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BACKGROUND: Flap hypoperfusion or ischemia-reperfusion (I/R) may occur during preparation-transposition procedures and by postoperative thrombotic complications. Behind the microcirculatory disturbances micro-rheological alterations are also supposed. OBJECTIVE: We aimed to investigate the groin flap I/R with following-up micro-rheological parameters. METHODS: Anesthetized rats were subjected to Control or I/R groups. Groin flaps were prepared bilaterally, pedicled on the superficial epigastric vessels. In the Control group the flaps were re-sutured after one hour, while in I/R group microvascular clips were applied on the pedicles for 60 minutes, then the flaps were repositioned. Besides daily wound control, before the operation and on the 1st, 3rd, 5th, 7th and 14th postoperative days blood samples were collected for testing red blood cell (RBC) deformability (rotational ektacytometry) and aggregation (light-transmission aggregometry). RESULTS: RBC deformability significantly worsened by the 3rd–7th postoperative day in I/R group. RBC aggregation enhanced significantly by the 1st day, in I/R group it remained elevated on the 3rd day as well. In a complicated case with unilateral flap necrosis, RBC deformability and aggregation worsening was outlined from its group (base, 1st, 3rd day). CONCLUSION: Wound healing affected micro-rheological parameters in the early postoperative period. Flap I/R exacerbated the alterations. The parameters markedly worsened in case of flap necrosis.

Ovarian tumor is one of the leading causes of cancer among women. Patients are diagnosed at an advanced stage, usually. There is a need for new specific and sensitive biomarkers. Mitochondrial DNA copy number change was observed in various cancers. Our aim was to detect mitochondrial DNA copy number in whole blood (wb-mtDNA) and in plasma (cell-free and exosome encapsulated mtDNA) in patients with serous epithelial ovarian tumor. DNA was isolated from EDTA blood and plasma obtained from 24 patients and 24 healthy controls. Exosomes were isolated from cell-free plasma, and exosome encapsulated DNA (exoDNA) was extracted. Quantitative-real-time PCR was performed with Human Mitochondrial DNA (mtDNA) Monitoring Primer Set. Kruskall‑Wallis and Mann‑Whitney U test were used for data analysis. Wb-mtDNA copy number was significantly different among healthy controls and patients in multiple comparison (p = 0.0090 considering FIGO stage independently, and p = 0.0048 considering early- and late-stage cancers). There was a significant decrease among early-stage, all advanced stage and all cancer patients (FIGO I: 32.5 ± 8.3, p = 0.0061; FIGO III + IV: 37.2 ± 13.7 p = 0.0139; FIGO I + III + IV: 35.6 ± 12.2, p = 0.0017) or FIGO III patients alone (32.8 ± 5.6, p = 0.00089) compared to healthy controls. We found significant increase in copy number in exosomal mtDNA in cancer patients (236.0 ± 499.0, p = 0.0155), advanced-stage cancer patients (333.0 ± 575.0, p = 0.0095), of FIGO III (362.0 ± 609.2, p = 0.0494), and FIGO IV (304.0 ± 585.0, p = 0.0393) patients alone but not in samples of FIGO I patients (10.0 ± 3.5, p = 0.3907). In multiple comparison the increase was significant considering early- and late-stage cancers (p = 0.0253). Cell-free mtDNA copy numbers were not increased significantly. We found the highest copy number of mtDNA in exosomes, followed by plasma and peripheral blood in late-stage cancer patients. We observed significant difference in wb-mtDNA copy number between healthy controls and both early- and late-stage cancer patients.

Mitochondrial DNA (mtDNA) copy number changes have been associated with various diseases. Several studies showed that mtDNA content in peripheral blood was associated with oxidative stress and cardiovascular disease. Atrial fibrillation (AF) is one of the severe cardiovascular diseases. We aimed to determine the mtDNA copy numbers in peripheral blood, in cell-free plasma and in exosomes of AF patients and healthy controls. Peripheral blood was drawn from 60 AF patients and 72 healthy controls. DNA was isolated from EDTA blood and plasma. Exosomes were isolated from cell-free plasma and then exosome encapsulated DNA (exoDNA) was extracted. Quantitative-real-time PCR was performed with Human Mitochondrial DNA (mtDNA) Monitoring Primer Set. Statistical analysis of the data was performed. We found statistically significant difference in mtDNA copy numbers in DNA isolated from peripheral whole blood, cell-free plasma and exosome samples of controls’ (44.4 ± 18.0, 27.2 ± 30.1, 11.5 ± 8.7), and patients’ group (43.4 ± 13.6, 26.2 ± 26.4, 14.5 ± 12.3). However there was no significant difference in mtDNA copy number between the two study groups either in peripheral blood, in cell-free plasma and in exosomes, and even in different sexes and ages. We found the highest copy number of mtDNA in peripheral blood, followed by plasma and exosomes. We did not find differences between patients and controls, neither age nor gender had effect on the mtDNA copy number. According to our results the mtDNA copy numbers did not differ in AF patients.

RATIONALE: Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known. OBJECTIVES: Our objective was to identify and characterize novel anti-inflammatory molecules from M. vaccae NCTC 11659. METHODS: We have purified and identified a unique anti-inflammatory triglyceride, 1,2,3-tri [Z-10-hexadecenoyl] glycerol, from M. vaccae and evaluated its effects in freshly isolated murine peritoneal macrophages. RESULTS: The free fatty acid form of 1,2,3-tri [Z-10-hexadecenoyl] glycerol, 10(Z)-hexadecenoic acid, decreased lipopolysaccharide-stimulated secretion of the proinflammatory cytokine IL-6 ex vivo. Meanwhile, next-generation RNA sequencing revealed that pretreatment with 10(Z)-hexadecenoic acid upregulated genes associated with peroxisome proliferator-activated receptor alpha (PPARα) signaling in lipopolysaccharide-stimulated macrophages, in association with a broad transcriptional repression of inflammatory markers. We confirmed using luciferase-based transfection assays that 10(Z)-hexadecenoic acid activated PPARα signaling, but not PPARγ, PPARδ, or retinoic acid receptor (RAR) α signaling. The effects of 10(Z)-hexadecenoic acid on lipopolysaccharide-stimulated secretion of IL-6 were prevented by PPARα antagonists and absent in PPARα-deficient mice. CONCLUSION: Future studies should evaluate the effects of 10(Z)-hexadecenoic acid on stress-induced exaggeration of peripheral inflammatory signaling, central neuroinflammatory signaling, and anxiety- and fear-related defensive behavioral responses.

