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Aims The aim of this study was to compare the efficacy, consumption and safety after piritramide administered either intramuscularly (IM) on demand or via patient-controlled intravenous analgesia (PCA) and to examine the impact of OPRM1 and ABCB1 gene polymorphisms on the drug efficacy/safety in both regimens. Methods One hundred and four patients scheduled for elective inguinal hernioplasty received piritramide with PCA or IM for postoperative pain management. We evaluated piritramide consumption, pain intensity using visual analogue scale (VAS) and adverse effects. Results Median (IQR) piritramide consumption was 18.5 (13.5-31.2) and 15.0 (15.0-15.0) mg in the PCA and IM groups, respectively (P=0.0092). The respective values of area under the VAS2-16-time curve were 40 and 280 mm.h (P=0.0027). Opioid-induced adverse effects were more frequent in the PCA than in the IM group. Variant OPRM1 allele was associated with decreased pain relief, increased opioid consumption and increased incidence of adverse effects, while ABCB1 polymorphisms showed no impact on the observed parameters. Conclusions We observed higher piritramide consumption, better pain relief and slightly worse safety profile in the PCA group compared with IM administration. Variant OPRM1 118G allele carriers required higher opioid dosing and suffered from more adverse effects, however, the differences between genotypes have been less pronounced in the PCA patients likely due to improved pain management via PCA.

In vitro studies are essential in pre-clinical research. While choice of cell lines is often driven by handling and cost-effectiveness, in-depth knowledge on specific characteristics is scant. Mesothelial cells, which interact with endothelial cells, are widely used in research, including cancer and drug development, but have not been comprehensively profiled. We therefore performed RNA sequencing of polarized, primary peritoneal (HPMC) and immortalized pleural mesothelial cells (MeT-5A), and compared them to endothelial cells from umbilical vein (HUVEC) and cardiac capillaries (HCMEC). Seventy-seven per cent of 12,760 genes were shared between the 4 cell lines, 1003 were mesothelial and 969 were endothelial cell specific. The transcripts reflected major differences between HPMC and MeT-5A in DNA-related processes, extracellular matrix, migration, proliferation, adhesion, transport, growth factor- and immune response, and between HUVEC and HCMEC in DNA replication, extracellular matrix and adhesion organization. Highly variable shared genes were related to six clusters, cell tissue origin and immortalization, but also cell migration capacity, cell adhesion, regulation of angiogenesis and response to hypoxia. Distinct, cell type specific biological processes were further described by cellular component-, molecular function- and Reactome pathway analyses. We provide crucial information on specific features of the most frequently used mesothelial and endothelial cell lines, essential for appropriate use.

The aim was to verify the impact of obesity on the long-term outcome of patients with severe SARS-CoV-2 ARDS.The retrospective study included patients admitted to the high-volume ECMO centre between March 2020 and March 2022. The impact of body mass index (BMI), co-morbidities and therapeutic measures on the short and 90-day outcomes was analysed.292 patients were included, of whom 119(40.8%) were treated with veno-venous ECMO cannulated mostly (73%) in a local hospital. 58.5% were obese (64.7% on ECMO), the ECMO was most frequent in BMI40(49%). The ICU mortality (36.8% for obese vs 33.9% for the non-obese, p = 0.58) was related to ECMO only for the non-obese (p = 0.04). The 90-day mortalities (48.5% obese vs 45.5% non-obese, p = 0.603) of the ECMO and non-ECMO patients were not significantly influenced by BMI (p = 0.47, p = 0.771, respectively). The obesity associated risk factors for adverse outcome were age50 (RR 2.14) and history of chronic immunosuppressive therapy (RR 2.11, p = 0.009). The higher dosage of steroids (RR 0.57, p = 0.05) associated with a better outcome.The high incidence of obesity was not associated with worse short and long-term outcomes. ECMO in obese patients together with the use of steroids in the later stage of ARDS may improve survival.

Altres ajuts: Deutsche Forschungsgemeinschaft (DFG) TRR221 (B11 Z02), TRR225 (B08) Clinical studies have suggested a potential involvement of endothelial dysfunction and damage in the development and severity of acute graft-versus-host disease (aGvHD). Accordingly, we found an increased percentage of apoptotic caspase 3 positive blood vessels in duodenal and colonic mucosa biopsies of patients with severe aGvHD. In murine experimental aGvHD, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGvHD target organs. During intestinal aGvHD, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter. As recent data demonstrated an association of endothelium-related factors and steroid refractory aGvHD (SR-aGvHD), we analyzed human biopsies and murine tissues from SR-aGvHD. We found extensive tissue damage but low levels of alloreactive T-cell infiltration in target organs, providing the rationale for T-cell independent SR-aGvHD treatment strategies. Consequently, we tested the endothelium-protective PDE5 inhibitor sildenafil, which reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced target organ damage during experimental SR-aGvHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGvHD. Therapeutic intervention by endothelium-protecting agents is an attractive approach for SR-aGvHD complementing current anti-inflammatory treatment options.

Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy.

Alternative branches of the classical renin–angiotensin–aldosterone system (RAS) represent an important cascade in which angiotensin 2 (AngII) undergoes cleavage via the action of the angiotensin-converting enzyme 2 (ACE2) with subsequent production of Ang(1-7) and other related metabolites eliciting its effects via Mas receptor activation. Generally, this branch of the RAS system is described as its non-canonical alternative arm with counterbalancing actions to the classical RAS, conveying vasodilation, anti-inflammatory, anti-remodeling and anti-proliferative effects. The implication of this branch was proposed for many different diseases, ranging from acute cardiovascular conditions, through chronic respiratory diseases to cancer, nonetheless, hypoxia is one of the most prominent common factors discussed in conjugation with the changes in the activity of alternative RAS branches. The aim of this review is to bring complex insights into the mechanisms behind the various forms of hypoxic insults on the activity of alternative RAS branches based on the different duration of stimuli and causes (acute vs. intermittent vs. chronic), localization and tissue (heart vs. vessels vs. lungs) and clinical relevance of studied phenomenon (experimental vs. clinical condition). Moreover, we provide novel insights into the future strategies utilizing the alternative RAS as a diagnostic tool as well as a promising pharmacological target in serious hypoxia-associated cardiovascular and cardiopulmonary diseases.

