The epigenetic state of IL-4-polarized macrophages enables inflammatory cistromic expansion and extended synergistic response to TLR ligands.

Prior exposure to microenvironmental signals could fundamentally change the response of macrophages to subsequent stimuli. It is believed that T helper-2 (Th2)-cell-type cytokine interleukin-4 (IL-4) and Toll-like receptor (TLR) ligand-activated transcriptional programs mutually antagonize each other, and no remarkable convergence has been identified between them. In contrast, here, we show that IL-4-polarized macrophages established a hyperinflammatory gene expression program upon lipopolysaccharide (LPS) exposure. This phenomenon, which we termed extended synergy, was supported by IL-4-directed epigenomic remodeling, LPS-activated NF-κB-p65 cistrome expansion, and increased enhancer activity. The EGR2 transcription factor contributed to the extended synergy in a macrophage-subtype-specific manner. Consequently, the previously alternatively polarized macrophages produced increased amounts of immune-modulatory factors both in vitro and in vivo in a murine Th2 cell-type airway inflammation model upon LPS exposure. Our findings establish that IL-4-induced epigenetic reprogramming is responsible for the development of inflammatory hyperresponsiveness to TLR activation and contributes to lung pathologies.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Block Synthesis and Step-Growth Polymerization of C-6-Sulfonatomethyl-Containing Sulfated Malto-Oligosaccharides and Their Biological Profiling.

Highly sulfated malto-oligomers, similar to heparin and heparan-sulfate, have good antiviral, antimetastatic, anti-inflammatory and cell growth inhibitory effects. Due to their broad biological activities and simple structure, sulfated malto-oligomer derivatives have a great therapeutic potential, therefore, the development of efficient synthesis methods for their production is of utmost importance. In this work, preparation of α-(1→4)-linked oligoglucosides containing a sulfonatomethyl moiety at position C-6 of each glucose unit was studied by different approaches. Malto-oligomeric sulfonic acid derivatives up to dodecasaccharides were prepared by polymerization using different protecting groups, and the composition of the product mixtures was analyzed by MALDI-MS methods and size-exclusion chromatography. Synthesis of lower oligomers was also accomplished by stepwise and block synthetic methods, and then the oligosaccharide products were persulfated. The antiviral, anti-inflammatory and cell growth inhibitory activity of the fully sulfated malto-oligosaccharide sulfonic acids were determined by in vitro tests. Four tested di- and trisaccharide sulfonic acids effectively inhibited the activation of the TNF-α-mediated inflammatory pathway without showing cytotoxicity.

BGP-15 Protects against Doxorubicin-Induced Cell Toxicity via Enhanced Mitochondrial Function.

Doxorubicin (DOX) is an efficacious and commonly used chemotherapeutic agent. However, its clinical use is limited due to dose-dependent cardiotoxicity. Several mechanisms have been proposed to play a role in DOX-induced cardiotoxicity, such as free radical generation, oxidative stress, mitochondrial dysfunction, altered apoptosis, and autophagy dysregulation. BGP-15 has a wide range of cytoprotective effects, including mitochondrial protection, but up to now, there is no information about any of its beneficial effects on DOX-induced cardiotoxicity. In this study, we investigated whether the protective effects of BGP-15 pretreatment are predominantly via preserving mitochondrial function, reducing mitochondrial ROS production, and if it has an influence on autophagy processes. H9c2 cardiomyocytes were pretreated with 50 µM of BGP-15 prior to different concentrations (0.1; 1; 3 µM) of DOX exposure. We found that BGP-15 pretreatment significantly improved the cell viability after 12 and 24 h DOX exposure. BGP-15 ameliorated lactate dehydrogenase (LDH) release and cell apoptosis induced by DOX. Additionally, BGP-15 pretreatment attenuated the level of mitochondrial oxidative stress and the loss of mitochondrial membrane potential. Moreover, BGP-15 further slightly modulated the autophagic flux, which was measurably decreased by DOX treatment. Hence, our findings clearly revealed that BGP-15 might be a promising agent for alleviating the cardiotoxicity of DOX. This critical mechanism appears to be given by the protective effect of BGP-15 on mitochondria.

Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

First Synthesis of DBU-Conjugated Cationic Carbohydrate Derivatives and Investigation of Their Antibacterial and Antifungal Activity.

The emergence of drug-resistant bacteria and fungi represents a serious health problem worldwide. It has long been known that cationic compounds can inhibit the growth of bacteria and fungi by disrupting the cell membrane. The advantage of using such cationic compounds is that the microorganisms would not become resistant to cationic agents, since this type of adaptation would mean significantly altering the structure of their cell walls. We designed novel, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)-derived amidinium salts of carbohydrates, which may be suitable for disturbing the cell walls of bacteria and fungi due to their quaternary ammonium moiety. A series of saccharide-DBU conjugates were prepared from 6-iodo derivatives of d-glucose, d-mannose, d-altrose and d-allose by nucleophilic substitution reactions. We optimized the synthesis of a d-glucose derivative, and studied the protecting group free synthesis of the glucose-DBU conjugates. The effect of the obtained quaternary amidinium salts against Escherichia coli and Staphylococcus aureus bacterial strains and Candida albicans yeast was investigated, and the impact of the used protecting groups and the sugar configuration on the antimicrobial activity was analyzed. Some of the novel sugar quaternary ammonium compounds with lipophilic aromatic groups (benzyl and 2-napthylmethyl) showed particularly good antifungal and antibacterial activity.

Comparison of the Modulating Effect of Anthocyanin-Rich Sour Cherry Extract on Occludin and ZO-1 on Caco-2 and HUVEC Cultures.

Increased permeability of the epithelial and endothelial cell layers results in the onset of pathogenic mechanisms. In both cell types, cell-cell connections play a regulatory role in altering membrane permeability. The aim of this study was to investigate the modulating effect of anthocyanin-rich extract (AC) on TJ proteins in inflammatory Caco-2 and HUVEC monolayers. Distribution of Occludin and zonula occludens-1 (ZO-1) were investigated by immunohistochemical staining and the protein levels were measured by flow cytometry. The mRNA expression was determined by quantitative real-time PCR. The transepithelial electrical resistance (TEER) values were measured during a permeability assay on HUVEC cell culture. As a result of inflammatory induction by TNF-α, redistribution of proteins was observed in Caco-2 cell culture, which was reduced by AC treatment. In HUVEC cell culture, the decrease in protein and mRNA expression was more dominant during inflammatory induction, which was compensated for by the AC treatment. Overall, AC positively affected the expression of the examined cell-binding structures forming the membrane on both cell types.

Effects of the Calix[4]arene Derivative Compound OTX008 on High Glucose-Stimulated ARPE-19 Cells: Focus on Galectin-1/TGF-ß/EMT Pathway.

Diabetic retinopathy (DR) is a neurovascular disease characterized by the reduction of retina integrity and functionality, as a consequence of retinal pigment epithelial cell fibrosis. Although galectin-1 (a glycan-binding protein) has been associated with dysregulated retinal angiogenesis, no evidence has been reported about galectin-1 roles in DR-induced fibrosis. ARPE-19 cells were cultured in normal (5 mM) or high glucose (35 mM) for 3 days, then exposed to the selective galectin-1 inhibitor OTX008 (2.5-5-10 µM) for 6 days. The determination of cell viability and ROS content along with the analysis of specific proteins (by immunocytochemistry, Western blotting, and ELISA) or mRNAs (by real time-PCR) were performed. OTX008 5 µM and 10 µM improved cell viability and markedly reduced galectin-1 protein expression in cells exposed to high glucose. This was paralleled by a down-regulation of the TGF-ß/, NF-kB p65 levels, and ROS content. Moreover, epithelial-mesenchymal transition markers were reduced by OTX008 5 µM and 10 µM. The inhibition of galectin-1 by OTX008 in DR may preserve retinal pigment epithelial cell integrity and functionality by reducing their pro-fibrotic phenotype and epithelial-mesenchymal transition phenomenon induced by diabetes.