Methotrexate functionalized silica-gelatin hybrid aerogel (SGM) was synthesized by the sol–gel method and co-gelation. The drug methotrexate (MTX) is covalently linked to the collagen molecules of the hybrid aerogel backbone by amide-bond. The characteristic MTX content of the functionalized hybrid aerogel is ca. 6 wt% by the dry weight. The micronization of SGM aerogel in water yields cell sized (d = 10–20 µm) particles. The cytotoxicity of these microparticles against tumor cell lines (SCC VII and HL-60) is unprecedentedly high, it is approximately equivalent to that of an equal dose of free (dissolved) MTX, as proved by in vitro experiments. Thus, the activity of MTX is intact after aerogel functionalization, and the mass specific cytotoxicity of SGM is high enough for medical applications. Drug release studies verified that MTX cannot be liberated from this drug delivery system solely by chemical hydrolysis, however, collagenase enzymatic activity releases MTX from the functionalized hybrid aerogel. The cytotoxicity of SGM towards various cancerous and non-cancerous cell lines correlates with the collagenase activities of cells. Therefore, conjugation with the hybrid aerogel provides a controlled release system for the antineoplastic agent MTX. The morphology of the delivery vehicle was chosen to adapt the size of cancer cells; thus the metastatic pathways of the tumor cells can get flooded.

Dietary food, depending on timing, amount and composition can influence gene expression in various tissues. Here, we investigated the effect of high-fat meal diets of different compositions on the gene expression pattern of human skeletal muscle. Gene expression data of skeletal muscle samples from human volunteers prior and 4 h after the consumption of high lipid-containing meal consisting of either saturated-, monounsaturated- or polyunsaturated fatty acids were downloaded from the public repository. List of 843 differently expressed genes (DEGs) was generated. Functional analysis revealed that circadian rhythm-, inflammation- and oxidative stress-related genes are highly overrepresented among the DEGs. The magnitude of gene expression changes significantly increases with the saturation level of the dietary fatty acids and the majority of the DEGs are upregulated. We propose that, by altering circadian clock gene expression and inducing inflammation and oxidative stress, high lipid intake can contribute to muscle function decay in the long run.

Myosin phosphatase (MP) holoenzyme is a Ser/Thr specific enzyme, which is the member of protein phosphatase type 1 (PP1) family and composed of a PP1 catalytic subunit (PP1c/PPP1CB) and a myosin phosphatase targeting subunit (MYPT1/PPP1R12A). PP1c is required for the catalytic activity of the holoenzyme, while MYPT1 regulates MP through targeting the holoenzyme to its substrates. Above the well-characterized function of MP, as the major regulator of smooth muscle contractility mediating the dephosphorylation of 20 kDa myosin light chain, accumulating data support its role in other, non-contractile functions. In this review, we summarize the scaffold function of MP holoenzyme and its roles in processes such as cell cycle, development, gene expression regulation and neurotransmitter release. In particular, we highlight novel interacting proteins of MYPT1 and pathophysiological functions of MP relevant to tumorigenesis, insulin resistance and neurodegenerative disorders. This article is part of a Special Issue entitled: Protein Phosphatases as Critical Regulators for Cellular Homeostasis edited by Prof. Peter Ruvolo and Dr. Veerle Janssens.

A hasnyálmirigy-adenocarcinoma az egyik legmagasabb halálozású daganat, medián ötéves túlélése 7-8%, korai diagnosztikája, eredményes kezelése kihívást jelent. A daganatos betegségekhez, így a pancreasrákhoz is gyakran társul a mikrobiom összetételének megváltozása (dysbiosis), amelyet onkobiózisnak nevezünk. Maga a pancreas is gyakran kolonizálódik (pl. többek között Helicobacter pylorival és Malassezia-fajokkal). Az onkobiomok összetétele eltér a rövid és hosszú túlélők esetén. Amennyiben pancreas-adenocarcinomás hosszú túlélők mikrobiomját ültetik hasnyálmirigy-adenocarcinoma állatmodellbe, az egerek túlélése meghosszabbodik. Az onkobiom modulálja a daganatos elfajuláshoz vezető gyulladásos folyamatot pancreas-adenocarcinomában. Az összefoglalóban a bakteriális metabolitok (rövid láncú zsírsavak, másodlagos epesavak, poliaminok, indolszármazékok) szerepét mutatjuk be a pancreas-adenocarcinoma patogenezisében, kitérve arra, hogy a betegségben a bakteriális metabolizmus és a bakteriális metabolom szabályozása is felborul. További metabolitok és anyagcsere-útvonalak felfedezése várható a közeljövőben, tovább bővítve ezen patogenetikai útvonal nyújtotta diagnosztikus és terápiás lehetőségeket hasnyálmirigy-adenocarcinomában.

LB KA

Cholesterol synthesis is a complex, coordinated process involving a series of enzymes. As of today, our understanding of subcellular localization of cholesterol biosynthesis enzymes is far from complete. Considering the complexity and intricacies of this pathway and the importance of functions of DHCR7, DHCR24 and EBP enzymes for human health, we undertook a study to determine their subcellular localization and co-localization. Using expression constructs and antibody staining in cell cultures and transgenic mice, we found that all three enzymes are expressed in ER and nuclear envelope. However, their co-localization was considerably different across the cellular compartments. Furthermore, we observed that in the absence of DHCR7 protein, DHCR24 shows a compensatory upregulation in a Dhcr(−/−) transgenic mouse model. The overall findings suggest that the sterol biosynthesis enzymes might not always work in a same functional complex, but that they potentially have different, multifunctional roles that go beyond the sterol biosynthesis pathway. Furthermore, the newly uncovered compensatory mechanism between DHCR7 and DHCR24 could be of importance for designing medications that would improve cholesterol production in patients with desmosterolosis and Smith-Lemli-Opitz syndrome.

Previously we described similarities and differences in the organization and molecular composition of an aggrecan based extracellular matrix (ECM) in three precerebellar nuclei, the inferior olive, the prepositus hypoglossi nucleus and the red nucleus of the rat associated with their specific cytoarchitecture, connection and function in the vestibular system. The aim of present study is to map the ECM pattern in a mesencephalic precerebellar nucleus, the pararubral area, which has a unique function among the precerebellar nuclei with its retinal connection and involvement in the circadian rhythm regulation. Using histochemistry and immunohistochemistry we have described for the first time the presence of major ECM components, the hyaluronan, aggrecan, versican, neurocan, brevican, tenascin-R (TN-R), and the HAPLN1 link protein in the pararubral area. The most common form of the aggrecan based ECM was the diffuse network in the neuropil, but each type of the condensed forms was also recognizable. Characteristic perineuronal nets (PNNs) were only recognizable with Wisteria floribunda agglutinin (WFA) and aggrecan staining around some of the medium-sized neurons, whereas the small cells were rarely surrounded by a weakly stained PNNs. The moderate expression of key molecules of PNN, the hyaluronan (HA) and HAPLN1 suggests that the lesser stability of ECM assembly around the pararubral neurons may allow quicker response to the modified neuronal activity and contributes to the high level of plasticity in the vestibular system.