Timely and accurate diagnosis of osteoporosis is essential for adequate therapy. Calcium isotope ratio (δ44/42Ca) determination has been suggested as a sensitive, noninvasive, and radiation-free biomarker for the diagnosis of osteoporosis, reflecting bone calcium balance. The quantitative diagnostic is based on the calculation of the δ44/42Ca difference between blood, urine, and bone. The underlying cellular processes, however, have not been studied systematically. We quantified calcium transport and δ44/42Ca fractionation during in vitro bone formation and resorption by osteoblasts and osteoclasts and across renal proximal tubular epithelial cells (HK-2), human vein umbilical endothelial cells (HUVECs), and enterocytes (Caco-2) in transwell systems and determined transepithelial electrical resistance characteristics. δ44/42Ca fractionation was furthermore quantified with calcium binding to albumin and collagen. Calcified matrix formed by osteoblasts was isotopically lighter than culture medium by −0.27 ± 0.03‰ within 5 days, while a consistent effect of activated osteoclasts on δ44/42Ca could not be demonstrated. A transient increase in δ44/42Ca in the apical compartment by 0.26‰ occured across HK-2 cells, while δ44/42Ca fractionation was small across the HUVEC barrier and absent with Caco-2 enterocytes, and with binding of calcium to albumin and collagen. In conclusion, δ44/42Ca fractionation follows similar universal principles as during inorganic mineral precipitation; osteoblast activity results in δ44/42Ca fractionation. δ44/42Ca fractionation also occurs across the proximal tubular cell barrier and needs to be considered for in vivo bone mineralization modeling. In contrast, the effect of calcium transport across endothelial and enterocyte barriers on blood δ44/42Ca should be low and is absent with physiochemical binding of calcium to proteins.

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.

Infectious peritonitis is a common complication in patients undergoing chronic peritoneal dialysis (PD), limiting the duration of PD as a modality for renal replacement therapy and increasing patient morbidity and mortality. Antimicrobial peptides (AMPs) serve critical roles in mucosal defense, but their expression and activity during peritonitis are poorly understood. We hypothesized that AMPs belonging to the Ribonuclease (RNase) A Superfamily are present in peritoneal fluid and increase during peritonitis in patients undergoing chronic PD. In the absence of peritonitis, we detected RNase 3, RNase 6, and RNase 7 in cell-free supernatants and viable cells obtained from peritoneal fluid of chronic PD patients. The cellular sources of these RNases were eosinophils (RNase 3), macrophages (RNase 6), and mesothelial cells (RNase 7). During peritonitis, RNase 3 increased 55-fold and RNase 7 levels increased 3-fold on average, whereas RNase 6 levels were unchanged. The areas under the receiver-operating characteristic curves for RNase 3 and RNase 7 were 0.99 (95% confidence interval (CI): 0.96–1.0) and 0.79 (95% CI: 0.64–0.93), respectively, indicating their potential as biomarkers of peritonitis. Discrete omental reservoirs of these RNases were evident in patients with end stage kidney disease prior to PD initiation, and omental RNase 3 reactive cells increased in patients undergoing PD with a history of peritonitis. We propose that constitutive and inducible pools of antimicrobial RNases form a network to shield the peritoneal cavity from microbial invasion in patients undergoing chronic PD.

Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies., Here, we demonstrate that hundreds to thousands of chemicals migrate into water from plastic products and that these leachates induce toxicity in vitro. This highlights that plastic chemicals leach under realistic conditions and, thus, can be a relevant contributor to the human exposome.

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane., This work is part of the IMPROVE-PD project that has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement number 812699. M.B. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 419826430. R.H. was supported by a research fellowship of the European Renal Association and European Dialysis and Transplant Association (ERA-EDTA). E.L. was supported by the ÚNKP-18-2 New National Excellence Program of the Ministry of Human Capacities, Hungary. E.L. and H.J. were supported by Jellinek-Harry scholarship. S.G.Z. acknowledges the Alexander von Humboldt Stiftung/Foundation for an Experienced Researcher Fellowship (2019–2021) and the International Peritoneal Dialysis Society (ISPD) for an International Cooperation Research Grant (2019–2021). C.P.S. has obtained funding from European Nephrology and Dialysis Institute (ENDI).

Background Rickettsialpox is a febrile illness caused by the mite-borne pathogen Rickettsia akari. Several cases of this disease are reported worldwide annually. Nevertheless, the relationship between the immunogenicity of R. akari and disease development is still poorly understood. Thus, misdiagnosis is frequent. Our study is aiming to identify immunogenic proteins that may improve disease recognition and enhance subsequent treatment. To achieve this goal, two proteomics methodologies were applied, followed by immunoblot confirmation. Results Three hundred and sixteen unique proteins were identified in the whole-cell extract of R. akari. The most represented protein groups were found to be those involved in translation, post-translational modifications, energy production, and cell wall development. A significant number of proteins belonged to amino acid transport and intracellular trafficking. Also, some proteins affecting the virulence were detected. In silico analysis of membrane enriched proteins revealed 25 putative outer membrane proteins containing beta-barrel structure and 11 proteins having a secretion signal peptide sequence. Using rabbit and human sera, various immunoreactive proteins were identified from which the 44 kDa uncharacterized protein (A8GP63) has demonstrated a unique detection capability. It positively distinguished the sera of patients with Rickettsialpox from other rickettsiae positive human sera. Conclusion Our proteomic analysis certainly contributed to the lack of knowledge of R. akari pathogenesis. The result obtained may also serve as a guideline for a more accurate diagnosis of rickettsial diseases. The identified 44 kDa uncharacterized protein can be certainly used as a unique marker of rickettsialpox or as a target molecule for the development of more effective treatment.

Long-term clinical outcome of peritoneal dialysis (PD) depends on adequate removal of small solutes and water. The peritoneal endothelium represents the key barrier and peritoneal transport dysfunction is associated with vascular changes. Alanyl-glutamine (AlaGln) has been shown to counteract PD-induced deteriorations but the effect on vascular changes has not yet been elucidated. Using multiplexed proteomic and bioinformatic analyses we investigated the molecular mechanisms of vascular pathology in-vitro (primary human umbilical vein endothelial cells, HUVEC) and ex-vivo (arterioles of patients undergoing PD) following exposure to PD-fluid. An overlap of 1813 proteins (40%) of over 3100 proteins was identified in both sample types. PD-fluid treatment significantly altered 378 in endothelial cells and 192 in arterioles. The HUVEC proteome resembles the arteriolar proteome with expected sample specific differences of mainly immune system processes only present in arterioles and extracellular region proteins primarily found in HUVEC. AlaGln-addition to PD-fluid revealed 359 differentially abundant proteins and restored the molecular process landscape altered by PD fluid. This study provides evidence on validity and inherent limitations of studying endothelial pathomechanisms in-vitro compared to vascular ex-vivo findings. AlaGln could reduce PD-associated vasculopathy by reducing endothelial cellular damage, restoring perturbed abundances of pathologically important proteins and enriching protective processes.

