces nematic and columnar liquid crystal (LC) phases as a function of concentration. We show that the LC temperature-concentration phase diagram of SSY can be obtained with a fair agreement with previous reports, that droplet hydration-dehydration can be reversibly controlled and automated, and that the simultaneous incubation of samples with different final water contents, corresponding to different phases, can be implemented. These methods can be further extended to study the assembly of diverse prebiotically relevant small molecules and to characterize their phase transitions.

The Stability and Efficency of CPB Cells Were Acclimated for Virus Proliferation.

Vaccinations are still the most effective means of preventing and controlling fish viral diseases, and cells are an important substrate for the production of a viral vaccine. Therefore, the rapid-stable growth and virus sensitivity of cells are urgently needed. Chinese perch brain 100th passage (CPB p100) were acclimated in a low serum with 5% FBS L-15 for 50 passages, then transferred to 8% FBS L-15 for 150 passages. Additionally, the morphology and cell type of CPB 300th passage (CPB p300) cells were identified. We analyzed the transfection efficiency and virus sensitivity of CPB p300 cells, and then optimized the conditions of ISKNV, SCRV, and LMBV multiplication in CPB cells. CPB p300 cells were more homogeneous, and the spread diameter (20-30) µm in CPB p300 cells became the dominant population. The doubling time of CPB p300 was 1.5 times shorter than that of CPB p100.However, multiplication rate of CPB p300 was 1.37 times higher than CPB p100. CPB p300 cells were susceptible to ISKNV, SCRV, and LMBV, and the optimal conditions of ISKNV, SCRV, and LMBV multiplication were simultaneous incubation, 0.6 × 105 cells/cm2 and MOI = 0.1; infection at 48 h, 0.8 × 105 cells/cm2 and MOI = 0.01; simultaneous incubation, 0.7 × 105 cells/cm2 and MOI = 0.05, respectively. The time and economic costs of ISKNV, SCRV, and LMBV multiplication in CPB p300 cells were significantly reduced. The acquisition of CPB p300 cells laid a good material foundation for the production of ISKNV, SCRV, and LMBV vaccines.

2,4-dinitrophenol enhances cisplatin and etoposide cytotoxic activity on prostate cancer cell line.

Including additional compounds that disturb the energy metabolism of cancer cells in advanced cancer therapy regimens may be an approach to overcome the problem of drug resistance and the therapeutic effectiveness of classic chemotherapeutics. One of the compounds that decouple oxidative phosphorylation, and thus alter the activity of energy-producing pathways, is 2,4-DNP (2,4- dinitrophenol). The aim of the study was to assess the ability of the 2,4-DNP to sensitize prostate cancer cells to the action of cisplatin and etoposide, or to intensify their action. The research was carried out on three prostate cancer cell lines (LNCaP, PC-3, DU-145. To assess the effect of cisplatin or etoposide with 2,4-DNP on prostate cancer cells, MTT assay, analysis of the cell cycle and apoptosis detection was performed. Oxidative stress was investigated by CellRox fluorescence staining and expression of genes related to antioxidant defence. In addition, analysis was conducted of the expression of genes related to cell cycle inhibition, transporters associated with multi-drug resistance and DNA repair. The study showed that the simultaneous incubation of 2,4-DNP with cisplatin or etoposide enhances the cytotoxic effect of the chemotherapeutic agent only in LNCaP cells (oxidative phenotype). The enhanced cytotoxic effect of chemotherapeutics by 2,4-DNP may be the result of disturbed redox balance, reduced ability of cells to repair DNA, and the oxidative metabolic phenotype of prostate cancer cells.

Painless photodynamic therapy for facial actinic keratoses: A retrospective cohort study of the post-treatment inflammatory response