Wound healing is a complex multiphase process which can be hampered by many factors including impaired local circulation, hypoxia, infection, malnutrition, immunosuppression, and metabolic dysregulation in diabetes. Redox dysregulation is a common feature of many skin diseases demonstrated by virtually all cell types in the skin with overproduction of reactive oxygen and nitrogen species. The objective of this study was to characterize the redox environment in wound fluids and sera from patients suffering from chronic leg ulcers (n=19) and acute wounds (bulla fluids from second degree burns; n=11) with serum data also compared to those from healthy volunteers (n=7). Significantly higher concentrations of TNF-α, interleukine-8, vascular endothelial growth factor, and lactate dehydrogenase (measure of cell damage) were found in fluids from chronic wounds compared to acute ones. The extent of protein carbonylation (measure of protein oxidation), lipid peroxidation, and tyrosine nitration (indicator of peroxynitrite production) was similar in acute and chronic wound fluids, while radical scavenging activity and glutathione (GSH) levels were elevated in chronic wound fluids compared to acute wounds. Sera were also assessed for the same set of parameters with no significant differences detected. Nitrotyrosine (the footprint of the potent oxidant peroxynitrite) and poly(ADP-ribose) (the product of the DNA damage sensor enzyme PARP-1) could be detected in wound biopsies. Our data identify multiple signs of redox stress in chronic wounds with notable differences. In chronic wounds, elevations in antioxidant levels/activities may indicate compensatory mechanisms against inflammation. The presence of nitrotyrosine and poly(ADP-ribose) in tissues from venous leg ulcers indicate peroxynitrite production and PARP activation in chronic wounds.

Metastatic studies on rats showed that after subrenal implantation of tumor cells under the capsule of the kidney or subhepatic implantation under Glisson's capsule of the liver generated primary tumors in these organs. It was assumed that tumor cells that escaped through the disrupted peripheral blood vessels of primary tumors entered the peritoneal cavity, crossed the diaphragm, and appeared in the thoracal, primarily in the parathymic lymph nodes. This explanation did not answer the question whether distant lymph nodes were reached via the blood stream from the primary tumor or through the thoracal lymphatic vessels. In this work, we investigated the metastatic pathway in C3H/HeJ mice, after direct intraperitoneal administration of murine SCC VII cells bypassing the hematogenic spread of tumor cells. The direct pathway was also mimicked by intraperitoneal injection of Pelican Ink colloidal particles, which appeared in the parathymic lymph nodes, similarly to the tumor cells that caused metastasis in the parathymic lymph nodes and in the thymic tissue. The murine peritoneal-parathymic lymph node route indicates a general mechanism of tumor progression from the abdominal effusion. This pathway starts with the growth of abdominal tumors, continues as thoracal metastasis in parathymic lymph nodes and may proceed as mammary lymph node metastasis.

Intrinsic optical signal imaging (IOSI) records tiny changes in optical reflection of the exposed cortical surface due to neuronal activation related local hemodynamic changes. Cortical electrophysiology performed in the very same visual cortical area may provide additional insight into the connectivity between functional domains. Our aim is to investigate the simultaneous use of IOSI and μECoG (micro-electrocorticography) techniques by introducing a transparent polymer based subdural microelectrode array into the optical recording chamber used for in vivo functional mapping experiments in anaesthetized cats. To demonstrate the feasibility of the combined optical-electrical recording, we have run several stimulus protocols and measured the evoked optical and electrical responses of the primary visual cortex in a synchronized manner. Optical quality of IOSI signals (609 nm) was also evaluated without the ECoG microelectrode as a reference. During the visual stimulus, local field potential on a 32-channel of the ECoG was simultaneously recorded and evaluated. Specific power density distribution was detected in wide band frequency range, and the distribution of a unique 80 Hz oscillation in gamma range occurring only as a response to stimuli was also found. This is the first demonstration of microelectrocorticography using PI/ITO/PI based microelectrode arrays in combination with in vivo IOSI.

Wound healing is a complex sequence of cellular and molecular processes such as inflammation, cell migration, proliferation and differentiation. ROCK is a widely investigated Ser/Thr kinase with important roles in rearranging the actomyosin cytoskeleton. ROCK inhibitors have already been approved to improve corneal endothelial wound healing. The purpose of this study was to investigate the functions of myosin phosphatase (MP or PPP1CB), a type-1 phospho-Ser/Thr-specific protein phosphatase (PP1), one of the counter enzymes of ROCK, in skin homeostasis and wound healing. To confirm our hypotheses, we applied tautomycin (TM), a selective PP1 inhibitor, on murine skin that caused the arrest of wound closure. TM suppressed scratch closure of HaCaT human keratinocytes without having influence on the survival of the cells. Silencing of, the regulatory subunit of MP (MYPT1 or PPP1R12A), had a negative impact on the migration of keratinocytes and it influenced the cell-cell adhesion properties by decreasing the impedance of HaCaT cells. We assume that MP differentially activates migration and differentiation of keratinocytes and plays a key role in the downregulation of transglutaminase-1 in lower layers of skin where no differentiation is required. MAPK Proteome Profiler analysis on human ex vivo biopsies with MYPT1-silencing indicated that MP contributes to the mediation of wound healing by regulating the Akt signaling pathway. Our findings suggest that MP plays a role in the maintenance of normal homeostasis of skin and the process of wound healing.

Background Micro-rheological relations of renal ischemia-reperfusion (I/R) have not been completely elucidated yet. Concerning anti-inflammatory agents, it is supposed that sigma-1 receptor agonist N,N-dimethyl-tryptamin (DMT) can be useful to reduce I/R injury. Objective To investigate the micro-rheological and metabolic parameters, and the effects of DMT in renal I/R in rats. Methods In anesthetized rats from median laparotomy both kidneys were exposed. In Control group (n = 6) no other intervention happened. In I/R group (n = 10) the right renal vessels were ligated and after 60 minutes the organ was removed. The left renal vessels were clamped for 60 minutes followed by 120-minute reperfusion. In I/R+DMT group (n = 10) DMT was administered 15 minutes before the ischemia. Blood samples were taken before/after ischemia and during the reperfusion for testing hematological, metabolic parameters, erythrocyte deformability and aggregation. Results Lactate concentration significantly increased and accompanied with decreased blood pH. Enhanced erythrocyte aggregation and impaired deformability were observed from the 30th minute of reperfusion. In I/R+DMT group we found diminished changes compared to the I/R group (lactate, pH, electrolytes, red blood cell deformability and aggregation). Conclusions Metabolic and micro-rheological parameters impair during renal I/R. DMT could reduce but not completely prevent the changes in this rat model.

