Phenotypic expression of ADAMTS13 in glomerular endothelial cells

Background

ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13^+/+) and deficient (Adamts13^−/−) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells.

Methodology/Principal Findings

Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13^+/+ mice but no staining was visible in tissue from Adamts13^−/− mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13^−/− mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF.

Conclusions/Significance

Glomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries.     

Complex Changes in von Willebrand Factor-Associated Parameters Are Acquired during Uncomplicated Pregnancy

Background

The coagulation protein von Willebrand Factor (VWF) is known to be elevated in pregnancy. However, the timing and nature of changes in VWF and associated parameters throughout pregnancy are not well understood.

Objectives

To better understand the changes in VWF provoked by pregnancy, we studied VWF-associated parameters in samples collected over the course of healthy pregnancies.

Methods

We measured VWF antigen (VWF:Ag), VWF propeptide (VWFpp), Factor VIII (FVIII), and ADAMTS13 activity in samples collected from 46 women during pregnancy and at non-pregnant baseline. We also characterized pregnant vs. non-pregnant VWF multimer structure in 21 pregnancies, and performed isoelectric focusing (IEF) of VWF in two pregnancies which had samples from multiple trimesters.

Results

VWF:Ag and FVIII levels were significantly increased during pregnancy. ADAMTS13 activity was unchanged. VWFpp levels increased much later in pregnancy than VWF:Ag, resulting in a progressive decrease in VWFpp:Ag ratios. FVIII:VWF ratios also decreased in pregnancy. Most pregnancies exhibited a clear loss of larger VWF multimers and altered VWF triplet structure. Further evidence of acquired VWF qualitative changes in pregnancy was found in progressive, reversible shifts in VWF IEF patterns over gestation.

Conclusions

These data support a new view of pregnancy in which VWF can acquire qualitative changes associated with advancing gestational age. Modeling supports a scenario in which both increased VWF production and doubling of the VWF half-life would account for the data observed. We propose that gestation induces a prolongation in VWF survival, which likely contributes to increased total VWF levels and altered VWF structure.     

Severe dengue is associated with consumption of von Willebrand factor and its cleaving enzyme ADAMTS-13

Background

Thrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition.

Methods and Principal Findings

In a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30 with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen (VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls (n = 17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died. There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with an acquired von Willebrand disease.

Conclusion

These findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop.     

Models for Prediction of Factor VIII Half-Life in Severe Haemophiliacs: Distinct Approaches for Blood Group O and Non-O Patients

Background

Von Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life.

Methodology

Standardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15–44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated.

Principal Findings

FVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5±2.6 h versus 14.3±3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%).

Conclusion

Our approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

Hemostatic Factors and Risk of Coronary Heart Disease in General Populations: New Prospective Study and Updated Meta-Analyses

Background

Activation of blood coagulation and fibrinolysis may be associated with increased risk of coronary heart disease. We aimed to assess associations of circulating tissue plasminogen activator (t-PA) antigen, D-dimer and von Willebrand factor (VWF) with coronary heart disease risk.

Design

Prospective case-control study, systematic review and meta-analyses.

Methods

Measurements were made in 1925 people who had a first-ever nonfatal myocardial infarction or died of coronary heart disease during follow-up (median 19.4 years) and in 3616 controls nested within the prospective population-based Reykjavik Study.

Results

Age and sex-adjusted odds ratios for coronary heart disease per 1 standard deviation higher baseline level were 1.25 (1.18, 1.33) for t-PA antigen, 1.01 (0.95, 1.07) for D-dimer and 1.11 (1.05, 1.18) for VWF. After additional adjustment for conventional cardiovascular risk factors, corresponding odds ratios were 1.07 (0.99, 1.14) for t-PA antigen, 1.06 (1.00, 1.13) for D-dimer and 1.08 (1.02, 1.15) for VWF. When combined with the results from previous prospective studies in a random-effects meta-analysis, overall adjusted odds ratios were 1.13 (1.06, 1.21) for t-PA antigen (13 studies, 5494 cases), 1.23 (1.16, 1.32) with D-dimer (18 studies, 6799 cases) and 1.16 (1.10, 1.22) with VWF (15 studies, 6556 cases).