IntroductionPhotodynamic therapy (PDT) is a safe, non-mutagenic, and non-scarring treatment for actinic keratoses (AK). Background‘Painless’ photodynamic therapy (p-PDT) is a regimen for AK that employs simultaneous aminolevulinate incubation and blue light illumination. The efficacy of p-PDT resembles that of traditional PDT, but detailed mechanisms of action for p-PDT are not well understood. MethodsTo characterize the inflammatory effects of the p-PDT procedure 48 h following treatment and determine the association of inflammation with precancer burden, we performed a retrospective cohort study of 104 patients with AK of face or scalp treated with p-PDT between 2017 and 2019. Patients self-reported their side effects 48 h following p-PDT and took photographs of their face and scalp. Photographs were edited to define seven anatomic regions, and erythema was scored by four investigators. ResultsNinety-eight patients provided photographs suitable for erythema evaluation. Most patients experienced 2 or more side effects and some pain 48 h post-procedure. Females experienced more pain (p = 0.01) and side effects (p = 0.002) compared to males. AK burden was positively associated with post p-PDT erythema response (p < 0.0001) at all sites, but particularly in the temples (p = 0.002) and supralabial area (p = 0.009). DiscussionThis study confirms a strong clinical inflammatory response after p-PDT. Severity of inflammation is positively associated with AK tumor burden, suggesting that post-treatment inflammation may be a pre-requisite for p-PDT efficacy. Interestingly, the results also identify certain gender-related differences in the severity of side effects experienced by patients post-PDT.

Clostridium botulinum C3bot mediated effects on cytokine-induced psoriasis-like phenotype in full-thickness skin model

Clostridium botulinum C3 exoenzyme (C3bot) exclusively inhibits RhoA, B and C by ADP-ribosylation and is therefore used as a cell-permeable tool for investigating the cellular role of these Rho-GTPases. Rho-GTPases represent a molecular switch integrating different receptor signalling to downstream cascades including transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton and cell proliferation. C3bot-induced inhibition of RhoA leads to reorganization of the actin cytoskeleton, morphological changes, and inhibition of cell proliferation as well as modulation of inflammatory response. In this study, we characterized the C3bot-mediated effects on a full-thickness skin model exhibiting a psoriasis-like phenotype through the addition of cytokines. Indeed, after the addition of cytokines, a decrease in epidermal thickness, parakeratosis, and induction of IL-6 was detected. In the next step, it was studied whether C3bot caused a reduction in the cytokine-induced psoriasis-like phenotypes. Basal addition of C3bot after cytokine induction of the full-thickness skin models caused less epidermal thinning and reduced IL-6 abundance. Simultaneous basal incubation with cytokines and C3bot, IL-6 abundance was inhibited, but epidermal thickness was only moderately affected. When C3bot was added apically to the skin model, IL-6 abundance was reduced, but no further effects on the psoriasis-like phenotype of the epidermis were observed. In summary, C3bot inhibits the cytokine-induced expression of IL-6 and thus may have an impact on the pro-inflammatory immune response in the psoriasis-like phenotype.

Simultaneous Quantification of Biomarkers for Bis-(2-chloroethyl) Sulfide and 1,2-Bis(2-chloroethylthio) Ethane Exposure in Human Urine at Trace Exposure Levels by Gas Chromatography Tandem Mass Spectrometry via Simultaneous Incubation and Extraction.

Sulfur mustard [HD; bis-(2-chloroethyl) sulfide] and other analogues are a kind of highly toxic vesicant and have been prohibited by the Organization for the Prohibition of Chemical Weapons (OPCW) since 1997. Exposures to HD could generate several adducts in the plasma and hydrolysis products in the urine, which are widely applied as biomarkers to identify HD exposure in forensic analysis. Several methods have been developed for the detection of related biomarkers. However, most methods are based on complex derivatization, and not enough attention is paid to HD analogues. A modified and convenient analytical method reported herein includes simultaneous incubation and organic solvent extraction. The biomarkers such as thiodiglycol and 1,2-bis (2-hydroxyethylthio) are transferred to HD and 1,2-bis(2-chloroethylthio) ethane via hydrochloric acid at the appropriate temperature. The analytes are analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS) with 2-chloroethyl ethyl sulfide (2-CEES) applied as the internal standard. The interday and intraday study according to FDA rules has been achieved to evaluate the accuracy and precision of the method. The two targets are detected with a good linearity (R2 > 0.99) in the concentration ranges from 5 to 1000 ng/mL and 10 to 1000 ng/mL, with small relative standard deviations (RSD ≤6.62% and RSD ≤6.93%) and favorable recoveries between 90.3 and 107.3% and between 89.4 and 108.7%, respectively. The established method can be used for retrospective detection of sulfur mustards in biological samples and successfully applied in the biomedical proficiency testing organized by the OPCW.

A New In Vitro Blood Flow Model for The Realistic Evaluation of Antimicrobial Surfaces.