Macrophages are able to differentiate into classically polarized (M1) or alternatively polarized (M2) states upon encountering pro-inflammatory cytokines such as interferon (IFN) γ or anti-inflammatory cytokines such as interleukin (IL) -4/IL-13, respectively. Moreover, macrophages are known to regulate lipid metabolism via multiple members of the nuclear hormone receptor family, including the retinoid X receptors (RXR). It has been also documented that cytokines are able to modulate macrophage responses to lipid signals but the nature of these interactions and the underlying mechanisms of these processes especially at the level of the chromatinized genome are not well understood. Previous work from our laboratory suggested that STAT6 is a facilitator of nuclear receptor mediated transcriptional activity acting at the genome level. This prompted us to investigate genome-wide DNA binding events and the development of cistromes in human CD14+ monocyte-derived macrophages upon exposure to IL-4. We determined the impact of IL-4 on the PU.1, RXR and STAT6 cistromes within the active enhancer regions marked by H3K27-acetylation using chromatin immunoprecipitation followed by deep sequencing and integrated bioinformatics analyses. We found that about 2/3rd of the IL-4 induced STAT6 peaks co-localized with RXR peaks. These STAT6/RXR co-peaks differed at least in part from the non-overlapping RXR peaks regarding the most enriched de novo transcription factor binding motifs. Interestingly, RXR-binding was not regulated at the STAT6/RXR co-bound enhancers following IL-4 stimulation, but differential enhancer interactions were observed between the IL-4/STAT6 and RXR signaling pathways acting in a gene selective manner. Our results suggest that there is a novel, so far uncharacterized cistromic crosstalk between RXR and STAT6 that is likely to contribute to the formation of the active enhancer repertoire, transcriptome and differential signal-specific gene regulation of polarized macrophages.

In this study, a mesoporous silica-aerogel with β-tricalcium phosphate (β-TCP-AE) was manufactured. The effect of β-TCP-AE on gene expressions (BMP2, BMP7, Runx2 and OSX) of SAOS-2 cells was tested...

The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumor evolution is poorly understood. Phenotypic drifts can be transmitted via inheritable transcriptional programs. Cell-type specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers. Here we have annotated the epigenome of 47 primary and metastatic estrogen-receptor (ERα)-positive breast cancer clinical specimens and inferred phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Shared regions contain a unique set of regulatory information including the motif for transcription factor YY1. We identify YY1 as a critical determinant of ERα transcriptional activity promoting tumor growth in most luminal patients. YY1 also contributes to the expression of genes mediating resistance to endocrine treatment. Finally, we used H3K27ac levels at active enhancer elements as a surrogate of intra-tumor phenotypic heterogeneity to track the expansion and contraction of phenotypic subpopulations throughout breast cancer progression. By tracking the clonality of SLC9A3R1-positive cells, a bona fide YY1-ERα-regulated gene, we show that endocrine therapies select for phenotypic clones under-represented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.

OBJECTIVE Fetal samples obtained by invasive techniques are prone to maternal cell contamination (MCC), which may lead to false genotyping results. Our aim was to determine 3 molecular genetic tests' sensitivity to MCC. METHOD By mixing experiments, 1%, 5%, 10%, 20%, 30%, and 40% MCC was simulated, and significant MCC levels were determined for Sanger DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and pyrosequencing, a next-generation sequencing method. RESULTS For Sanger sequencing, the limit of sensitivity to MCC was 5% to 30%. For MLPA, a higher proportion of MCC (≥40%) was shown to lead to diagnostic uncertainty. In contrast, pyrosequencing proved to be very sensitive to MCC, detecting a proportion as low as 1%. CONCLUSION In the case of Sanger sequencing, sensitivity to MCC was variable, while for MLPA, only high levels of MCC proved to be significant. Although the next-generation sequencing method was sensitive to low-level MCC, if MCC level is determined in parallel, accurate quantification of allelic ratios can help to interpret the diagnostic results. Knowledge of significant MCC levels allows correct prenatal diagnosis even if samples are not purely of fetal origin and repeated sampling can be avoided in many of the cases.

Biobanks operating at ambient temperatures would dramatically reduce the costs associated with standard cryogenic storage. In the present study, we used lyophilization to stabilize unfractionated human cells in a dried state at room temperature and tested the yield and integrity of the isolated RNA by microfluidic electrophoresis, RT-qPCR and RNA sequencing. RNA yields and integrity measures were not reduced for lyophilized cells (unstored, stored for two weeks or stored for two months) compared to their paired controls. The abundance of the selected mRNAs with various expression levels, as well as enhancer-associated RNAs and cancer biomarker long non-coding RNAs (MALAT1, GAS5 and TUG1), were not significantly different between the two groups as assessed by RT-qPCR. RNA sequencing data of three lyophilized samples stored for two weeks at room temperature revealed a high degree of similarity with their paired controls in terms of the RNA biotype distribution, cumulative gene diversity, gene body read coverage and per base mismatch rate. Among the 28 differentially expressed genes transcriptional regulators, as well as certain transcript properties suggestive of a residual active decay mechanism were enriched. Our study suggests that freeze-drying of human cells is a suitable alternative for the long-term stabilization of total RNA in whole human cells for routine diagnostics and high-throughput biomedical research.

Redox regulation has been proposed to control various aspects of carcinogenesis, cancer cell growth, metabolism, migration, invasion, metastasis and cancer vascularization. As cancer has many faces, the role of redox control in different cancers and in the numerous cancer-related processes often point in different directions. In this review, we focus on the redox control mechanisms of tumor cell destruction. The review covers the tumor-intrinsic role of oxidants derived from the reduction of oxygen and nitrogen in the control of tumor cell proliferation as well as the roles of oxidants and antioxidant systems in cancer cell death caused by traditional anticancer weapons (chemotherapeutic agents, radiotherapy, photodynamic therapy). Emphasis is also put on the role of oxidants and redox status in the outcome following interactions between cancer cells, cytotoxic lymphocytes and tumor infiltrating macrophages. Keywords: Cancer, Redox regulation, Natural killer cells, Cytotoxic lymphocytes, Chemotherapeutics, Free radicals, Antioxidants

Data provided here are related to the research article entitled as ‘A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening’. Here we describe data related to the investigation of the properties of the His6-MBP-VSQNY↓PIVQ-mApple recombinant protein substrate and its interactions with Ni-NTA magnetic beads, including the dependence of substrate attachment on incubation time and concentration. Data on the folding efficiency and conformational stability of the recombinant substrate assessed by tryptic digestion are also presented. We describe here the changes of fluorescent properties and binding abilities upon treatments commonly used for stopping enzymatic reactions: trichloroacetic acid (TCA) or heat treatment. Keywords: Recombinant fusion protein substrate, Protease assay, Fluorescent protein

More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field.