The negative influence of non-ionizing electromagnetic radiation on organisms, including humans, has been discussed widely in recent years. This paper deals with the methodology of examining possible harmful effects of mobile phone radiation, focusing on in vivo and in vitro laboratory methods of investigation and evaluation and their main problems and difficulties. Basic experimental parameters are summarized and discussed, and recent large studies are also mentioned. For the laboratory experiments, accurate setting and description of dosimetry are essential; therefore, we give recommendations for the technical parameters of the experiments, especially for a well-defined source of radiation by Software Defined Radio.

Cardiovascular disease (CVD) is the leading cause of increased mortality in patients with CKD and is further aggravated by peritoneal dialysis (PD). Children are devoid of preexisting CVD and provide unique insight into specific uremia- and PD-induced pathomechanisms of CVD. We obtained peritoneal specimens from children with stage 5 CKD at time of PD catheter insertion (CKD5 group), children with established PD (PD group), and age-matched nonuremic controls (n=6/group). We microdissected omental arterioles from tissue layers not directly exposed to PD fluid and used adjacent sections of four arterioles per patient for transcriptomic and proteomic analyses. Findings were validated in omental and parietal arterioles from independent pediatric control (n=5), CKD5 (n=15), and PD (n=15) cohorts. Transcriptomic analysis revealed differential gene expression in control versus CKD5 arterioles and in CKD5 versus PD arterioles. Gene ontology analyses revealed activation of metabolic processes in CKD5 arterioles and of inflammatory, immunologic, and stress-response cascades in PD arterioles. PD arterioles exhibited particular upregulation of the complement system and respective regulatory pathways, with concordant findings at the proteomic level. In the validation cohorts, PD specimens had the highest abundance of omental and parietal arteriolar C1q, C3d, terminal complement complex, and phosphorylated SMAD2/3, a downstream effector of TGF-β. Furthermore, in the PD parietal arterioles, C1q and terminal complement complex abundance correlated with the level of dialytic glucose exposure, abundance of phosphorylated SMAD2/3, and degree of vasculopathy. We conclude that PD fluids activate arteriolar complement and TGF-β signaling, which quantitatively correlate with the severity of arteriolar vasculopathy.

Life-saving renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. However, the pathophysiological mechanisms involved are incompletely understood, limiting identification of therapeutic targets. We report that addition of lithium chloride (LiCl) to PDF is a translatable intervention to counteract PDF-induced mesothelial cell death, peritoneal membrane fibrosis, and angiogenesis. LiCl improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most consistently counter-regulated by LiCl. In vitro and in vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like up-regulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGF-β-independent activation of TGF-β-regulated targets. In contrast, αB-crystallin knockdown decreased VEGF expression and early mesothelial-to-mesenchymal transition. LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically up-regulated in response to PDF and increased in peritoneal mesothelial cells from biopsies from pediatric patients undergoing PD, correlating with markers of angiogenesis and fibrosis. LiCl-supplemented PDF promoted morphological preservation of mesothelial cells and the submesothelial zone in a mouse model of chronic PD. Thus, repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy.

The study provides an up-to-date overview of pathogenesis, functional and structural changes in normal tension glaucoma (NTG) and its differences from high tension glaucomas (HTG). The authors point to less known facts which make both diagnostic groups different. First of all, there are electrophysiological findings that verify pathology in the complete visual pathway in HTG in contrast to NTG where the retinal ganglion cell response is relatively normal but the abnormalities are in the visual pathway. This corresponds to the findings of functional magnetic resonance imaging of the brain with a significant decrease in activity in HTG compared to NTG. We found a higher decrease in activity in HTG following application of the colour paradigm compared to NTG where we did not see a similar difference. We also investigated the central corneal thickness (CCT) in both diagnostic groups. We did not find a statistically significant difference. However, we found the effect of CCT on progression of the changes in visual fields in HTG. In relation to suspicion of abnormally low cerebrospinal pressure and a possible cerebrovascular fluid flow disturbance in NTG, we examined the optic nerve thickness (OND) and optic nerve sheath diameter (OSD) at a distance of 4, 8, 16 and 20mm from the posterior pole of the eye. In the comparison with the healthy population, we did not find any abnormalities except for the width of the optic chiasma that was markedly lower in NTG. In relation to a possible impairment of cerebral perfusion we determined the degrees of cerebral atrophy using magnetic resonance imaging by measuring the bicaudate ratio (BCR) and white matter lesions using the Fazekas scale. We did not find a difference between HTG and NTG in BCR. We found statistically significant changes in BCR which correlated with the changes in visual fields. The higher values of the pattern defect were associated with increased brain atrophy (BCR). We did not detect similar relations in the Fazekas scale. We found a significant difference in this parameter among NTG, HTG and a control group. We found the most advanced changes in the patients with HTG. Conclusion: In HTG, impairment of retinal ganglion cells and subsequently also their axons, including visual cortex occurs because of a high intraocular pressure. In NTG, the retinal ganglion cells are relatively normal like the visual cortex, but alteration occurs in their axons. The cause is not a high intraocular pressure but most probably ischemia.

Encapsulating peritoneal sclerosis (EPS) is a life-threatening complication of long-term peritoneal dialysis (PD), which may even occur after patients have switched to hemodialysis (HD) or undergone kidney transplantation. The incidence of EPS varies across the globe and increases with PD vintage. Causative factors are the chronic exposure to bioincompatible PD solutions, which cause long-term modifications of the peritoneum, a high peritoneal transporter status involving high glucose concentrations, peritonitis episodes, and smoldering peritoneal inflammation. Additional potential causes are predisposing genetic factors and some medications. Clinical symptoms comprise signs of intestinal obstruction and a high peritoneal transporter status with incipient ultrafiltration failure. In radiological, macro-, and microscopic studies, a massively fibrotic and calcified peritoneum enclosed the intestine and parietal wall in such cases. Empirical treatments commonly used are corticosteroids and tamoxifen, which has fibrinolytic properties. Immunosuppressants like azathioprine, mycophenolate mofetil, or mTOR inhibitors may also help with reducing inflammation, fibrin deposition, and collagen synthesis and maturation. In animal studies, N-acetylcysteine, colchicine, rosiglitazone, thalidomide, and renin-angiotensin system (RAS) inhibitors yielded promising results. Surgical treatment has mainly been performed in severe cases of intestinal obstruction, with varying results. Mortality rates are still 25–55% in adults and about 14% in children. To reduce the incidence of EPS and improve the outcome of this devastating complication of chronic PD, vigorous consideration of the risk factors, early diagnosis, and timely discontinuation of PD and therapeutic interventions are mandatory, even though these are merely based on empirical evidence.