Device-associated bloodstream infections can cause serious medical problems and cost-intensive post-infection management, defining a need for more effective antimicrobial coatings. Newly developed coatings often show reduced bacterial colonization and high hemocompatibility in established in vitro tests, but fail in animal studies or clinical trials. The poor predictive power of these models is attributed to inadequate representation of in vivo conditions. Here, we present a new single-pass blood flow model, with simultaneous incubation of the test surface with bacteria and freshly-drawn human blood. The flow model is validated by comparative analysis of a recently developed set of anti-adhesive and contact-killing polymer coatings, and the corresponding uncoated polycarbonate surfaces. The results confirm the model's ability to differentiate the antimicrobial activities of the studied surfaces. Blood activation data correlate with bacterial surface coverage: low bacterial adhesion is associated with low inflammation and hemostasis. Shear stress correlates inversely with bacterial colonization, especially on anti-adhesive surfaces. The introduced model is concluded to enable the evaluation of novel antimicrobial materials under in vivo-like conditions, capturing interactions between bacteria and biomaterials surfaces in the presence of key components of the ex vivo host response. This article is protected by copyright. All rights reserved.

Direct inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 by per- and polyfluoroalkyl substances: Structure-activity relationship and in silico docking analysis

Per- and polyfluoroalkyl substances (PFAS) are persistent in the environment and may disrupt the endocrine system. Our previous study showed that perfluorooctanoic acid (PFOA, C8) and perfluorooctanesulfonic acid (PFOS, C8S) can inhibit 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity leading to an active glucocorticoid accumulation. In this study, we extended investigation for 17 PFAS, including carboxylic and sulfonic acids, with different carbon-chain lengths, to determine their inhibitory potency and structure-activity relationship in human placental and rat renal 11β-HSD2. C8-C14 PFAS at 100 μM significantly inhibited human 11β-HSD2 with a potency as C10 (half-maximal inhibitory concentration, IC50, 9.19 μM) > C11 (15.09 μM) > C12 (18.43 μM) > C9 (20.93 μM) > C13 (124 μM) > C14 (147.3 μM) > other C4-C7 carboxylic acids, and C8S > C7S = C10S > other sulfonic acids. For rat 11β-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11β-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11β-HSD2 but not rat 11β-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11β-HSD2. Docking analysis showed that all PFAS bound to the steroid-binding site and carbon-chain length determined the potency of inhibition, with the optimal molecular length (12.6 Å) for potent inhibitors PFDA and PFOS, which is comparable to the molecular length (12.7 Å) of the substrate cortisol. The length between 8.9 and 17.2 Å is the probable threshold molecular length to inhibit human 11β-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11β-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11β-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11β-HSD2.

A New Organotypic 3D Slice Culture of Mouse Meibomian Glands Reveals Impact of Melanocortins

The meibomian glands (MGs) within the eyelids produce a lipid-rich secretion that forms the superficial layer of the tear film. Meibomian gland dysfunction (MGD) results in excessive evaporation of the tear film, which is the leading cause of dry eye disease (DED). To develop a research model similar to the physiological situation of MGs, we established a new 3D organotypic slice culture (OSC) of mouse MGs (mMGs) and investigated the effects of melanocortins on exocrine secretion. Tissue viability, lipid production and morphological changes were analyzed during a 21-day cultivation period. Subsequently, the effects on lipid production and gene expression were examined after stimulation with a melanocortin receptor (MCR) agonist, α-melanocyte-stimulating hormone (α-MSH), and/or an MCR antagonist, JNJ-10229570. The cultivation of mMGs OSCs was possible without impairment for at least seven days. Stimulation with the MCR agonists induced lipid production in a dose-dependent manner, whereas this effect was tapered with the simultaneous incubation of the MCR antagonist. The new 3D OSC model is a promising approach to study the (patho-) physiological properties of MG/MGD while reducing animal studies. Therefore, it may accelerate the search for new treatments for MGD/DED and lead to new insights, such as that melanocortins likely stimulate meibum production.

