The effects of nitric oxide on prostanoid production and release by human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) express and synthesize both constitutive and inducible nitric oxide synthase (NOS) and cyclo-oxygenase (COX) enzymes, and have been extensively used as an in vitro model to investigate the role of these enzymes in the patho-physiology of placenta-fetal circulation. In this study we investigated the role of NO in regulating prostanoid production and release from HUVEC. Both untreated and IL-1beta-treated HUVEC were exposed to various NOS inhibitors and NO donors in short-term (1 or 3 hours) experiments, and the effects on prostanoid production were evaluated through the measurement of prostaglandins (PG) I2, E2 and F2alpha released in the incubation medium. We found that the inhibition of inducible NOS but not endothelial NOS antagonizes the IL-1beta-induced increase in PGI2 release. However, NOS inhibitors do not modify baseline PGI2 production. Pharmacological levels of NO, obtained with various NO donors, inhibit basal and IL-1beta-stimulated PG release.     

Synthesis and cholinergic affinity of diastereomeric and enantiomeric isomers of 1-methyl-2-(2-methyl-1,3-dioxolan-4-yl)- pyrrolidine, 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine and of Their iodomethylates

Four out of the eight possible stereoisomers of 1-methyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidine, 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine and the corresponding iodomethylates have been synthesised. They were formally derived from hybridisation of potent though unselective agonists studied before, such as 1,3-dioxolane 1 and 1,3-oxathiolane 2, with the structure of nicotine. It was expected that, by exalting the molecular complexity of the parent compounds, in particular through stereochemical complication in the proximity of the critical cationic head of the molecule, the chance to find agonists able to discriminate among cholinergic receptors subtypes would increase. The relative and absolute configuration of the compounds obtained has been established by means of NMR spectroscopy and X-ray crystallography. In preliminary studies, their binding affinity has been evaluated on rat brain nicotinic and muscarinic receptors. While none of the compounds showed any nicotinic affinity up to the dose of 10 microM, most of the iodomethylates were endowed with promising affinity for the muscarinic receptors.     

The surprising composition of the salivary proteome of preterm human newborn

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β(4) and β(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.     

Amendments promote the development of Lolium perenne in soils affected by historical copper smelting operations

The Puchuncaví valley, central Chile, has been exposed to aerial emissions from a copper smelter. Nowadays, soils in the surroundings are sparsely-vegetated, acidic, and metal-contaminated, and their remediation is needed to reduce environmental risks. We assessed effectiveness of lime, fly ash, compost, and iron grit as amendments to immobilize Cu in soils and promote plant growth. Amended soils were cultivated with Lolium perenne for 60 days under controlled conditions. Total dissolved Cu and Cu2+ activity in the soil solution, ryegrass biomass, and Cu accumulation in plant tissues were measured. Addition of lime and fly ash decreased Cu concentrations and Cu2+ activity in the soil solution, increased plant biomass, and reduced shoot Cu concentration below 22 mg kg(-1) (the phytotoxicity threshold for the species). The most effective amendment with respect to the shoot biomass yield was a combination of lime and compost. Water content of the substrate and the K accumulation were positively correlated with the compost application rate. Compost combined with iron grit decreased dissolved Cu concentrations during the period of highest solubility, i.e., during the first 60 days after the compost application. However, iron grit incorporation into soils amended with lime and compost decreased the shoot biomass of ryegrass.     

Loss of the BMP antagonist, SMOC-1, causes Ophthalmo-acromelic (Waardenburg Anophthalmia) syndrome in humans and mice

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to ～10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.     

Tools to support policy decisions related to treatment strategies and surveillance of Schistosomiasis japonica towards elimination

Background

Appropriate diagnostics to monitor disease trends and assess the effectiveness and impact of interventions are essential for guiding treatment strategies at different thresholds of schistosomiasis transmission and for certifying elimination. Field validation of these assays is urgently needed before they can be adopted to support policy decisions of the national programme for control and elimination of schistosomiasis in P.R. China. We compared the efficacy and utility of different immunoassays in guiding control strategies and monitoring the endemic status of S. japonicum infections towards elimination.

Methodology/Principal Findings

A cross-sectional survey was conducted in seven villages with different transmission intensities settings to assess the performance and utility of three immunoassays, e.g., an indirect hemagglutination assay (IHA_JX), an enzyme linked immunosorbent assay (ELISA_SZ), and a dot immunogold filtration assay (DIGFA_SH). 6,248 individuals aged 6–65 years old who gave consent and supplied their stool and blood samples were included for data analysis. Results showed that ELISA_SZ performed significantly higher sensitivity (95.45%, 95%CI: 92.94–97.97%) than IHA_JX (87.59%, 95%CI: 83.51–91.49%) and DIGFA_SH (79.55%, 95%CI: 74.68–84.41%), especially in subgroups with very low infection intensity. The specificity of ELISA_SZ, IHA_JX, DIGFA_SH in 6–9 year olds with occasional exposure was nearly 90%. DIGFA_SH performed the highest screening efficacy for patients among three assays with overall positive predicative value of 13.07% (95%CI: 11.42–14.72%). We found a positive correlation of antibody positive rate of IHA_JX with results of stool examination in age strata (r = 0.70, P<0.001). Seropositivity of IHA_JX in children aged 6–9 years old showed an excellent correlation with prevalence of schistosome infection in the seven communities (r = 0.77, P<0.05).