A hypoxic microenvironment is one of the predominant reasons for incomplete response to melanoma treatment. Vemurafenib, which targets the mutated BRAF-V600 kinase, improves melanoma patient survival, however, resistance invariably develops. The present study evaluated the effect of hypoxia on three BRAF-V600E mutant melanoma cell lines, M14, A375 and 518A2, treated with vemurafenib. Compared with the other two cell lines, hypoxic vemurafenib-treated A375 cells exhibited an enhanced cell proliferation rate and migratory capacity compared with normoxic vemurafenib-treated A375 cells. Immunoblotting analyses revealed that the expression levels of hypoxia inducible factor (HIF)1α and carbonic anhydrase IX were reduced in vemurafenib‑treated M14 and 518A2 cells, however, not in A375 cells. The expression levels of the mitogen‑activated protein kinase, Janus kinase-signal transducer and activator of transcription, and phosphatidylinositol-4,5-bisphosphate 3‑kinase signaling pathway proteins revealed a cell‑type specific response to vemurafenib and hypoxia. Knockdown experiments of HIF1α performed in hypoxic A375 cells decreased the expression of phosphorylated (p‑)protein kinase B, which was restored following vemurafenib treatment, and increased the expression of p‑extracellular‑signal‑regulated kinases. Therefore, three melanoma cell lines responded to vemurafenib under hypoxia in a cell type‑specific manner, suggesting that a subset of cells provides a treatment-resistant pool, from which disease relapse may originate. These data confirmed that vemurafenib may be successful in treating the proliferating cells, whereas the non‑proliferating subpopulation must be addressed by a combination of vemurafenib with other treatment strategies.

Peritoneal dialysis (PD) is limited by glucose-mediated peritoneal membrane (PM) fibrosis, angiogenesis, and ultrafiltration failure. Influencing PM integrity by pharmacologically targeting sodium-dependent glucose transporter (SGLT)-mediated glucose uptake has not been studied. In this study, wildtype C57Bl/6N mice were treated with high-glucose dialysate via an intraperitoneal catheter, with or without addition of selective SGLT2 inhibitor dapagliflozin. PM structural changes, ultrafiltration capacity, and peritoneal equilibration testing (PET) status for glucose, urea, and creatinine were analyzed. Expression of SGLT and facilitative glucose transporters (GLUT) was analyzed by real-time PCR, immunofluorescence, and immunohistochemistry. Peritoneal effluents were analyzed for cellular and cytokine composition. We found that peritoneal SGLT2 was expressed in mesothelial cells and in skeletal muscle. Dapagliflozin significantly reduced effluent transforming growth factor (TGF-&beta, ) concentrations, peritoneal thickening, and fibrosis, as well as microvessel density, resulting in improved ultrafiltration, despite the fact that it did not affect development of high-glucose transporter status. In vitro, dapagliflozin reduced monocyte chemoattractant protein-1 release under high-glucose conditions in human and murine peritoneal mesothelial cells. Proinflammatory cytokine release in macrophages was reduced only when cultured in high-glucose conditions with an additional inflammatory stimulus. In summary, dapagliflozin improved structural and functional peritoneal health in the context of high-glucose PD.

Besides natural and acquired mechanisms of resistance, bacteria can cope with presence of antibiotics by using complex mechanisms such as persistence or tolerance. The main purpose of this study was to evaluate the suitability of newly developed Tolerance Disk Test (TDtest) (Gefen et al., 2017) to detect persistent or tolerant bacterial cells in clinical isolates of Staphylococcus aureus. The principle of the test is to resuscitate the subpopulation of persistent or tolerant bacterial cells following a disk diffusion test by glucose. Results of the TDtest were evaluated using time killing experiments for three pairs of consecutive S. aureus isolates from lower respiratory airway samples of three cystic fibrosis patients with chronic staphylococcal infections. TDtest enabled semi-quantitative detection of persistent or tolerant bacterial populations in all analyzed isolates for oxacillin, vancomycin, and ciprofloxacin to which isolates studied were susceptible. Therefore, TDtest is a promising method for rapidly determining persistence/tolerance in clinical isolates of S. aureus.

WOS: 000439138700024 PubMed ID: 29776755 The effect of peritoneal dialysates with low-glucose degradation products on peritoneal membrane morphology is largely unknown, with functional relevancy predominantly derived from experimental studies. To investigate this, we performed automated quantitative histomorphometry and molecular analyses on 256 standardized peritoneal and 172 omental specimens from 56 children with normal renal function, 90 children with end-stage kidney disease at time of catheter insertion, and 82 children undergoing peritoneal dialysis using dialysates with low-glucose degradation products. Follow-up biopsies were obtained from 24 children after a median peritoneal dialysis of 13 months. Prior to dialysis, mild parietal peritoneal inflammation, epithelial-mesenchymal transition and vasculopathy were present. After up to six and 12 months of peritoneal dialysis, blood microvessel density was 110 and 93% higher, endothelial surface area per peritoneal volume 137 and 95% greater, and submesothelial thickness 23 and 58% greater, respectively. Subsequent peritoneal changes were less pronounced. Mesothelial cell coverage was lower and vasculopathy advanced, whereas lymphatic vessel density was unchanged. Morphological changes were accompanied by early fibroblast activation, leukocyte and macrophage infiltration, diffuse podoplanin presence, epithelial mesenchymal transdifferentiation, and by increased proangiogenic and profibrotic cytokine abundance. These transformative changes were confirmed by intraindividual comparisons. Peritoneal microvascular density correlated with peritoneal small-molecular transport function by uni- and multivariate analysis. Thus, in children on peritoneal dialysis neutral pH dialysates containing low-glucose degradation products induce early peritoneal inflammation, fibroblast activation, epithelial-mesenchymal transition and marked angiogenesis, which determines the PD membrane transport function. European Training and Research in Peritoneal Dialysis (EuTRiPD) Programme - European Union within the Marie Curie Scheme [287813]; Medical Faculty of the University of Heidelberg; Fresenius Medical Care (Bad Homburg, Germany); ERA-EDTA; KfH Foundation for Preventive Medicine; Baxter; Fresenius Medical Care MB was supported by the European Training and Research in Peritoneal Dialysis (EuTRiPD) Programme, a project funded by the European Union within the Marie Curie Scheme (287813). BS was supported by the Medical Faculty of the University of Heidelberg. Further support was received from Fresenius Medical Care (Bad Homburg, Germany), ERA-EDTA, and the KfH Foundation for Preventive Medicine. FS, BAW, and CPS obtained further research funding from Baxter and Fresenius Medical Care. We are grateful to Dr. E. Herpel and Mr. J. Moyers from Tissue Bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany) and Institute of Pathology (Heidelberg University Hospital) for technical assistance.