Abstract: Autoinflammatory diseases are disorders of the innate immune system characterized by recurrent systematic inflammation and serious complications. Dysregulation of inflammasome and overproduction of interleukin-1 play a major role in the pathogenesis of autoinflammatory diseases. The diagnosis of these rare conditions rely on recognising the pattern of presentation and differential diagnosis. Manifestations may include fever, rash, serositis (pleuritis and peritonitis), arthritis, meningitis and uveitis. Secondary amyloidosis may complicate longstanding disease. Advances in our understanding of the molecular and pathophysiological basis of the autoinflammatory diseases have resulted in new treatment strategies. Early diagnosis and effective therapy are critical to prevent irreversible organ damage. The purpose of this review is to describe the major clinical, genetic, and therapeutic features of the most common autoinflammatory syndromes. Orv Hetil. 2018; 159(23): 898–907.

Glutamate is the most abundant neurotransmitter of the central nervous system, as the majority of neurons use glutamate as neurotransmitter. It is also well known that this neurotransmitter is not restricted to synaptic clefts, but found in the extrasynaptic regions as ambient glutamate. Extrasynaptic glutamate originates from spillover of synaptic release, as well as from astrocytes and microglia. Its concentration is magnitudes lower than in the synaptic cleft, but receptors responding to it have higher affinity for it. Extrasynaptic glutamate receptors can be found in neuronal somatodendritic location, on astroglia, oligodendrocytes or microglia. Activation of them leads to changes of neuronal excitability with different amplitude and kinetics. Extrasynaptic glutamate is taken up by neurons and astrocytes mostly via EAAT transporters, and astrocytes, in turn metabolize it to glutamine. Extrasynaptic glutamate is involved in several physiological phenomena of the central nervous system. It regulates neuronal excitability and synaptic strength by involving astroglia; contributing to learning and memory formation, neurosecretory and neuromodulatory mechanisms, as well as sleep homeostasis.The extrasynaptic glutamatergic system is affected in several brain pathologies related to excitotoxicity, neurodegeneration or neuroinflammation. Being present in dementias, neurodegenerative and neuropsychiatric diseases or tumor invasion in a seemingly uniform way, the system possibly provides a common component of their pathogenesis. Although parts of the system are extensively discussed by several recent reviews, in this review I attempt to summarize physiological actions of the extrasynaptic glutamate on neuronal excitability and provide a brief insight to its pathology for basic understanding of the topic.

Background: Clinical treatment of heart failure is still suffering from limited efficacy and unfavorable side effects. The recently developed group of agents, the myosin motor activators, act directly on cardiac myosin resulting in an increased force generation and prolongation of contraction. The lead molecule, omecamtiv mecarbil is now in human 3 stage. In addition to the promising clinical data published so far, there are new in vitro results indicating that the effect of omecamtiv mecarbil on contractility is rate-dependent. Furthermore, omecamtiv mecarbil was shown to activate cardiac ryanodine receptors, an effect that may carry proarrhythmic risk. Methods: These new results, together with the controversial effects of the drug on cardiac oxygen consumption, are critically discussed in this review in light of the current literature on omecamtiv mecarbil. Results: In therapeutically relevant concentrations the beneficial inotropic effect of the agent is not likely affected by these new results - in accordance with the good clinical data. At supratherapeutic concentrations, however, activation of cardiac ryanodine receptors may increase arrhythmia propensity, and the stronger effect on diastolic than systolic cell shortening, observed at higher pacing frequencies, may decrease or offset the inotropic effect of omecamtiv mecarbil. Conclusion: Further studies with definitely supratherapeutical concentrations of omecamtiv mecarbil should be designed to map the actual risk of these potentially harmful side-effects.

In surgical pathophysiology ischemia-reperfusion, inflammatory processes, sepsis, vascular interventions, tissue trauma, shock, all mean conditions in which hemorheological parameters show alterations. Despite of numerous clinical and experimental studies, the in vivo hemorheology is not completely understood yet, and several fundamental questions still need to be answered. Investigating these issues, experimental surgical models are important, in point of view of the translational research as well. In this paper we aimed to make an attempt on summarizing the possible factors and conditions that might have an effect on hemorheological results in experimental surgical studies. Hemorheological parameters show alterations in surgical pathophysiological processes in a complex way. However, the changes are dominantly non-specific. Standardized experimental conditions, related to the experimental animal (species, animal welfare) anesthesia-medications, operation, sampling and, if applicable, conditions of the postoperative period, are inevitable for a safe assessment of valuable (hemorheological) results. Parallel investigations - such as microcirculatory monitoring, imaging techniques, other laboratory methods, histomorphology- have great importance, together with individual analysis of changes, for a better understanding of the changes and for comparability with clinical results.

Abstract: Ten years have passed since the publication of the DEWS Report that summarized the information based on scientific literature concerning dry eye disease. Hundreds of papers have been published since then and time has come for a new summary. Organized by the Tear Film & Ocular Surface Society, 12 working groups summerized former and recent data. The DEWS II Report was created. The authors of the present publication summarize the most important changes in definition, classification, diagnostics, and therapy concerning dry eye disease. They also disclose the relevant changes on which the non-ophthalmologist specialists have to be informed. The DEWS II Report published by TFOS consists of 11 chapters. Completely new chapters deal with the role of sensation/pain and iatrogenic dry eyes. Orv Hetil. 2018; 159(20): 775–785.

Aralkyl and aryl selenoglycosides as well as glycosyl selenocarboxylate derivatives were assayed on the activity of protein phosphatase-1 (PP1) and -2A (PP2A) catalytic subunits (PP1c and PP2Ac) in search of compounds for PP1c and PP2Ac effectors. The majority of tested selenoglycosides activated both PP1c and PP2Ac by ∼2–4-fold in a phosphatase assay with phosphorylated myosin light chain substrate when the hydroxyl groups of the glycosyl moiety were acetylated, but they were without any effects in the non-acetylated forms. A peptide from the myosin phosphatase target subunit-1 (MYPT123–38) that included an RVxF PP1c-binding motif attenuated activation of PP1c by 2-Trifluoromethylbenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β- d -glucopyranoside (TFM-BASG) and 4-Bromobenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β- d -glucopyranoside (Br-BASG). MYPT123–38 stimulated PP2Ac and contributed to PP2Ac activation exerted by either Br-BASG or TFM-BASG. Br-BASG and TFM-BASG suppressed partially binding of PP1c to MYPT1 in surface plasmon resonance based binding experiments. Molecular docking predicted that the hydrophobic binding surfaces in PP1c for interaction with either the RVxF residues of PP1c-interactors or selenoglycosides are partially overlapped. Br-BASG and TFM-BASG caused a moderate increase in the phosphatase activity of HeLa cells in 1 h, and suppressed cell viability in 24 h incubations. In conclusion, our present study identified selenoglycosides as novel activators of PP1 and PP2A as well as provided insights into the structural background of their interactions establishing a molecular model for future design of more efficient phosphatase activator molecules.

