First-line therapy in atypical hemolytic uremic syndrome: consideration on infants with a poor prognosis.

BackgroundAtypical hemolytic uremic syndrome (aHUS) is a rare and heterogeneous disorder. The first line treatment of aHUS is plasma therapy, but in the past few years, the recommendations have changed greatly with the advent of eculizumab, a humanized monoclonal anti C5-antibody. Although recent recommendations suggest using it as a primary treatment for aHUS, important questions have arisen about the necessity of immediate use of eculizumab in all cases. We aimed to draw attention to a specific subgroup of aHUS patients with rapid disease progression and high mortality, in whom plasma therapy may not be feasible.MethodsWe present three pediatric patients of acute complement-mediated HUS with a fatal outcome. Classical and alternative complement pathway activity, levels of complement factors C3, C4, H, B and I, as well as of anti-factor H autoantibody and of ADAMTS13 activity were determined. The coding regions of CFH, CFI, CD46, THBD, CFB and C3 genes were sequenced and the copy number of CFI, CD46, CFH and related genes were analyzed.ResultsWe found severe activation and consumption of complement components in these patients, furthermore, in one patient we identified a previously not reported mutation in CFH (Ser722Stop), supporting the diagnosis of complement-mediated HUS. These patients were not responsive to the FFP therapy, and all cases had fatal outcome.ConclusionTaking the heterogeneity and the variable prognosis of atypical HUS into account, we suggest that the immediate use of eculizumab should be considered as first-line therapy in certain small children with complement dysregulation

Vasodilator Phosphostimulated Protein (VASP) Protects Endothelial Barrier Function During Hypoxia

The endothelial barrier controls the passage of solutes from the vascular space. This is achieved through active reorganization of the actin cytoskeleton. A central cytoskeletal protein involved into this is vasodilator-stimulated phosphoprotein (VASP). However, the functional role of endothelial VASP during hypoxia has not been thoroughly elucidated. We determined endothelial VASP expression through real-time PCR (Rt-PCR), immunhistochemistry, and Western blot analysis during hypoxia. VASP promoter studies were performed using a PGL3 firefly luciferase containing plasmid. Following approval by the local authorities, VASP−/− mice and littermate controls were subjected to normobaric hypoxia (8% O2, 92% N2) after intravenous injection of Evans blue dye. In in vitro studies, we found significant VASP repression in human microvascular and human umbilical vein endothelial cells through Rt-PCR, immunhistochemistry, and Western blot analysis. The VASP promoter construct demonstrated significant repression in response to hypoxia, which was abolished when the binding of hypoxia-inducible factor 1 alpha was excluded. Exposure of wild-type (WT) and VASP−/− animals to normobaric hypoxia for 4 h resulted in an increase in Evans blue tissue extravasation that was significantly increased in VASP−/− animals compared to WT controls. In summary, we demonstrate here that endothelial VASP holds significant importance for endothelial barrier properties during hypoxia

Polymorphic variants of SCN1A and EPHX1 influence plasma carbamazepine concentration, metabolism and pharmacoresistance in a population of Kosovar Albanian epileptic patients