ПРИМЕНЕНИЕ МЕТОДА ЯМР C ПАРАМАГНИТНЫМ ДОПИНГОМ ДЛЯ ОЦЕНКИ АПОПЛАСТНОГО ПЕРЕНОСА ВОДЫ В КОРНЯХ ИНТАКТНЫХ РАСТЕНИЙ ПРИ ДЕЙСТВИИ АБИОТИЧЕСКИХ СТРЕССОВ

In this work, the methodological approach based on low-field NMR using the GdDTPA paramagnetic complex for the qualitative assessment of water transport along the apoplastic (extracellular) pathway in the roots of intact wheat plants is proposed. This approach consists in measuring the spin-spin relaxation times of the water magnetization in the roots during simultaneous incubation of the roots in a solution of the paramagnetic Gd-DTPA complex and the impact of a stress factor on the plants. During root incubation, this complex spreads only along the root apoplast system and shortens the apoplast water relaxation times. GdDTPA does not penetrate into the cells and, accordingly, does not change the intracellular water relaxation times. Thus, the rate of decrease in the relaxation times of the magnetization of apoplast water, which directly depends on the intensity of water transfer through the apoplast, can be used to determine the relative contribution of water transfer through the root apoplast during stress exposure. A twofold increase in the concentration of carbon dioxide in the aerial parts of plants was used as an abiotic factor presumably influencing the transfer of water along the root apoplast. Using this methodological approach, it was shown that increase in the CO2 concentration in the leaf zone of wheat plants to 800 ppm leads to decrease in the rate of water transfer through the root apoplast by 2–2.5 times compared with the control at ambient CO2 concentration of 400 ppm.

An endoplasmic reticulum-targeting fluorescent probe for the visualization of the viscosity fluctuations during ferroptosis in live cells

Ferroptosis is an unique iron-dependent cell death form and currently has been shown to closely relate with ER. Revealing the viscosity fluctuations of ER during ferroptosis is of great significance not only to monitor the occurrence and development of iron poisoning, but also to deeply understand the biological effects of ER in ferroptosis. Herein, we present an ER-targeting fluorescent probe (PV1) to detect viscosity changes of ER during ferroptosis. PV1 utilized a rotatable C–C bond to connect the two rigid π-systems, and responded viscosity by the regulation of the coplanarity of these two planes. PV1 displayed desirable sensitivity and selectivity to viscosity. The biological imaging results suggested that PV1 mainly distributed at ER in live cells, and the viscosity of ER exhibited an evident increase in the process of erastin-induced ferroptosis. After the simultaneous incubation of cells with erastin and Fer-1 or VE, the viscosity of ER showed no marked change, and it suggested that the erastin-induced ferroptosis could be inhibited by Fer-1 and VE. We expect that the developed probe could provide a feasible and rapid method for the in-depth study of the ferroptosis-based disease treatment and drug design.

The role of IKK-2/NF-κB signaling cascade in the activation of peritoneal macrophages by humic acids of peat

Introduction . Macrophages occupy a special place in the formation and coordination of the immune response, their population is characterized by heterogeneity, however, two main types are distinguished among them: pro-inflammatory (classically activated) macrophages M1, which are activated by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) of bacteria, produce pro-inflammatory cytokines, nitric oxide (NO) and initiate an immune response, and anti-inflammatory (alternatively activated) macrophages M2, which are activated by such cytokines as interleukin 1 (IL-1), IL-10 or IL-13 and are associated with suppression of the immune response and tissue healing. The proinflammatory properties of macrophages are most often associated with the activation of an intracellular signaling cascade with the participation of the IKK-2 kinase complex and nuclear factor-κB (NF-κB), which is the most important signal transduction molecule. Previously, the authors showed that humic acids (HA) of peat cause the appearance of pro-inflammatory properties in macrophages; however, the intracellular mechanisms of such activation have not been studied. Aim . Study of the contribution of the intracellular signaling cascade IKK-2/NF-κB to the activation of pro-inflammatory properties of macrophages by humic acids of peat. Materials and methods . The studies were carried out using cultural methods and western blotting. Results and discussion . It was shown that the addition of the IKK-2 inhibitor to the macrophage culture reduced the ability of HA to stimulate the production of nitric oxide by the cells. Western blotting revealed that after 30 minutes simultaneous incubation of HA with macrophages, the content of the NF-κB p65 molecule in the cells increased, after 60 minutes it decreased to the values of intact control, and after 17 hours it decreased 3 times compared to the control level. However, the IKK-2 inhibitor after a 30-minutes incubation did not have a blocking effect on the IKK kinase, whereas in an hour of the co-incubation of the cells with the inhibitor and substances, a decrease in the level of NF-κB p65 was observed. This trend continued in 17 hours. Conclusion. The stimulating effect of peat humic acids on macrophages is realized, among other things, due to the activation of the IKK-2 kinase complex with further release of the p65 subunit of the NF-κB molecule. The dependence of the degree of NF-κB activation on the period of interaction of the cell with activating agents is traced. HA start the intracellular signaling cascade of the nuclear factor NF-κB already within the first 30 minutes.