The effect of air plasma exposure time on the surface energies and acid-base characteristics of polydimethylsiloxane (PDMS) particles was studied. Polymerized PDMS powder was radio frequency induced air plasma irradiated for 2–10 s with the power of 500 W. The efficiency of the plasma treatments was investigated by a new generation inverse gas chromatograph, a surface energy analyzer. The dispersive component of surface energy was determined by the Dorris-Gray method describing the van der Waals interactions, while the specific component of surface energy expressed the surface ability for Lewis acid-base interactions. It was demonstrated that the air plasma treatment did not affect the dispersive and acidic parts of the surface energy and the change of surface hydrophilicity was caused by the raise of the electron-donor ability of the PDMS surface, characterized by van Oss-Good-Chaudhury’s base number. The optimal plasma treatment time was found to be 5 s. Analysis of the specific surface energy and acid-base characteristics with respect to exposure time showed that partial to complete hydrophobic recovery occurred within 38 h.

Background Intestinal ischemia-reperfusion (I/R) is a potentially life-threatening situation and its pathomechanism is not fully understood yet. Objective To investigate the early micro-rheological, microcirculatory and morphological consequences of intestinal I/R in a rat model. Methods CD rats were anesthetized and subjected to Control (n = 7) or I/R (n = 7) groups. Left femoral artery cannulation and median laparotomy were performed. In the I/R group the superior mesenteric artery was clamped for 30 minutes. Blood samples were taken before (Base) and after the ischemia, at the 30th, 60th and 120th minutes of the reperfusion (R-30, R-60, R-120). Hematological parameters, erythrocyte deformability and aggregation were determined. On the jejunum, the liver and the right kidney laser Doppler flowmetry tests were completed. At the end of experiment histological samples were taken. Results Hematocrit, leukocyte and platelet counts increased during the reperfusion. Erythrocyte deformability worsened versus Control. All erythrocyte aggregation index values of I/R group increased gradually. Intestinal microcirculatory blood flux units (BFU) did not recover completely after ischemia, at R-30 liver BFU values were lower, and kidney values decreased by R-120. Histology showed signs of I/R injury. Conclusions Micro-rheological parameters may show early and significant deterioration during the reperfusion that might contribute further to microcirculatory alterations.

Aspergillus fumigatus is an opportunistic pathogen, the leading cause of invasive and disseminated aspergillosis in systemic immunocompromised patients, and an important cause of mortality. The aim of the present study was to adapt a pulmonary aspergillosis murine model, to determine pathodynamical parameters quantitatively, and to follow the progression of fungal infection in vivo. The nasal inoculation of Aspergillus conidia in mice previously subjected to immunosuppression with cyclophosphamide (CP) turned out to be a more suitable model than that of immunosuppressed with hydrocortisone (HC). The following parameters were found to correlate quantitatively with the progress of the infection: (i) survival rate, (ii) weight loss of mice, (iii) infected focal plaque size, (iv) hyphal density, (v) hyphal length distribution of A. fumigatus, and the (vi) the histopathological status and scores. These parameters will be essential elements for the development of antifungal drugs and therapies, and important for the investigation of the pathogenicity in different strains of A. fumigatus.

PACAP is a neuropeptide with diverse functions in various organs, including reproductive system. It is present in the testis in high concentrations, and in addition to the stage-specific expression within the seminiferous tubules, PACAP affects spermatogenesis and the functions of Leydig and Sertoli cells. Mice lacking endogenous PACAP show reduced fertility, but the possibility of abnormalities in spermatogenic signaling has not yet been investigated. Therefore, we performed a detailed morphological analysis of spermatozoa, sperm motility and investigated signaling pathways that play a role during spermatogenesis in knockout mice. No significant alterations were found in testicular morphology or motility of sperm in homozygous and heterozygous PACAP-deficient mice in spite of the moderately increased number of severely damaged sperms. However, we found robust changes in mRNA and/or protein expression of several factors that play an important role in spermatogenesis. Protein kinase A expression was markedly reduced, while downstream phospho-ERK and p38 were elevated in knockout animals. Expression of major transcription factors, such as Sox9 and phospho-Sox9, was decreased, while that of Sox10, as a redundant factor, was increased in PACAP-deficient mice. The reduced phospho-Sox9 expression was partly due to increased expression and activity of phosphatase PP2A in knockout mice. Targets of Sox transcription factors, such as collagen type IV, were reduced in knockout mice. In summary, our results show that lack of PACAP leads to disturbed signaling in spermatogenesis, which could be a factor responsible for reduced fertility in PACAP knockout mice, and further support the role of PACAP in reproduction.

Background Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis. Results In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 – 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance. Conclusions Our results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing. Electronic supplementary material The online version of this article (10.1186/s12864-018-4544-x) contains supplementary material, which is available to authorized users.

Assessment of intracranial vessels includes transcranial Doppler (TCD). TCD performance requires intact temporal acoustic windows (TAW). Failure of TAW (TAWF) is present in 8–20% of people. There have been no reports on TAWF in rheumatoid arthritis (RA). Altogether, 62 female RA patients were included. Among them, 20 were MTX-treated and biologic-free, 20 received infliximab, and 22 tocilizumab. The controls included 60 non-RA women. TAWF, temporal bone thickness, and texture were determined by ultrasound and CT. BMD and T-scores of multiple bones were determined by DEXA. Several bone biomarkers were assessed by ELISA. In RA, 54.8% of the patients had TAWF on at least one side. Neither TAW could be identified in 34% of RA subjects. In contrast, only 20.0% of control subjects had TAWF on either or both sides (p

The transcription factor RUNX3 is a prominent regulator of multiple hematopoietic cell lineages. Gene loss of function studies demonstrated the unique and essential roles of this master regulator in differentiated lymphoid and myeloid cells. As a complementary approach, RUNX3 was upregulated in various leukocyte subsets to probe the instructive role of this 'multilineage'-specific transcription factor. In this report, we overview the immunomodulatory functions of RUNX3 within the hematopoietic compartment to gain insight into the consequences of Runx3 deletion or overexpression in committed immune cells. Genetic studies revealed the essential role of RUNX3 in Langerhans cell development. Moreover, this transcription factor is necessary for the differentiation and maintenance of the cytotoxic CD8+ T cells. In addition, T helper, natural killer, and B cells are also influenced by RUNX3. Importantly, the ectopic expression of Runx3 enhances the immunogenicity of cytotoxic T cells and pluripotent stem-cell-derived dendritic cells, suggesting that this protein can be applied in cell-based immunotherapies.