Effects of Hydroxypropyl-Beta-Cyclodextrin on Cultured Brain Endothelial Cells.

The application of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) in the treatment of the rare cholesterol and lipid storage disorder Niemann-Pick disease type C opened new perspectives in the development of an efficient therapy. Even if the systemic administration of HPBCD was found to be effective, its low permeability across the blood-brain barrier (BBB) limited the positive neurological effects. Nevertheless, the cellular interactions of HPBCD with brain capillary endothelial cells have not been investigated in detail. In this study, the cytotoxicity, permeability, and cellular internalization of HPBCD on primary rat and immortalized human (hCMEC/D3) brain capillary endothelial cells were investigated. HPBCD shows no cytotoxicity on endothelial cells up to 100 µM, measured by impedance kinetics. Using a fluorescent derivative of HPBCD (FITC-HPBCD) the permeability measurements reveal that on an in vitro triple co-culture BBB model, FITC-HPBCD has low permeability, 0.50 × 10-6 cm/s, while on hCMEC/D3 cell layers, the permeability is higher, 1.86 × 10-5 cm/s. FITC-HPBCD enters brain capillary endothelial cells, is detected in cytoplasmic vesicles and rarely localized in lysosomes. The cellular internalization of HPBCD at the BBB can help to develop new strategies for improved HPBCD effects after systemic administration.

Cellular Effects of Cyclodextrins: Studies on HeLa Cells.

Cyclodextrins are high molecular weight, hydrophilic, cyclic, non-reducing oligosaccharides, applied as excipients for the improvement of the solubility and permeability of insoluble active pharmaceutical ingredients. On the other hand, beta-cyclodextrins are used as cholesterol sequestering agents in life sciences. Recently, we demonstrated the cellular internalization and intracellular effects of cyclodextrins on Caco-2 cells. In this study, we aimed to further investigate the endocytosis of (2-hydroxylpropyl)-beta-(HPBCD) and random methylated-beta-cyclodextrin (RAMEB) to test their cytotoxicity, NF-kappa B pathway induction, autophagy, and lysosome formation on HeLa cells. These derivatives were able to enter the cells; however, major differences were revealed in the inhibition of their endocytosis compared to Caco-2 cells. NF-kappa B p65 translocation was not detected in the cell nuclei after HPBCD or RAMEB pre-treatment and cyclodextrin treatment did not enhance the formation of autophagosomes. These cyclodextrin derivates were partially localized in lysosomes after internalization.

Enhanced Antioxidant and Anti-Inflammatory Effects of Self-Nano and Microemulsifying Drug Delivery Systems Containing Curcumin.

Turmeric has been used for decades for its antioxidant and anti-inflammatory effect, which is due to an active ingredient isolated from the plant, called curcumin. However, the extremely poor water-solubility of curcumin often limits the bioavailability of the drug. The aim of our experimental work was to improve the solubility and thus bioavailability of curcumin by developing self-nano/microemulsifying drug delivery systems (SN/MEDDS). Labrasol and Cremophor RH 40 as nonionic surfactants, Transcutol P as co-surfactant and isopropyl myristate as the oily phase were used during the formulation. The average droplet size of SN/MEDDS containing curcumin was between 32 and 405 nm. It was found that the higher oil content resulted in larger particle size. The drug loading efficiency was between 93.11% and 99.12% and all formulations were thermodynamically stable. The curcumin release was studied at pH 6.8, and the release efficiency ranged between 57.3% and 80.9% after 180 min. The results of the MTT cytotoxicity assay on human keratinocyte cells (HaCaT) and colorectal adenocarcinoma cells (Caco-2) showed that the curcumin-containing preparations were non-cytotoxic at 5 w/v%. According to the results of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase (SOD) assays, SNEDDS showed significantly higher antioxidant activity. The anti-inflammatory effect of the SN/MEDDS was screened by enzyme-linked immunosorbent assay (ELISA). SNEDDS formulated with Labrasol as surfactant, reduced tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) levels below 60% at a concentration of 10 w/w%. Our results verified the promising use of SN/MEDDS for the delivery of curcumin. This study demonstrates that the SN/MEDDS could be promising alternatives for the formulation of poorly soluble lipophilic compounds with low bioavailability.