Experimental and theoretical studies on the anti‐amyloidogenic and destabilizing effects of pyrogallol against human insulin protein

One of the major problems caused by repeated subcutaneous insulin injections in patients with diabetes is insulin amyloidosis. Understanding the molecular mechanism of amyloid fibril formation of insulin and finding effective compounds to inhibit or eliminate them is very important, and extensive research has been done on it. In this study, the anti-amyloidogenic and destabilizing effects of the pyrogallol, as a phenolic compound, on human insulin protein were investigated by CR absorbance, ThT and ANS fluorescence, FTIR spectroscopy, and atomic force microscopy. According to the obtained results, the formation of amyloid fibrils at pH2.0 and 50°C was confirmed by CR, ThT, ANS, and FTIR assays. Microscopic images also showed the twisted and long structures of amyloid fibrils. Simultaneous incubation of the protein with pyrogallol at different concentrations reduced the intensities of CR, ThT, and ANS in a dose-dependent manner, and no trace of fibrillar structures was observed in the microscopic images. FTIR spectroscopy also showed that the position of the amide I band in the spectrum of samples containing pyrogallol was shifted. Based on the findings of this study, it can be concluded that pyrogallol can be effective in preventing and suppressing human insulin amyloid fibrils. PRACTICAL APPLICATIONS: In recent years, finding a strategy for the treatment of amyloid diseases has been considered by many researchers. Targeting protein aggregates by small organic molecules such as polyphenols is one of the most desirable and effective strategies to prevent and improve amyloid disease, which has received much attention in recent years. 1,2,3-Trihydroxybenzene, commonly known as pyrogallol (Py), is a phenolic compound like other natural polyphenols that are present in human food sources, including fruits and vegetables, and a variety of edible and medicinal plants. So far, many beneficial activities for pyrogallol such as anti-cancer, antioxidant, antibacterial, antiviral, and antifungal have been reported in various studies. Since various studies have shown that natural polyphenols have special properties to prevent amyloid disease, the present study could be useful in advancing the design purposes of new anti-amyloid drugs in the future.

Direct effects of anti‐inflammatory medication on barrier function in intestinal epithelial cells

An impaired intestinal epithelial barrier (IEB) is a hallmark in the pathogenesis of inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn’s disease. Most of the IBD patients are treated with anti‐inflammatory reagents including glucocorticoids, anti TNFα (tumor necrosis factor alpha) antibodies and mesasalazine. While the Immune‐modulating effects of those therapies are well documented, we hypothesized whether there are direct effects of those reagents on enterocytes and the IEB.To address this hypothesis we used Caco2 cells as well as 2D and 3D murine organoids and performed analysis of intestinal epithelial barrier function. Inflammation‐induced alterations were mimicked using cytomix for 24h (TNFα, Interleukin 1 beta and Interferon gamma). The enterocytes were treated with 100ng/ml anti‐TNFα antibody Infliximab, 1mM prednisolone or 5mM mesasalazine, respectively following the challenge with cytomix. Epithelial permeability as revealed by measurements of 4kDa FITC Dextran flux and of transepithelial electric resistance showed that Infliximab and a pre‐incubation with prednisolone for 24h attenuated inflammation‐induced breakdown of the IEB. This was not the case after application of mesasalazine or a simultaneous incubation of prednisolone. The functional changes in intestinal epithelial barrier function following incubation with Cytomix were paralleled by alterations in the expression of “leaky” tight junction protein Claudin2, as revealed by Western blots. Furthermore, immunostainings demonstrated an altered distribution of barrier sealing proteins such as Claudin1, Desmoglein2 and E‐Cadherin. All of these effects were blocked by infliximab and prednisolone.In summary, our data indicate that infliximab and prednisolone have direct effects on the IEB that may contribute to the efficacy in the treatment of IBD.