Aim The present study aimed to evaluate the effects of gene variants in key genes influencing pharmacokinetic and pharmacodynamic of carbamazepine (CBZ) on the response in patients with epilepsy. Materials & Methods Five SNPs in two candidate genes influencing CBZ transport and metabolism, namely ABCB1 or EPHX1, and CBZ response SCN1A (sodium channel) were genotyped in 145 epileptic patients treated with CBZ as monotherapy and 100 age and sex matched healthy controls. Plasma concentrations of CBZ, carbamazepine-10,11-epoxide (CBZE) and carbamazepine-10,11-trans dihydrodiol (CBZD) were determined by HPLC-UV-DAD and adjusted for CBZ dosage/kg of body weight. Results The presence of the SCN1A IVS5-91G>A variant allele is associated with increased epilepsy susceptibility. Furthermore, carriers of the SCN1A IVS5-91G>A variant or of EPHX1 c.337T>C variant presented significantly lower levels of plasma CBZ compared to carriers of the common alleles (0.71±0.28 vs 1.11±0.69 μg/mL per mg/Kg for SCN1A IVS5-91 AA vs GG and 0.76±0.16 vs 0.94±0.49 μg/mL per mg/Kg for EPHX1 c.337 CC vs TT; PG showed a reduced microsomal epoxide hydrolase activity as reflected by a significantly decreased ratio of CBZD to CBZ (0.13±0.08 to 0.26±0.17, pT SNP and SCN1A 3148A>G variants were not associated with significant changes in CBZ pharmacokinetic. Patients resistant to CBZ treatment showed increased dosage of CBZ (657±285 vs 489±231 mg/day; P<0.001) but also increased plasma levels of CBZ (9.84±4.37 vs 7.41±3.43 μg/mL; P<0.001) compared to patients responsive to CBZ treatment. CBZ resistance was not related to any of the SNPs investigated. Conclusions The SCN1A IVS5-91G>A SNP is associated with susceptibility to epilepsy. SNPs in EPHX1 gene are influencing CBZ metabolism and disposition. CBZ plasma levels are not an indicator of resistance to the therapy

Global Functional Analyses of Cellular Responses to Pore-Forming Toxins

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs

Copper Induced Lysosomal Membrane Destabilisation in Haemolymph Cells of Mediterranean Green Crab (Carcinus aestuarii, Nardo, 1847) from the Narta Lagoon (Albania)

ABSTRACTDestabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA). Crabs collected in Narta Lagoon, Vlora (Albania) during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT) and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05), indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05) than in control group, demonstrating the presence of stress on the animals. These results showed thatC. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota

Accurate and precise determination of silver isotope fractionation in environmental samples by multicollector-ICPMS