Background Ultrafiltration decline is a progressive issue for patients on chronic peritoneal dialysis (PD) and can be caused by peritoneal angiogenesis induced by PD fluids. A recent pediatric trial suggests better preservation of ultrafiltration with bicarbonate versus lactate buffered fluid; underlying molecular mechanisms are unknown. Methods Angiogenic cytokine profile, tube formation capacity and Receptor Tyrosine Kinase translocation were assessed in primary human umbilical vein endothelial cells following incubation with bicarbonate (BPDF) and lactate buffered (LPDF), pH neutral PD fluid with low glucose degradation product content and lactate buffered, acidic PD fluid with high glucose degradation product content (CPDF). Peritoneal biopsies from age-, PD-vintage- and dialytic glucose exposure matched, peritonitis-free children on chronic PD underwent automated histomorphometry and immunohistochemistry. Results In endothelial cells angiopoietin-1 mRNA and protein abundance increased 200% upon incubation with BPDF, but decreased by 70% with LPDF as compared to medium control; angiopoietin-2 remained unchanged. Angiopoietin-1/Angiopoietin-2 protein ratio was 15 and 3-fold increased with BPDF compared to LPDF and medium. Time-lapse microscopy with automated network analysis demonstrated less endothelial cell tube formation with BPDF compared to LPDF and CPDF incubation. Receptor Tyrosine Kinase translocated to the cell membrane in BPDF but not in LPDF or CPDF incubated endothelial cells. In children dialyzed with BPDF peritoneal vessels were larger and angiopoietin-1 abundance in CD31 positive endothelium higher compared to children treated with LPDF. Conclusion Bicarbonate buffered PD fluid promotes vessel maturation via upregulation of angiopoietin-1 in vitro and in children on dialysis. Our findings suggest a molecular mechanism for the observed superior preservation of ultrafiltration capacity with bicarbonate buffered PD fluid with low glucose degradation product content.

Human peritoneal mesothelial cells (HPMC) secrete phosphatidylcholines (PC) which form a lipid bilayer lining the peritoneum. They prevent frictions and adhesions and act as a barrier to the transport of water-soluble solutes while permitting water flux. PC may play an essential role in peritoneal integrity and function, the role of PD induced HPMC senescence on PC homeostasis, however, is unknown. HPMC cell lines were isolated from four non-uremic patients. Expression of the three PC synthesis genes (rt-PCR), and cellular storage and secretion of PC (ESI-mass-spectrometry) were analyzed in young and senescent HPMC (>Hayflick-limit). Senescent cells displayed significantly altered morphology; flow cytometry demonstrated extensive staining for senescence-associated beta galactosidase. Nine different PC were detected in HPMC with palmitoyl-myristoyl phosphatidylcholine (PMPC) being most abundant. In senescent HPMC mRNA expression of the three key PC synthesis genes was 1.5-, 2.4- and 6-fold increased as compared to young HPMC, with the latter, phosphatidylcholine cytidylyltransferase, being rate limiting. Intracellular storage of the nine PC was 75–450 % higher in senescent vs. young HPMC, PC secretion rates were 100–300 % higher. Intracellular PC concentrations were not correlated with the PC secretion rates. Electron microscopy demonstrated lamellar bodies, the primary storage site of PC, in senescent but not in young cells. Senescent HPMC store and secrete substantially more PC than young cells. Our findings indicate a novel protective mechanism, which should counteract peritoneal damage induced by chronic exposure to PD fluids.

Peritoneal dialysis (PD) is a life-saving form of renal replacement therapy for those with end-stage kidney disease. Mesothelial cells (MCs) line the peritoneal cavity and help define peritoneal response to treatment-associated injury, a major reason for treatment failure. miRNAs are important regulators, but their roles in peritoneal fibrosis are largely unknown. In this study, miR-21 was one of the most abundant miRNAs in primary MCs, and was up-regulated by the profibrotic cytokine transforming growth factor-β1 and in PD effluent-derived MCs exhibiting mesenchymal phenotypic change. Increased miR-21 was found in peritoneal membrane biopsy specimens from PD patients compared to healthy controls (PD biocompatible, 5.86×, P = 0.0001; PD conventional, 7.09×, P n = 11 per group). In PD effluent from a cohort of 230 patients, miR-21 was higher in those receiving the therapy long-term compared to new starters ( n = 230, miR-21 3.26×, P = 0.001) and associated with icodextrin use ( R = 0.52; 95% CI, 0.20–0.84), peritonitis count ( R = 0.16; 95% CI, 0.03–0.29), and dialysate cytokines. miR-21 down-regulated programmed cell death 4 and programmed cell death 4 protein was decreased in peritoneal membrane biopsy specimens from PD patients compared to healthy controls. New miR-21 targets were identified that may be important during PD fibrogenesis. These data identify miR-21 as an important effector of fibrosis in the peritoneal membrane, and a promising biomarker in the dialysis effluent for membrane change in patients receiving PD.

Fibrotic thickening of the peritoneum develops in patients receiving peritoneal dialysis (PD) for renal failure. For unknown reasons, however, in some patients it progresses to extensive fibrosis that compromises dialysis capacity of the peritoneum. It is increasingly clear that fibroblasts display large heterogeneity not only between but also within tissues. Differential surface expression of thymocyte differentiation antigen 1 (Thy-1) has been shown to identify functionally distinct fibroblast subsets in several organs. Here, we isolated Thy-1

42-year old patient was presented to our clinic with a fever lasting for seven months and a ten month history of subcutaneous nodules on all extremities and trunk. Further examination revealed anaemia, lymphocytopenia and elevation of inflammatory parameters and liver enzymes. Authors comment their difficulties in differential diagnostic process. The bone marrow biopsy and reappraisal of subcutaneous lesions confirmed idiopathic lobular panniculitis, known as Weber-Christian disease. A combined immunosuppressive therapy was followed by improvement of clinical state as well as laboratory parameters (Tab. 2, Fig. 3, Ref. 11).

Background Endothelial dysfunction is an early manifestation of cardiovascular disease (CVD) and consistently observed in patients with chronic kidney disease (CKD). We hypothesized that CKD is associated with systemic damage to the microcirculation, preceding macrovascular pathology. To assess the degree of “uremic microangiopathy”, we have measured microvascular density in biopsies of the omentum of children with CKD. Patients and Methods Omental tissue was collected from 32 healthy children (0–18 years) undergoing elective abdominal surgery and from 23 age-matched cases with stage 5 CKD at the time of catheter insertion for initiation of peritoneal dialysis. Biopsies were analyzed by independent observers using either a manual or an automated imaging system for the assessment of microvascular density. Quantitative immunohistochemistry was performed for markers of autophagy and apoptosis, and for the abundance of the angiogenesis-regulating proteins VEGF-A, VEGF-R2, Angpt1 and Angpt2. Results Microvascular density was significantly reduced in uremic children compared to healthy controls, both by manual imaging with a digital microscope (median surface area 0.61% vs. 0.95%, p

We analyzed the VDR TaqI (rs731236) gene polymorphism in children with and those without dental caries.A total of 388 subjects, 153 caries-free (with decayed/missing/filled teeth [DMFT] = 0) and 235 children with dental caries (DMFT ≥1), were genotyped by the TaqMan method.Although no significant differences in VDR TaqI allele and genotype frequencies between caries-free and caries-affected children were detected, a significant association between this polymorphism and gingivitis was found (p0.05).In contrast to previous studies from China and Turkey, the VDR TaqI gene variant cannot be used as a marker for identification of Czech children with increased dental caries risk.