In dentistry, it is necessary to increase the bonding strength between various bioinert zirconia ceramics and the luting cement in order to extend the lifetime of the restoration. As a new method, an ion source was used to implant silicon ions close to the surface of the zirconium-dioxide on nanoscale to improve chemical bonding in between the bonding agent and the zirconium-dioxide. This paper summarizes the main results of a unique technique to functionalize biomaterial surfaces with silicon ions produced by Electron Cyclotron Resonance Ion Source (ECRIS).

During the perinatal adaptation process N2BA titin isoforms are switched for N2B titin isoforms leading to an increase in cardiomyocyte passive tension (Fpassive). Here we attempted to reveal how titin isoform composition and oxidative insults (i.e. sulfhydryl (SH)-group oxidation or carbonylation) influence Fpassive of left ventricular (LV) cardiomyocytes during rat heart development. Moreover, we also examined a hypothetical protective role for titin associated small heat shock proteins (sHSPs), Hsp27 and αB-crystallin in the above processes. Single, permeabilized LV cardiomyocytes of the rat (at various ages following birth) were exposed either to 2,2'-dithiodipyridine (DTDP) to provoke SH-oxidation or Fenton reaction reagents (iron(II), hydrogen peroxide (H2O2), ascorbic acid) to induce protein carbonylation of cardiomyocytes in vitro. Thereafter, cardiomyocyte force measurements for Fpassive determinations and Western immunoblot assays were carried out for the semiquantitative determination of oxidized SH-groups or carbonyl-groups of titin isoforms and to monitor sHSPs' expressions. DTDP or Fenton reagents increased Fpassive in 0- and 7-day-old rats to relatively higher extents than in 21-day-old and adult animals. The degrees of SH-group oxidation or carbonylation declined with cardiomyocyte age to similar extents for both titin isoforms. Moreover, the above characteristics were mirrored by increasing levels of HSP27 and αB-crystallin expressions during cardiomyocyte development. Our data implicate a gradual build-up of a protective mechanism against titin oxidation through the upregulation of HSP27 and αB-crystallin expressions during postnatal cardiomyocyte development.

A mucosal oxidative burst is a hallmark response to pollen exposure that promotes allergic inflammatory responses. Reactive species constituents of oxidative stress signal via the modification of cellular molecules including nucleic acids. One of the most abundant forms of oxidative genomic base damage is 8-oxo-7,8-dihydroguanine (8-oxoG), which is removed from DNA by 8-oxoguanine DNA glycosylase 1 (OGG1). OGG1 in complex with 8-oxoG acts as a GDP-GTP exchange factor and induces acute inflammation; however, the mechanism(s) by which OGG1 signaling regulates allergic airway inflammation is not known. Here, we postulate that the OGG1 signaling pathway differentially altered the levels of small regulatory RNAs and increased the expression of T helper 2 (Th2) cytokines in ragweed pollen extract (RWPE)-challenged lungs. To determine this, the lungs of sensitized mice expressing or lacking OGG1 were challenged with RWPE and/or with OGG1’s excision product 8-oxoG. The responses in lungs were assessed by next-generation sequencing, as well as various molecular and histological approaches. The results showed that RWPE challenge induced oxidative burst and damage to DNA and activated OGG1 signaling, resulting in the differential expression of 84 micro-RNAs (miRNAs), which then exacerbated antigen-driven allergic inflammation and histological changes in the lungs. The exogenous administration of the downregulated let-7b-p3 mimetic or inhibitors of upregulated miR-23a or miR-27a decreased eosinophil recruitment and mucus and collagen production via controlling the expression of IL-4, IL-5, and IL-13. Together, these data demonstrate the roles of OGG1 signaling in the regulation of antigen-driven allergic immune responses via differential expression of miRNAs upstream of Th2 cytokines and eosinophils.

The ageing population ought to be celebrated as evidence for the efficacy of modern medicine, but the challenge that this demographic shift presents for 21st century healthcare systems, with increasing numbers of people living with multi-morbidity and frailty, cannot be ignored. There is therefore a need to ensure that all healthcare professionals grasp the basic principles of care of older people. In this paper, we make a case for the development of pan-European education and training standards for the field of geriatric medicine. Firstly, the challenges which face the implementation and delivery of geriatric medicine in a systematic way across Europe are described – these include, but are not limited to; variance in geriatric medicine practice across Europe, insecurity of the specialty in some countries and significant heterogeneity in geriatric medicine training programs across Europe. The opportunities for geriatric medicine are then presented and we consider how engendering core geriatric medicine competencies amongst nongeriatricians has potential to bridge existing gaps in service provision across Europe. Finally, we consider how work can proceed to teach sufficient numbers of doctors and health professionals in the core knowledge, skills and attitudes required to do this. To safeguard the future of the specialty across Europe, we contend that there is a need to strive towards harmonisation of post-graduate geriatric medicine training across Europe, through the establishment of pan-European education and training standards in the specialty.

BACKGROUND Decrease or loss in splenic filtration function may influence the hemorheological state. OBJECTIVE To follow-up the long-term effects of splenectomy, spleen autotransplantation and spleen resections on red blood cell aggregation in a canine model. METHODS Beagle dogs were subjected to control (n = 6), splenectomy (SE, n = 4), spleen autotransplantation (AU, Furka's spleen-chip method, n = 8) or partial and subtotal spleen resection (n = 4/each) groups, and followed-up for 18 postoperative (p.o.) months. Erythrocyte aggregation was determined in parallel by light-transmittance aggregometry (Myrenne MA-1 aggregometer) and syllectometry (LoRRca). RESULTS Erythrocyte aggregation decreased three months after splenectomy, with lower aggregation index and elongated aggregation time. It was more or less associated with relatively lower hematocrit and fibrinogen concentration. However, in autotransplantated animals a relatively higher fibrinogen did not increase the aggregation markedly. Spleen resection resulted in the most controversial red blood cell aggregation findings, and it seems, that the degree of the resection is an influencing factor. CONCLUSIONS Splenectomy alters erythrocyte aggregation, spleen autotransplantation can be useful to preserve filtration function. However, the degree of restoration shows individual differences with a kind of 'functional periodicity'. Spleen resection controversially influences erythrocyte aggregation parameters. The subtotal resection is supposed to be worse than spleen autotransplantation.