Topical Dosage Formulation of Lyophilized Philadelphus coronarius L. Leaf and Flower: Antimicrobial, Antioxidant and Anti-Inflammatory Assessment of the Plant.

Philadelphus coronarius is a versatile plant and its use in folk medicine has a long tradition; however, scientifically, the medical utilization of the herb is a less explored research field. The aim of our study was to identify and determine the quantity of the bioactive compounds of both the leaf and the flower and prepare a lyophilized product of them, from which medical ointments were formulated, since the topical application of P. coronarius has also not been studied. In vitro drug release, texture analysis and biocompatibility experiments were carried out, as well as the investigation of microbiological, antioxidant and anti-inflammatory properties. According to our results the composition and the selected excipients of the ointments have a great impact on the drug release, texture and bioavailability of the preparation. During the microbiological testing, the P. coronarius leaf was effective against Escherichia coli and Staphylococcus aureus, but it did not significantly decrease IL-4 production when it was tested on HaCaT cells. P. coronarius is a promising herb, and its topical application in antimicrobial therapy can be a useful addition to modern medical therapy.

Formulation, Characterization and Permeability Studies of Fenugreek (Trigonella foenum-graecum) Containing Self-Emulsifying Drug Delivery System (SEDDS).

Fenugreek is used as a spice and a traditional herbal medicine for a variety of purposes, given its antidiabetic and antioxidant effects. Self-emulsifying drug delivery systems (SEDDS) of herbal drugs are targets of extensive research aiming to increase bioavailability and stability. The study's objective was to formulate SEDDS containing Trigonella foenum-graecum extract to improve the stability of herbal extract and to increase their permeability through a Caco-2 monolayer. A characterized fenugreek dry extract was used for the formulations, while the SEDDS properties were examined by particle size analysis and zeta potential measurements. Permeability assays were carried out on Caco-2 cell monolayers, the integrity of which was monitored by follow-up trans-epithelial electric resistance measurements (TEER). Cytocompatibility was tested by the MTT method, and an indirect dissolution test was performed, using DPPH antioxidant reagent. Two different SEDDS compositions were formulated from a standardized fenugreek dry extract at either the micro- or the nanoemulsion scale with sufficient stability, enhanced bioavailability of the compounds, and sustained release from HPMC capsules. Based on our results, a modern, non-toxic, cytocompatible fenugreek SEDDS formulation with high antioxidant capacity was developed in order to improve the permeability and bioavailability of all components.

Preterm Intraventricular Hemorrhage-Induced Inflammatory Response in Human Choroid Plexus Epithelial Cells.

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.

Complexation with Random Methyl-ß-Cyclodextrin and (2-Hidroxypropyl)-ß-Cyclodextrin Enhances In Vivo Anti-Fibrotic and Anti-Inflammatory Effects of Chrysin via the Inhibition of NF-κB and TGF-ß1/Smad Signaling Pathways and Modulation of Hepatic Pro/Anti-Fibrotic miRNA.

Chrysin (CHR) is a natural flavonoid with a wide range of pharmacological activities, including hepatoprotection, but poor water solubility. By including water-soluble hydroxypropyl (HPBCD) and randomly methylated (RAMEB) ß-cyclodextrin, we aimed to increase its biodisponibility and the effectiveness of the antifibrotic effects of chrysin at oral administration. Liver fibrosis in mice was induced in 7 weeks by CCl4 i.p. administration, and afterwards treated with 50 mg/kg of CHR-HPBCD, CHR-RAMEB, and free chrysin. CCl4 administration increased hepatic inflammation (which was augmented by the upregulation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB), tumor necrosis factor (TNF)-α, and interleukin 6 (IL-6) and induced fibrosis, as determined using histopathology and electron microscopy. These results were also confirmed by the upregulation of Collagen I (Col I) and matrix metalloproteinase (MMP) expression, which led to extracellular fibrotic matrix proliferation. Moreover, the immunopositivity of alpha-smooth muscle actin (a-SMA) in the CCl4 group was evidence of hepatic stellate cell (HSC) activation. The main profibrotic pathway was activated, as confirmed by an increase in the transforming growth factor- ß1 (TGF-ß1) and Smad 2/3 expression, while Smad 7 expression was decreased. Treatment with CHR-HPBCD and CHR-RAMEB considerably reduced liver injury, attenuated inflammation, and decreased extracellular liver collagen deposits. CHR-RAMEB was determined to be the most active antifibrotic complex. We conclude that both nanocomplexes exert anti-inflammatory effects and antifibrotic effects in a considerably stronger manner than for free chrysin administration.