High precision silver isotope ratios in environmental samples were determined by multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). Purification of Ag from sample matrixes was performed by a two stage tandem column setup with use of anion and cation exchange resin, sequentially. It was found that 1% HNO3 and 3% HCl was efficient to stabilize Ag in the final purified sample digests prior to MC-ICPMS determination. Pd at 2 \u3bcg g 121 was added to both sample and Ag standard solution as a common doping matrix as well as an internal standard for mass bias correction. Mass discrimination and instrument drift were corrected by a combination of internal normalization with Pd and standard-sample-standard bracketing, without assuming identical mass bias for Pd and Ag. NIST SRM 978a (silver isotopic standard reference material) was used for method validation and subjected to column separation and sample preparation processes. A value of 120.003 \ub1 0.010 \u2030 for \u3b4107/109Ag (mean and 2SD, n = 4) was obtained, confirming accurate results can be obtained using the proposed method. To the best of our knowledge, this is the first report on \u3b4107/109Ag variations in environmental samples. Significant differences in Ag isotope ratios were found among NIST SRM 978a standard, sediment CRM PACS-2, domestic sludge SRM 2781, industrial sludge 2782, and the fish liver CRM DOLT-4. The sediment CRM PACS-2 has a very small negative \u3b4107/109Ag value of 120.025 \ub1 0.012 \u2030 (2SD, n = 4). The domestic sludge SRM 2781 has a negative \u3b4107/109Ag value of 120.061 \ub1 0.010 \u2030 (2SD, n = 4), whereas industrial sludge SRM 2782 has a positive \u3b4107/109Ag value of +0.044 \ub1 0.014 \u2030 (2SD, n = 4), which may indicate the contribution of Ag from different anthropogenic inputs. DOLT-4 has a much larger negative value of 120.284 \ub1 0.014 \u2030 (2SD, n = 4), possibly caused by biological processes. These observations confirm that Ag isotope fractionation may provide a useful tool for fingerprinting sources of Ag in the environment and for studying a wide variety of chemical and biological processes in nature. High precision of better than \ub10.015 \u2030 (2SD, n = 4) obtained in real sample matrixes makes the present method well suited for monitoring small Ag isotope fractionation in nature.Des rapports isotopiques de haute pr\ue9cision de l\u2019argent dans des \ue9chantillons pr\ue9lev\ue9s dans l\u2019environnement sont obtenus par la spectrom\ue9trie de masse \ue0 source \ue0 plasma inductif et collection d\u2019ions multiples (MC-ICP-MS). Une purification de l\u2019Ag \ue0 partir de matrices de pr\ue9l\ue8vement est effectu\ue9e au moyen d\u2019un montage en tandem de deux colonnes; l\u2019une contenant un \ue9changeur d\u2019anions et l'autre, un \ue9changeur de cations. On constate que l\u2019utilisation de HNO3 \ue0 1 % et d\u2019HCL \ue0 3 % est efficace pour stabiliser l\u2019Ag dans les \ue9chantillons finaux dig\ue9r\ue9s et purifi\ue9s avant le dosage par la MC ICP-MS. On ajoute du Pd \ue0 une concentration de 2 \u3bcg g 121 aux deux \ue9chantillons et \ue0 la solution \ue9talon d\u2019Ag comme matrice de dopage commune, de m\ueame qu\u2019un \ue9talon interne pour la correction du biais massique. On a corrig\ue9 la discrimination de masse et la d\ue9rive de l\u2019instrument par la combinaison d\u2019une normalisation interne avec le Pd et d\u2019un encadrement \ue9talon-\ue9chantillon-\ue9talon, sans pr\ue9tendre une justesse de masse identique pour le Pd et l\u2019Ag. Le NIST SRM 978a (mat\ue9riau de r\ue9f\ue9rence \ue9talon d\u2019isotopes d\u2019argent) a \ue9t\ue9 utilis\ue9 pour valider la m\ue9thode et soumis aux processus de s\ue9paration par colonne et de pr\ue9paration d\u2019\ue9chantillons. On a obtenu une valeur \u3b4107/109Ag de 120,003 \ub1 0,010 \u2030 (moyenne et 2ET, n = 4), confirmant qu\u2019il est possible d\u2019obtenir des r\ue9sultats pr\ue9cis au moyen de la m\ue9thode propos\ue9e. Autant que nous sachions, c\u2019est la premi\ue8re fois que l\u2019on fait \ue9tat des variations de \u3b4107/109Ag dans des \ue9chantillons pr\ue9lev\ue9s dans l\u2019environnement. Nous avons constat\ue9 des diff\ue9rences importantes dans les rapports isotopiques de l\u2019Ag entre l\u2019\ue9talon NIST SRM 978a, le s\ue9diment CRM PACS-2, la boue domestique SRM 2781, la boue industrielle SRM 2782 et le foie de poisson CRM DOLT-4. Le s\ue9diment CRM PACS-2 poss\ue8de une tr\ue8s petite valeur \u3b4107/109Ag n\ue9gative de 120,025 \ub1 0,012 \u2030 (2ET, n = 4). La boue domestique SRM 2781 poss\ue8de une valeur \u3b4107/109Ag n\ue9gative de 120,061 \ub1 0,010 \u2030 (2ET, n = 4), alors que la boue industrielle SRM 2782 poss\ue8de une valeur \u3b4107/109Ag positive de +0,044 \ub1 0,014 \u2030 (2ET, n = 4), ce qui peut indiquer la contribution de l\u2019Ag provenant de diff\ue9rents apports anthropiques. Le DOLT-4 a une valeur n\ue9gative beaucoup plus importante de 120,284 \ub1 0,014 \u2030 (2ET, n = 4), probablement attribuable \ue0 des processus biologiques. Ces observations confirment que le fractionnement des isotopes de l\u2019Ag peut constituer un outil utile pour caract\ue9riser des sources d\u2019Ag dans l\u2019environnement et \ue9tudier une grande vari\ue9t\ue9 de processus chimiques et biologiques dans la nature. La haute pr\ue9cision sup\ue9rieure \ue0 \ub10,015 \u2030 (2ET, n = 4) obtenue dans des matrices de pr\ue9l\ue8vement r\ue9elles rend la pr\ue9sente m\ue9thode bien adapt\ue9e au suivi du faible fractionnement isotopique de l\u2019Ag dans la nature.Peer reviewed: YesNRC publication: Ye