Natural Polyphenols May Normalize Hypochlorous Acid-Evoked Hemostatic Abnormalities in Human Blood

During pathogen invasion, activated neutrophils secrete myeloperoxidase (MPO), which generates high local concentrations of hypochlorous acid (HOCl), a strong antimicrobial agent. Prolonged or uncontrolled HOCl production may, however, affect hemostasis, manifesting in inhibition of platelet aggregation and thrombus formation and in elevated fibrin density and attenuated fibrinolysis. In this report, we investigated whether three plant-derived polyphenols with well-known antioxidant properties, i.e., quercetin (Que), epigallocatechin gallate (EGCG), and resveratrol (Resv), at concentrations not affecting platelet responses per se, may normalize particular aspects of hemostasis disturbed by HOCl. Specifically, Que (5–25 μM) and EGCG (10–25 μM) abolished HOCl-evoked inhibition of platelet aggregation (assessed by an optical method), while the simultaneous incubation of platelet-rich plasma with Resv (10–25 μM) enhanced the inhibitory effect of HOCl. A similar effect was observed in the case of thrombus formation under flow conditions, evaluated in whole blood by confocal microscope. When plasma samples were incubated with HOCl, a notably higher density of fibrin (recorded by confocal microscope) was detected, an effect that was efficiently normalized by Que (5–25 μM), EGCG (10–25 μM), and Resv (5–25 μM) and which corresponded with the normalization of the HOCl-evoked prolongation of fibrinolysis, measured in plasma by a turbidimetric method. In conclusion, this report indicates that supplementation with Que and EGCG may be helpful in the normalization of hemostatic abnormalities during inflammatory states associated with elevated HOCl production, while the presence of Resv enhances the inhibitory action of HOCl towards platelets.

Cisplatin - A more Efficient Drug in Combination with Radionuclides?

The combination of conventional chemotherapeutic drugs with radionuclides or external radiation is discussed for a long period of time. The major advantage of a successful combination therapy is the reduction of severe side effects by decreasing the needed dose and simultaneously increasing therapeutic efficiency. In this study, pUC19 plasmid DNA was incubated with the cytostatic drug cisplatin and additionally irradiated with 99mTc, 188Re and 223Ra. To verify the contribution of possibly excited platinum atoms to the emission of Auger electrons we determined DNA damages, such as single- and double strand breaks. The threshold concentration value of cisplatin, which was tolerated by pUC19 plasmid DNA was determined to be 18-24 nM. Nevertheless, even at higher dose values (>100 Gy) and simultaneous incubation of cisplatin to 200 ng plasmid DNA, no significant increase in the number of induced single- and double-strand breaks was obtained, compared to the damage solely caused by the radionuclides. We thereby conclude that there is no direct dependence of the mechanism of strand break induction to the absence or presence of platinum atoms attached to the DNA. Reported increasing DNA damages in therapy approaches on a cellular level strongly depend on the study design and are mainly influenced by repair mechanisms in living cells. Nevertheless, the use of radioactive cisplatin, containing the Auger electron emitter 191Pt, 193mPt or 195mPt, is a bright prospect for future therapy by killing tumor cells combining two operating principles: a cytostatic drug and a radiopharmaceutical at the same time.

Genotoxicity of chromium (III) and cobalt (II) and interactions between them

Abstract Introduction. Chromium and cobalt are essential trace elements that are required only in a small amount, otherwise their excess can cause toxic effects. Aim. The aim of this study was to determine the effects of chromium (III) and cobalt (II) and their combinations on genotoxicity in human fibroblasts cells (BJ). Material and methods. In this work, comet and micronucleus assays were used. The BJ cells were exposed to chromium chloride and cobalt chloride at concentration ranges from 100 to 1400 µM. Mixtures of these elements were prepared so as to examine interactions between them. Results. The present study shows the genotoxic effects of chromium (III) and cobalt (II) and their mixtures on BJ cells. In the comet assay, no comets were observed at the lowest concentrations; in the higher, a significant increase in their percentage was observed. In the other assay (formation of micronuclei), a statistically significant increase in the number of cells with micronuclei was observed in the BJ cells spiked with cobalt chloride and chromium chloride. In the case of simultaneous incubation of chromium chloride at 200 µM and cobalt chloride at 1000 µM in the BJ line, antagonism was observed. However, the interaction of chromium chloride at the 1000 µM and cobalt chloride at 200 µM leads to synergism between the studied elements. Conclusions. Cobalt (II) and chromium (III) show genotoxic properties, they induce breaks in double and single-stranded DNA and they cause formation of AP-sites that do not have purine or pyrimidine bases.