Formulation of Novel Liquid Crystal (LC) Formulations with Skin-Permeation-Enhancing Abilities of Plantago lanceolata (PL) Extract and Their Assessment on HaCaT Cells.

Exposure to reactive oxygen species can easily result in serious diseases, such as hyperproliferative skin disorders or skin cancer. Herbal extracts are widely used as antioxidant sources in different compositions. The importance of antioxidant therapy in inflammatory conditions has increased. Innovative formulations can be used to improve the effects of these phytopharmacons. The bioactive compounds of Plantago lanceolata (PL) possess different effects, such as anti-inflammatory, antioxidant, and bactericidal pharmacological effects. The objective of this study was to formulate novel liquid crystal (LC) compositions to protect Plantago lanceolata extract from hydrolysis and to improve its effect. Since safety is an important aspect of pharmaceutical formulations, the biological properties of applied excipients and blends were evaluated using assorted in vitro methods on HaCaT cells. According to the antecedent toxicity screening evaluation, three surfactants were selected (Gelucire 44/14, Labrasol, and Lauroglycol 90) for the formulation. The dissolution rate of PL from the PL-LC systems was evaluated using a Franz diffusion chamber apparatus. The antioxidant properties of the PL-LC systems were evaluated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and malondialdehyde (MDA) assessments. Our results suggest that these compositions use a nontraditional, rapid-permeation pathway for the delivery of drugs, as the applied penetration enhancers reversibly alter the barrier properties of the outer stratum corneum. These excipients can be safe and highly tolerable thus, they could improve the patient's experience and promote adherence.

Process Optimization for the Continuous Production of a Gastroretentive Dosage Form Based on Melt Foaming.

Several drugs have poor oral bioavailability due to low or incomplete absorption which is affected by various effects as pH, motility of GI, and enzyme activity. The gastroretentive drug delivery systems are able to deal with these problems by prolonging the gastric residence time, while increasing the therapeutic efficacy of drugs. Previously, we developed a novel technology to foam hot and molten dispersions on atmospheric pressure by a batch-type in-house apparatus. Our aim was to upgrade this technology by a new continuous lab-scale apparatus and confirm that our formulations are gastroretentive. At first, we designed and built the apparatus and continuous production was optimized using a Box-Behnken experimental design. Then, we formulated barium sulfate-loaded samples with the optimal production parameters, which was suitable for in vivo imaging analysis. In vitro study proved the low density, namely 507 mg/cm3, and the microCT record showed high porosity with 40 µm average size of bubbles in the molten suspension. The BaSO4-loaded samples showed hard structure at room temperature and during the wetting test, the complete wetting was detected after 120 min. During the in vivo study, the X-ray taken showed the retention of the formulation in the rat stomach after 2 h. We can conclude that with our device low-density floating formulations were prepared with prolonged gastric residence time. This study provides a promising platform for marketed active ingredients with low bioavailability.

Reduced miR-26b Expression in Megakaryocytes and Platelets Contributes to Elevated Level of Platelet Activation Status in Sepsis.