Background The aim of this study was to compare four automated urinalysis systems: the Iris iQ200 Sprint (Iris Diagnostics, U.S.A.) combined with the Arkray AUTION MAX AX 4030, Iris + AUTION, Arkray AU 4050 (Arkray Global Business, Inc., Japan), Dirui FUS 2000 (Dirui Industrial Co., P.R.C.), and Menarini sediMAX (Menarini, Italy). Methods Urine concentrations of protein and glucose (Iris, Dirui) were compared using reference quantitative analysis on an Abbott Architect c16000. Leukocytes, erythrocytes, epithelia, and casts (Iris, Arkray, Diuri, Menarini) were compared to urine sediment under reference light microscopy, Leica DM2000 (Leica Microsystems GmbH, Germany) with calibrated FastRead plates (Biosigma S.r.l., Italy), using both native and stained preparations. Results Total protein and glucose levels were measured using the Iris + AUTION system with borderline trueness, while the Dirui analysis revealed worse performances for the protein and glucose measurements. True classifications of leukocytes and erythrocytes were above 85% and 72%, respectively. Kappa statistics revealed a nearly perfect evaluation of leukocytes for all tested systems; the erythrocyte evaluation was nearly perfect for the Iris, Dirui and Arkray analyzers and substantial for the Menarini analyzer. The epithelia identification was connected to high false negativity (above 15%) in the Iris, Arkray, and Menarini analyses. False-negative casts were above 70% for all tested systems. Conclusions The use of automated urinalysis demonstrated some weaknesses and should be checked by experienced laboratory staff using light microscopy.

The peritoneum plays an essential role in preventing abdominal frictions and adhesions and can be utilized as a dialysis membrane. Its physiological ultrastructure, however, has not yet been studied systematically. 106 standardized peritoneal and 69 omental specimens were obtained from 107 patients (0.1–60 years) undergoing surgery for disease not affecting the peritoneum for automated quantitative histomorphometry and immunohistochemistry. The mesothelial cell layer morphology and protein expression pattern is similar across all age groups. Infants below one year have a thinner submesothelium; inflammation, profibrotic activity and mesothelial cell translocation is largely absent in all age groups. Peritoneal blood capillaries, lymphatics and nerve fibers locate in three distinct submesothelial layers. Blood vessel density and endothelial surface area follow a U-shaped curve with highest values in infants below one year and lowest values in children aged 7–12 years. Lymphatic vessel density is much lower and again highest in infants. Omental blood capillary density correlates with parietal peritoneal findings, whereas only few lymphatic vessels are present. The healthy peritoneum exhibits major thus far unknown particularities, pertaining to functionally relevant structures and subject to substantial changes with age. The reference ranges established here provide a framework for future histomorphometric analyses and peritoneal transport modeling approaches.

The assessment of percutaneous absorption of molecules is a very important step in the evaluation of any dermal or transdermal drug delivery system. In order to perform percutaneous drug absorption studies, it is essential that the methods are standardized and that the integrity of the skin is monitored and maintained to ensure that the data obtained are valid and relevant. Reproducible data on percutaneous absorption in humans are as well required to predict the systemic risk from dermal exposure to chemicals, such as hazardous substances at the workplace, agrochemicals and cosmetic ingredients. In vitro and animal models provide important tools for screening a series of drug formulations, evaluation of skin permeation enhancing properties and mechanism of action of the carrier systems and estimation of rank of skin transport for a series of drug molecules. In this review, we have summarized in vitro testing of skin absorption using static Franz-type diffusion cells.

The aim of our study was to evaluate possible effect of ABCB1, and OPRM1 polymorphisms on the efficacy and safety of remifentanil in women undergoing elective cesarean section under general anesthesia. Women received remifentanil (1 microg/kg i.v.) 30 s prior to the induction to standardized general anesthesia. The ABCB1 (rs2032582, rs1045642) and OPRM1 (rs1799971) polymorphisms were analyzed from maternal peripheral blood. The basal hemodynamic and demographic parameters in the study population (n=54) were similar in all the subgroups. The median +/- SD increase of systolic blood pressure at 5 min from the baseline was practically completely abolished in homozygous carriers of ABCB1 variants in comparison with wild-type subjects -2.67+/-25.0 vs. 16.57+/-15.7 mm Hg, p0.05 for rs2032582, and 2.00+/-23.9 vs. 22.13+/-16.8 mm Hg, p0.05, for rs1045642, respectively. While no neonate belonging to ABCB1 wild-type homozygous or OPRM1 variant carrying mothers needed any resuscitative measure, 10.5 % of the neonates belonging to OPRM1 wild-type homozygous mothers received resuscitative support similarly as 11.1 %, and 12.5 % of neonates of mothers carrying variants of rs2032582, and rs1045642, respectively. Decreased stabilizing effects of remifentanil on maternal hemodynamics has been observed in ABCB1 wild type mothers, while the adaptation of the neonates was clinically worse in OPRM1 wild type, and ABCB1 variant allele carriers.

The aim of this study was to detect the prevalence of the polymorphisms of growth arrest—specific gene 6 (Gas6; Gas6 c. 834 + 7G > A) in patients with sticky platelet syndrome (SPS). Sticky platelet syndrome is a hereditary, autosomal dominant thrombophilia characterized by platelet hyperaggregation after low concentrations of platelet inducers—adenosine diphosphate (ADP) and epinephrine (EPI). The cause of SPS still remains unknown, but in recent years it was suggested that Gas6 protein may have a potential role in the pathogenesis of SPS. To assess the Gas6 polymorphisms (Gas6 c. 834 + 7G > A), 128 patients with SPS were included in the study and examined by polymerase chain reaction (PCR) method. GG genotype was detected in 63 (49.2%) patients, GA genotype in 53 (41.4%) patients, and AA genotype in 12 (9.4%) patients. The results in controls did not differ significantly compared to patients with SPS. Our findings did not prove allele A to be less associated with thrombosis and that ‘‘prothrombotic’’ allele G may be associated with higher risk of thrombosis. We cannot support the idea that Gas6 protein and Gas6 polymorphisms may be associated with thrombosis in SPS.