Conclusions

Concentrations of t-PA antigen, D-dimer and VWF may be more modestly associated with first-ever CHD events than previously reported. More detailed analysis is required to clarify whether these markers are causal risk factors or simply correlates of coronary heart disease.     

Classifying leukemia types with chromatin conformation data

Background

Although genetic or epigenetic alterations have been shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed.

Results

Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data.

Conclusions

Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes.     

Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide

Background

Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca²⁺-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca²⁺-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.

Principal Findings

Ca²⁺-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s⁻¹ (37°C) to 0.80±0.14 s⁻¹ (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca²⁺-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.

Conclusions

We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.     

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells

Background

Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells.

Methods

We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins.

Results

Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling.

Conclusion

Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.     

Storage of factor VIII variants with impaired von Willebrand factor binding in Weibel-Palade bodies in endothelial cells

Background

Point mutations resulting in reduced factor VIII (FVIII) binding to von Willebrand factor (VWF) are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood.

Methodology/Principal Findings

We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser) to severe (Tyr1680Phe, Ser2119Tyr) VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH.

Conclusions

Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo.     

Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry

Background

An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce.

Methodology

Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry.

Principal Findings

A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%).

Conclusions

Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.     

The GPI anchor signal sequence dictates the folding and functionality of the Als5 adhesin from Candida albicans

Background

Proteins destined to be Glycosylphosphatidylinositol (GPI) anchored are translocated into the ER lumen completely before the C-terminal GPI anchor attachment signal sequence (SS) is removed by the GPI-transamidase and replaced by a pre-formed GPI anchor precursor. Does the SS have a role in dictating the conformation and function of the protein as well?

Methodology/Principal Findings

We generated two variants of the Als5 protein without and with the SS in order to address the above question. Using a combination of biochemical and biophysical techniques, we show that in the case of Als5, an adhesin of C. albicans, the C-terminal deletion of 20 amino acids (SS) results in a significant alteration in conformation and function of the mature protein.

Conclusions/Significance

We propose that the locking of the conformation of the precursor protein in an alternate conformation from that of the mature protein is one probable strategy employed by the cell to control the behaviour and function of proteins intended to be GPI anchored during their transit through the ER.     

Green tea polyphenol treatment is chondroprotective,anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model

Introduction

Epigallocatechin 3-gallate (EGCG), a polyphenol present in green tea, was shown to exert chondroprotective effects in vitro. In this study, we used a posttraumatic osteoarthritis (OA) mouse model to test whether EGCG could slow the progression of OA and relieve OA-associated pain.

Methods

C57BL/6 mice were subjected to surgical destabilization of the medial meniscus (DMM) or sham surgery. EGCG (25 mg/kg) or vehicle control was administered daily for 4 or 8 weeks by intraperitoneal injection starting on the day of surgery. OA severity was evaluated using Safranin O staining and Osteoarthritis Research Society International (OARSI) scores, as well as by immunohistochemical analysis to detect cleaved aggrecan and type II collagen and expression of proteolytic enzymes matrix metalloproteinase 13 (MMP-13) and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). Real-time PCR was performed to characterize the expression of genes critical for articular cartilage homeostasis. During the course of the experiments, tactile sensitivity testing (von Frey test) and open-field assays were used to evaluate pain behaviors associated with OA, and expression of pain expression markers and inflammatory cytokines in the dorsal root ganglion (DRG) was determined by real-time PCR.