In sepsis, platelets may become activated via toll-like receptors (TLRs), causing microvascular thrombosis. Megakaryocytes (MKs) also express these receptors; thus, severe infection may modulate thrombopoiesis. To explore the relevance of altered miRNAs in platelet activation upon sepsis, we first investigated sepsis-induced miRNA expression in platelets of septic patients. The effect of abnormal Dicer level on miRNA expression was also evaluated. miRNAs were profiled in septic vs. normal platelets using TaqMan Open Array. We validated platelet miR-26b with its target SELP (P-selectin) mRNA levels and correlated them with clinical outcomes. The impact of sepsis on MK transcriptome was analyzed in MEG-01 cells after lipopolysaccharide (LPS) treatment by RNA-seq. Sepsis-reduced miR-26b was further studied using Dicer1 siRNA and calpain inhibition in MEG-01 cells. Out of 390 platelet miRNAs detected, there were 121 significantly decreased, and 61 upregulated in sepsis vs. controls. Septic platelets showed attenuated miR-26b, which were associated with disease severity and mortality. SELP mRNA level was elevated in sepsis, especially in platelets with increased mean platelet volume, causing higher P-selectin expression. Downregulation of Dicer1 generated lower miR-26b with higher SELP mRNA, while calpeptin restored miR-26b in MEG-01 cells. In conclusion, decreased miR-26b in MKs and platelets contributes to an increased level of platelet activation status in sepsis.

Formulation of Creams Containing Spirulina Platensis Powder with Different Nonionic Surfactants for the Treatment of Acne Vulgaris.

Natural products used in the treatment of acne vulgaris may be promising alternative therapies with fewer side effects and without antibiotic resistance. The objective of this study was to formulate creams containing Spirulina (Arthrospira) platensis to be used in acne therapy. Spirulina platensis belongs to the group of micro algae and contains valuable active ingredients. The aim was to select the appropriate nonionic surfactants for the formulations in order to enhance the diffusion of the active substance and to certify the antioxidant and antibacterial activity of Spirulina platensis-containing creams. Lyophilized Spirulina platensis powder (SPP) was dissolved in Transcutol HP (TC) and different types of nonionic surfactants (Polysorbate 60 (P60), Cremophor A6:A25 (CR) (1:1), Tefose 63 (TFS), or sucrose ester SP 70 (SP70)) were incorporated in creams as emulsifying agents. The drug release was evaluated by the Franz diffusion method and biocompatibility was tested on HaCaT cells. In vitro antioxidant assays were also performed, and superoxide dismutase (SOD) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays were executed. Antimicrobial activities of the selected compositions were checked against Staphylococcus aureus (S. aureus) and Cutibacteriumacnes (C. acnes) (formerly Propionibacterium acnes) with the broth microdilution method. Formulations containing SP 70 surfactant with TC showed the most favorable dissolution profiles and were found to be nontoxic. This composition also showed significant increase in free radical scavenger activity compared to the blank sample and the highest SOD enzyme activity was also detected after treatment with the cream samples. In antibacterial studies, significant differences were observed between the treated and control groups after an incubation time of 6 h.

In Vitro Tests of FDM 3D-Printed Diclofenac Sodium-Containing Implants.

One of the most promising emerging innovations in personalized medication is based on 3D printing technology. For use as authorized medications, 3D-printed products require different in vitro tests, including dissolution and biocompatibility investigations. Our objective was to manufacture implantable drug delivery systems using fused deposition modeling, and in vitro tests were performed for the assessment of these products. Polylactic acid, antibacterial polylactic acid, polyethylene terephthalate glycol, and poly(methyl methacrylate) filaments were selected, and samples with 16, 19, or 22 mm diameters and 0%, 5%, 10%, or 15% infill percentages were produced. The dissolution test was performed by a USP dissolution apparatus 1. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye (MTT)-based prolonged cytotoxicity test was performed on Caco-2 cells to certify the cytocompatibility properties. The implantable drug delivery systems were characterized by thermogravimetric and heatflow assay, contact angle measurement, scanning electron microscopy, microcomputed tomography, and Raman spectroscopy. Based on our results, it can be stated that the samples are considered nontoxic. The dissolution profiles are influenced by the material properties of the polymers, the diameter, and the infill percentage. Our results confirm the potential of fused deposition modeling (FDM) 3D printing for the manufacturing of different implantable drug delivery systems in personalized medicine and may be applied during surgical interventions.