UNLABELLED The human multidrug resistance gene (MDR1) is encoding the transmembrane transporter P-glycoprotein (P-gp) which plays an important role in the efflux of various drugs and thus is potentially influencing the drug-treatment outcome. It has been indicated that the level of P-gp activity may be affected by the presence of single nucleotide polymorphisms (SNP) in the gene which led to the studies estimating MDR1-SNP frequencies in various populations. Here, we have investigated the occurrence of seven SNP in the MDR1 gene for the first time in Slovak population using multiplex SNaPshot genotyping method. The allelic frequencies of the most common gene variants, i.e. 1236C>T, 2677G>T, 2677G>A and 3435C>T were estimated to be 42.5%, 43.5%, 2%, and 44.5%, respectively. We found that the most prevalent haplotype in Slovak population is 1236C-2677G-3435C occurring in 42.2% of individuals. Our preliminary data show that it is reasonable and feasible to utilize MDR1 genotypes and haplotypes in Slovak patients, e.g. those with acute myeloid leukemia, in order to adjust the individual effective drug dosage and predict the patient's response to the treatment as well as the treatment outcome. KEYWORDS MDR1 gene, P-glycoprotein, polymorphisms, MDR1 haplotypes, Slovak population.

Objective. Severe fetomaternal transplacental hemorrhage increases the risk of fetal anemia. In the third trimester, the syncytiotrophoblast becomes thinner, especially in areas where it comes into intimate contact with villous capillaries, and forms a vasculosyncytial membrane. Our aim was to determine whether ABO compatibility puts the fetus at a greater risk of severe fetomaternal hemorrhage. Design. Case study. Setting. A tertiary care center. Sample and methods. Between 2003 and 2007, we evaluated eight cases of severe fetomaternal transfusion. The Kleihauer-Betke test was used for diagnosis of fetomaternal hemorrhage. We evaluated blood group compatibility between the mother and fetus and assessed the perinatal outcome. The Fischer’s factorial test was used for testing a hypothesis. Results. The incidence of adverse outcomes following transplacental hemorrhage was 75% (six of eight). There were two perinatal deaths and four infants were affected by posthypoxic damage of varying severity. Fetomaternal ABO compatibility was present in seven of the eight cases. The risk of severe fetomaternal hemorrhage was significantly increased when there was ABO compatibility between the mother and fetus. This was associated with a very poor perinatal outcome. Conclusion. We recommend that resuscitation in utero by intrauterine transfusion should be considered before the 33rd week of gestation in cases of severe fetal anemia. In later gestation, urgent cesarean section is required with adequate resuscitation of the newborn.

Basic part of the current standard treatment of organophosphate (OP) agent poisoning is administration of cholinesterase reactivators. It includes different types of oximes with a similar basic structure differing by the number of pyridinium rings and by the position of the oxime group in the pyridinium ring. Oximes hydrolytically cleave the organophosphates from acetylcholinesterase (AChE), restoring enzymatic function. This reactivation of AChE is dependent on the type of the agent and, on the reactivator used. From the common oximes, mono- and bisquaternary pyridinium oximes are more or less frequently used in clinical practice such as pralidoxime, obidoxime, trimedoxime, and HI-6. Though there are data on a good therapeutic effects of reactivators, some attempts to undermine the role of reactivators as effective antidotes against OP poisoning have been made. Some arguments on the necessity of their administration following OP poisoning are discussed with the aim to resolve the question on their effective use, possible repeated administration in the treatment of OP poisoning, their peripheral and central effects including questions on their penetration through the blood brain barrier as well as a possibility to achieve their effective concentration for AChE reactivation in the brain. Reactivation of cholinesterases in the peripheral and central nervous system is described and it is underlined its importance for the survival or death of the organism poisoned with OP. An universality of oximes able to reactivate AChE inhibited by all OP is questioned and trends (molecular modelling using neural network, structure-activity relationship, combination of reactivation and anticholinergic properties in one molecule) for future research are characterized.

Dental caries is a multifactorial, infectious disease where genetic predisposition plays an important role. Insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) has very recently been associated with caries in Polish children. The aim of this study was to analyze ACE I/D polymorphism in a group of caries-free children versus subjects affected by dental caries in the Czech population.In this case-control study, 182 caries-free children (with decayed/missing/filled teeth, DMFT = 0), 561 subjects with dental caries (DMFT ≥1) aged 13-15 years and 220 children aged 2-6 years with early childhood caries (ECC, dmft ≥1) were included. Genotype determination of ACE I/D polymorphism in intron 16 was based on the TaqMan method.Although no significant differences in the allele or genotype frequencies between the caries-free children and those affected by dental caries were observed, statistically significant differences between the children with DMFT = 0 and the subgroup of 179 patients with high caries experience (DMFT ≥4; p0.01 and p0.05, respectively) were detected. The comparison of DD versus II+ID genotype frequencies between the patients with DMFT ≥1 or DMFT ≥4 and healthy children also showed significant differences (31.5% or 35.6% vs. 23.6%, p0.05 or p0.01, respectively). A gender-based analysis identified a significant difference in the DD versus II+ID genotype frequencies only in girls (p0.05). In contrast, no significant association of ACE I/D polymorphism with ECC in young children was found (p0.05).ACE I/D polymorphism may be associated with caries in permanent but not primary dentition, especially in girls in the Czech population.

Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment.

Dental caries is one of the most frequent multifactorial diseases. Among the numerous factors influencing the risk of caries, genetics plays a substantial role, with heritability ranging from 40 to 60%. Gene variants affecting taste preference and glucose transport were recently associated with caries risk. The aim of this study was to analyze two common polymorphisms in the sweet taste receptor (TAS1R2) and glucose transporter (GLUT2) genes in children with dental caries and healthy controls in the Czech population.A total of 637 unrelated Caucasian children, aged 11-13 years, were included in this case-control study. One hundred and fifty-five subjects were caries-free (with decayed/missing/filled teeth, DMFT = 0) and 482 children were caries-affected (DMFT ≥ 1). The TAS1R2 (Ile191Val, rs35874116) and GLUT2 (Thr110Ile, rs5400) genotypes were determined using the 5' nuclease TaqMan® assay for allelic discrimination.Compared with subjects with the common Thr allele, carriers of the Ile allele of GLUT2 had significantly more frequently dental caries (p0.05, OR = 1.639, 95% CI: 1.089-2.466). Similarly, children with the Val allele for the TAS1R2 Ile191Val polymorphism were more frequently affected by caries than children who carried the Ile allele (p0.05, OR = 1.413, 95% CI: 1.014-1.969). In contrast, no significant associations between GLUT2 and/or TAS1R2 polymorphisms and fillings were found, but allele frequencies of the TAS1R2 variant were marginally significantly different between children with DMFT = 0 and DMFT ≥1 (p = 0.053, OR = 1.339, 95% CI: 0.996-1.799). However, no significant interaction between both genes and risk of dental caries was found.In conclusion, GLUT2 and TASR1 polymorphisms may influence the risk of caries in the Czech population.