Conclusions/Significance

Studies suggest that ELISA_SZ could be used to guide selective chemotherapy in moderate or low endemic regions. IHA_JX could be used to as a surveillance tool and for certifying elimination of schistosomiasis through monitoring children as a sentinel population.     

Evaluation of immunoassays for the diagnosis of Schistosoma japonicum infection using archived sera

Background

With a national program initiated recently to reduce transmission of Schistosoma japonicum in the People''s Republic of China (P.R. China), there is an urgent need for accessible, quality-assured diagnostics for case detection, surveillance, and program monitoring of chemotherapy efficacy and other control interventions in areas of low endemicity. We compared the performance of nine immunodiagnostic tests developed in P.R. China for detection of antibodies against S. japonicum and established their priority for further assessment in field settings.

Methodology/Principal Findings

Using the Kato-Katz technique as the reference standard, 240 well-characterized archived serum specimens (100 positive and 140 negative) were evaluated in nine immunological tests developed in P.R. China. The enzyme-linked immunoelectrotransfer blot assay (EITB), which uses an adult worm extract of S. japonicum, supplied by the Center of Disease Control and Prevention, USA, was also evaluated. The sensitivity and specificity of each test were determined and the reproducibility of each test was assessed by evaluating operator-to-operator and run-to-run variation. In addition the simplicity of use for the end-user was evaluated. All tests showed good sensitivities ranging from 92.0% (95% confidence interval (CI): 86.7–97.3%) to 98.0% (95% CI: 95.3–100.0%). The test specificities varied from 70.0% (95% CI: 62.4–77.6%) to 97.1% (95% CI: 94.4–99.9%). All tests showed excellent reproducibility with a discordant rate in the range of 0–10.0% for operator-to-operator variation and run-to-run variation. All tests, except one magnetic particle-based enzyme-linked immunosorbent assay, were found to be easy to use, especially the dot immunogold filtration assays.

Conclusions/Significance

Most evaluated tests had acceptable performance characteristics and could make an impact on the schistosomiasis control programs in P.R. China. Three tests with the highest sensitivity, specificity and greatest ease of use, were selected for further evaluation in field settings.     

Protein-protein interaction heterogeneity of plasma apolipoprotein A1 in nephrotic syndrome

Apolipoprotein A1 (apoA1) is a component of the high density lipoproteins (HDL) that regulates the transport of cholesterol between the liver and peripheral cells and modulates the removal of any excess of cholesterol from membranes. Any variation in apoA1 composition may modify the plasma lipid profile and be involved in atherogenesis. We investigated apoA1 composition in plasma of 6 children with nephrotic syndrome, a condition characterized by high levels of cholesterol in plasma and by a high risk to develop atherosclerosis. Non-denaturing two-dimensional electrophoresis (Nat/SDS-PAGE), mass spectrometry, western blot and pull down experiments were done to characterize proteins and define putative interactions. ApoA1 was resolved in 12 variants, 6 of which had a slightly lower molecular weight (18-19 KDa) and migrated on the same axes of the β chain of haptoglobin (Hp). Low molecular weight apoA1 were observed in carriers of different Hp haplotypes (including one homozygous for the rare ββα1α1) ruling out any contaminant effect of co-migration of apoA1 with Hp α2 chain. Overall, apoA1 isoforms were much more present in plasma of nephrotic patients compared to a normal profile. These findings show that apoA1 plasma in nephrotic syndrome is heterogeneous in terms of molecular weight. Low molecular weight fragments lack internal structural domains and likely form macro-aggregates with Hp. Fragmentation and transport of apoA1 may be involved in the general disorder of lipid metabolism that characterizes nephrotic syndrome.     

The Staphylococcus aureus peptidoglycan protects mice against the pathogen and eradicates experimentally induced infection

Staphylococcus aureus, in spite of antibiotics, is still a major human pathogen causing a wide range of infections. The present study describes the new vaccine A170PG, a peptidoglycan-based vaccine. In a mouse model of infection, A170PG protects mice against a lethal dose of S. aureus. Protection lasts at least 40 weeks and correlates with increased survival and reduced colonization. Protection extends into drug-resistant (MRSA or VISA) and genetically diverse clinical strains. The vaccine is effective when administered - in a single dose and without adjuvant - by the intramuscular, intravenous or the aerosol routes and induces active as well as passive immunization. Of note, A170PG also displays therapeutic activity, eradicating staphylococci, even when infection is systemic. Sustained antibacterial activity and induction of a strong and rapid anti-inflammatory response are the mechanisms conferring therapeutic efficacy to A170PG.     

Homocysteinylated albumin promotes increased monocyte-endothelial cell adhesion and up-regulation of MCP1, Hsp60 and ADAM17

Rationale

The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties.

Objective

Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels.

Methods and Results

Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2.

Conclusions

Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.