T cells expressing CD19-specific chimeric antigen receptors (CARs) with endodomains that encode a signaling domain derived from CD3ζ and CD28 or 41BB have potent antitumor activity in early-phase clinical studies for B-cell malignancies. Besides CD19-specific CARs, other approaches are actively being pursued to redirect T cells to CD19, including recombinant bispecific T-cell engager (BiTE) proteins or T cells genetically modified to express BiTEs [engager (ENG) T cells]. As BiTEs provide no costimulation, we investigated here if provision of costimulation through CD28 and 41BB enhances the effector function of CD19-ENG T cells. CD19-ENG T cells expressing CD80 and 41BBL on their cell surface (CD19-ENG.41BBL/CD80 T cells) were generated by retroviral transduction. CD19-ENG.41BBL/CD80 T cells retained their antigen specificity and had superior effector function compared with both unmodified T cells and CD19-ENG T cells expressing either CD80, 41BBL, or no costimulatory molecule, as judged by cytokine (IFNγ and IL2) production, T-cell proliferation, and their ability to sequentially kill target cells. In vivo, CD19-ENG.41BBL/CD80 T cells had superior antileukemia activity in the BV173 xenograft model, resulting in a survival advantage in comparison to CD19-ENG T cells. Thus, provision of costimulation is critical for the effector function of ENG T cells. Cancer Immunol Res; 5(10); 860–70. ©2017 AACR.

Background Atopic eczema (AE) is a chronic inflammatory and pruritic skin disease. There is still an unmet need for topical anti-inflammatory and anti-pruritic substances exhibiting an excellent safety profile. The endocannabinoid system is known to regulate various aspects of cutaneous barrier and immune functions, thus targeting it may be a valid approach for alleviating the symptoms of AE. Objective To assess the putative efficacy of Echinacea purpurea-derived alkylamides (Ec. extract) activating cannabinoid (CB)-2 receptors in exerting anti-inflammatory effects and alleviating symptoms of AE. Methods In vitro anti-inflammatory efficiency was investigated by monitoring the effects of Ec. extract on poly-(I:C)-induced pro-inflammatory cytokine expression (Q-PCR) and release (ELISA) of HaCaT keratinocytes. Irritancy and sensitization potential (assessed by Human Repeat Insult Patch Test; Clinical trial 1); clinical efficiency in alleviating symptoms of AE (Clinical trial 2) as well as effects on human skin structure and lipid content (Clinical trial 3 followed by transmission electron microscopy and HPTLC) were investigated in randomized double blind clinical trials. Results Ec. extract significantly reduced mRNA expression as well as release of poly-(I:C)-induced pro-inflammatory cytokines (IL-6 and IL-8) in keratinocytes. Thus, not surprisingly, the well-tolerated (Clinical trial 1) Ec. extract-based cream reduced local SCORAD statistically significantly, not only compared to baseline, but also compared to the comparator (Clinical trial 2). Of great importance, besides the in vitro anti-inflammatory effects, administration of the Ec. extract-based cream also resulted in significantly higher levels of overall epidermal lipids, ceramide EOS (ω-esterified fatty acid + sphingosine sphingoid base), and cholesterol at Day 15 compared to baseline as well as significantly greater numbers of intercellular lipid lamellae in the intercellular space (Clinical trial 3). Conclusion The investigated Ec. extract shows great potential in alleviating cutaneous symptoms of AE, and by exerting remarkable anti-inflammatory actions and restoring the epidermal lipid barrier, it will be very likely a well-tolerated, powerful novel ingredient for the adjuvant therapy of AE.

Data obtained by genotyping single nucleotide polymorphisms (SNPs) related to high-density lipoprotein cholesterol (HDL-C) levels were utilized in Genetic Risk Score [unweighted (GRS) and weighted (wGRS)] computation on Hungarian general and Roma populations. The selection process of the SNPs as well as the results obtained are published in our research article (Piko et al., 2017) [1]. Linkage analyses were performed by study groups. Study populations were stratified by quintiles of weighted Genetic Risk Score. Multivariate linear regression analyses were performed using Genetic Risk Scores and HDL-C levels as dependent variables; and ethnicity, sex and age as independent variables. The study subjects were categorized into quintiles according their wGRS values. Associations of Genetic Risk Scores with plasma HDL-C levels (as a continuous variable) were observed in both populations. Finally, the two populations were merged and analyzed together by multivariate logistic regression where reduced plasma HDL-C level was the dependent variable; while ethnicity, age and sex were the independent ones.

Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated., Graphical abstract fx1, Highlights • MtROS have versatile role in fine-tuning the type I IFN responses of pDCs. • MtROS affect the TLR- and RIG-I-mediated type I IFN responses in pDCs oppositely. • MtROS decrease the TLR-induced early phase of type I IFN production in pDCs. • MtROS enhance the RIG-I-mediated late phase of type I IFN production in pDCs.

Comprehensive molecular characterization of and novel therapeutic approaches to glioblastoma have been explored as a result of advancements in biotechnologies. In this study, we aimed to bring basic research discoveries closer to clinical practice and ultimately incorporate molecular classification into the routine histopathological evaluation of grade IV gliomas. Integrated results of genome-wide sequencing, transcriptomic and epigenomic analyses by The Cancer Genome Atlas Network defined the classic, proneural, neural and mesenchymal subtypes of this tumor. In a retrospective cohort, we analyzed selected subgroup-defining molecular markers in formalin-fixed paraffin-embedded surgical specimens by immunohistochemistry. Quantitative and qualitative scores of marker expression were tested in hierarchical cluster analyses to evaluate segregations of the molecular subgroups, which then were correlated with clinical parameters including patients' age, gender and overall survival. Our study has confirmed the separation of molecular glioblastoma subgroups with clear trends regarding clinical correlations. Future analyses in a larger, prospective cohort using similar methods are expected to facilitate the development of a molecular diagnostic panel that may complement routine histological work up and support prognostication as well as treatment decisions in glioblastoma.

The outcome for advanced stage hepatocellular carcinoma (HCC) remains poor, highlighting the need for novel therapies. Genetically modified mesenchymal stem cells (MSCs) are actively being explored as cancer therapeutics due to their inherent ability to migrate to tumor sites. We reasoned that MSCs can be genetically modified to redirect T cells to Glypican-3 (GPC3)+ HCC, and genetically modified these with viral vectors encoding a GPC3/CD3 bispecific T cell engager (GPC3-ENG), a bispecifc T cell engager specific for an irrelevant antigen (EGFRvIII), and/or costimulatory molecules (CD80 and 41BBL). Coculture of GPC3+ cells, GPC3-ENG MSCs, and T cells resulted in T cell activation, as judged by interferon γ (IFNγ) production and killing of tumor cells by T cells. Modification of GPC3-ENG MSCs with CD80 and 41BBL was required for antigen-dependent interleukin-2 (IL-2) production by T cells and resulted in faster tumor cell killing by redirected T cells. In vivo, GPC3-ENG MSCs ± costimulatory molecules had antitumor activity in the HUH7 HCC xenograft model, resulting in a survival advantage. In conclusion, MSCs genetically modified to express GPC3-ENG ± costimulatory molecules redirect T cells to GPC3+ tumor cells and have potent antitumor activity. Thus, further preclinical exploration of our modified approach to GPC3-targeted immunotherapy for HCC is warranted., Graphical Abstract