There is a significant phenotypic variance among cystic fibrosis (CF) patients. Due to the role of TGF-beta1 in fibrotic processes we investigated its role in CF pathogenesis. TGF-beta 1 codons 10 and 25 were genotyped in 118 Czech CF patients and 268 controls by PCR-ARMS. Difference between CF and controls was found at codon 10, lower frequency of T/T homozygotes, and codon 25, higher frequency of G/C heterozygotes. We did not prove the association of TGF-beta1 polymorphisms and lung function in CF, however, the TT (codon 10)/GG (codon 25) genotype was preferentially associated with CF-related liver disease and diabetes. Independent of the TGF-beta1 genotype, production of cytokine was higher in patients than in controls with the notable exception of very low levels in Burkholderia cepacia complex colonized patients. In CF, both extremes, highest or lowest TGF-beta 1 production, were associated with impaired lung function.

Organophosphorus compounds such as nerve agents inhibit, practically irreversibly, cholinesterases by their phosphorylation in the active site of these enzymes. Current antidotal treatment used in the case of acute nerve agent intoxications consists of combined administration of anticholinergic drug (usually atropine) and acetylcholinesterase (AChE, EC 3.1.1.7) reactivator (HI-6, obidoxime, pralidoxime), which from a chemical view is a derivative from the group of pyridinium or bispyridinium aldoximes (commonly called "oxime"). Oximes counteract acetylcholine increase, resulting from AChE inhibition. In the human body environment these compounds are powerful nucleophiles and are able to break down the bond between AChE and nerve agent molecule. This process leads to renewal of enzyme functionality -- to its reactivation. The usefulness of oxime in the reactivation process depends on its chemical structure and on the nerve agent whereby AChE is inhibited. Due to this fact, selection of suitable reactivator in the treatment of intoxications is very important. In our work, we have compared differences in the in vitro inhibition potency of VX and Russian VX on rat, pig and human brain, and subsequently we have tested reactivation of rat brain cholinesterase inhibited by these agents using oxime HI-6, obidoxime, pralidoxime, trimedoxime and methoxime. The results showed that no major differences in the reactivation process of both VX and Russian VX-inhibited cholinesterase. The similarity in reactivation was caused by analogous chemical structure of either nerve agent; and that oxime HI-6 seems to be the most effective reactivator tested, which confirms that HI-6 is currently the most potent reactivator of AChE inhibited by nerve agents. The results obtained in our study should be considered in the future development of new AChE reactivators.

The morbidity and mortality rates in patients with cystic fibrosis (CF) are significantly affected by infections with Burkholderia cepacia complex. In a Czech CF Centre, the prevalence of the infection reached up to 30 %, with the majority of patients found to be infected with Burkholderia cenocepacia (formerly genomovar III of the Burkholderia cepacia complex). Since B. cenocepacia is associated with patient-to-patient transmission and epidemic outbreaks among CF patients, this study sought to examine the epidemiological relatedness between the Czech isolates belonging to the genomovar-homogeneous group. Eighty-three clinical isolates recovered from 67 CF patients were analysed using a random amplified polymorphic DNA (RAPD) assay and macrorestriction typing (SpeI and XbaI) followed by PFGE. A single predominant banding pattern shared by multiple isolates was detected, although SpeI-generated PFGE results yielded a higher rate of inter-pattern variability in comparison to the more uniform RAPD and XbaI-generated PFGE results for this clone. Both typing systems also showed that only three out of 67 patients harboured strains distinct from the major strain type. The dominant clone was characterized by PCR positivity for the B. cepacia epidemic strain marker, PCR negativity for the cable pilin subunit gene and close genetic relatedness to the epidemic strain of RAPD 01 type previously identified in Canada.

Antineutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability-increasing protein (BPI) were repeatedly found in cystic fibrosis (CF) patients. We analyzed the effect of BPI-ANCA in inhibiting neutrophil-mediated killing of Pseudomonas aeruginosa. The bactericidal effect expressed as percentage of killed bacteria after 1 h incubation with neutrophils was 55% when the neutrophils were pretreated with normal human serum, ranged from 49 to 63% with the sera from control BPI-ANCA-negative groups and sharply decreased to the mean 30.5% (range 8-51%) in the presence of BPI-ANCA. Furthermore, the effect mediated by BPI-ANCA was dose dependent and reflected the titer of BPI-ANCA in tested sera.

We developed a nested PCR assay that detects the recA gene of the Burkholderia cepacia complex in sputum. The product of the first PCR round is also used to identify the genomovar of the pathogen. The protocol achieves high sensitivity and specificity with simple interpretation of genomovar status.

One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1α protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules.

Although most of the pertinent data on the sequence-directed processes leading to genome rearrangements (GRs) have come from studies on somatic tissues, little is known about GRs in the germ line of patients with hereditary disorders. This study aims at identifying DNA motifs and higher order structures of genome architecture, which can result in losses and gains of genetic material in the germ line. We first identified candidate motifs by studying 112 pathogenic germ-line GRs in hereditary colorectal cancer patients, and subsequently created an algorithm, termed recombination type ratio, which correctly predicts the propensity of rearrangements with respect to homologous versus nonhomologous recombination events.

The Large genomic rearrangements (LGR) in BRCA1/2 represent a substantial proportion of disease-causing changes. In our pilot study we demonstrate the specific case of the Slovak breast/ovarian cancer family, where BRCA1 analysis revealed the discrepancy of SNPs haplotypes and primarily indicated the presence of LGR. Initially, the analysis of all exons of BRCA1 was based on the combination of SSCP and sequencing techniques. After the abnormal SNPs haplotypes identification, MLPA analysis was performed. The results were finally proved with array-comparative genomic hybridization (array-CGH). The hemizygous status of 9 SNPs identificated in BRCA1 indicated a possible occurrence of LGR. The MLPA results showed reduction of peaks levels for each BRCA1 exon. Array-CGH method displays a single deletion signal for BRCA1 gene among set of 287 gene probes. Totally, 8 members of the family where analysed, in 3 of them the deletion was confirmed. Two of the LGR carriers suffered with unilateral and bilateral breast cancer respectively; the third carrier of the LGR was affected with an ovarian cancer. We have discovered rare BRCA1 germline LGR affecting complete gene. According to our knowledge, this is the first Slovak family with LGR in BRCA1 gene and second time, when the complete deletion of BRCA1 gene was described worldwide. Concerning the family history it is evident, that clinical effect of LGR is comparable with small deletions/insertions and substitutions and leads to the loss-off gene function. In conclusion, it is also important to note that DNA analysis of BRCA1/2 genes should be performed in all affected members of breast/ovarian cancer families concurrently, since the discrepancy in the SNPs haplotypes may indicate the presence of LGR.