Results

Four and eight weeks after DMM surgery, the cartilage in EGCG-treated mice exhibited less Safranin O loss and cartilage erosion, as well as lower OARSI scores compared to vehicle-treated controls, which was associated with reduced staining for aggrecan and type II collagen cleavage epitopes, and reduced staining for MMP-13 and ADAMTS5 in the articular cartilage. Articular cartilage in the EGCG-treated mice also exhibited reduced levels of Mmp1, Mmp3, Mmp8, Mmp13,Adamts5, interleukin 1 beta (Il1b) and tumor necrosis factor alpha (Tnfa) mRNA and elevated gene expression of the MMP regulator Cbp/p300 interacting transactivator 2 (Cited2). Compared to vehicle controls, mice treated with EGCG exhibited reduced OA-associated pain, as indicated by higher locomotor behavior (that is, distance traveled). Moreover, expression of the chemokine receptor Ccr2 and proinflammatory cytokines Il1b and Tnfa in the DRG were significantly reduced to levels similar to those of sham-operated animals.

Conclusions

This study provides the first evidence in an OA animal model that EGCG significantly slows OA disease progression and exerts a palliative effect.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0508-y) contains supplementary material, which is available to authorized users.     

The structure of the NPC1L1 N-terminal domain in a closed conformation

Background

NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels.

Principal Findings

Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent.

Conclusion

The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.     

A randomized controlled phase Ib trial of the malaria vaccine candidate GMZ2 in African children

Background

GMZ2 is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP) that mediates an immune response against the blood stage of the parasite. Two previous phase I clinical trials, one in naïve European adults and one in malaria-exposed Gabonese adults showed that GMZ2 was well tolerated and immunogenic. Here, we present data on safety and immunogenicity of GMZ2 in one to five year old Gabonese children, a target population for future malaria vaccine efficacy trials.

Methodology/Principal Findings

Thirty children one to five years of age were randomized to receive three doses of either 30 µg or 100 µg of GMZ2, or rabies vaccine. GMZ2, adjuvanted in aluminum hydroxide, was administered on Days 0, 28 and 56. All participants received a full course of their respective vaccination and were followed up for one year. Both 30 µg and 100 µg GMZ2 vaccine doses were well tolerated and induced antibodies and memory B-cells against GMZ2 as well as its antigenic constituents MSP3 and GLURP. After three doses of vaccine, the geometric mean concentration of antibodies to GMZ2 was 19-fold (95%CI: 11,34) higher in the 30 µg GMZ2 group than in the rabies vaccine controls, and 16-fold (7,36) higher in the 100 µg GMZ2 group than the rabies group. Geometric mean concentration of antibodies to MSP3 was 2.7-fold (1.6,4.6) higher in the 30 µg group than in the rabies group and 3.8-fold (1.5,9.6) higher in the 100 µg group. Memory B-cells against GMZ2 developed in both GMZ2 vaccinated groups.

Conclusions/Significance

Both 30 µg as well as 100 µg intramuscular GMZ2 are immunogenic, well tolerated, and safe in young, malaria-exposed Gabonese children. This result confirms previous findings in naïve and malaria-exposed adults and supports further clinical development of GMZ2.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00703066","term_id":"NCT00703066"}}NCT00703066     

Evaluating the Potential Bioactivity of a Novel Compound ER1626

Background

ER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626.

Method

MTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis.

Results

ER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC₅₀ of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells.

Conclusion

In conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.     

Y-SNPs do not indicate hybridisation between European aurochs and domestic cattle

Background

Previous genetic studies of modern and ancient mitochondrial DNA have confirmed the Near Eastern origin of early European domestic cattle. However, these studies were not able to test whether hybridisation with male aurochs occurred post-domestication. To address this issue, Götherström and colleagues (2005) investigated the frequencies of two Y-chromosomal haplotypes in extant bulls. They found a significant influence of wild aurochs males on domestic populations thus challenging the common view on early domestication and Neolithic stock-rearing. To test their hypothesis, we applied these Y-markers on Neolithic bone specimens from various European archaeological sites.

Methods and Findings

Here, we have analysed the ancient DNA of 59 Neolithic skeletal samples. After initial molecular sexing, two segregating Y-SNPs were identified in 13 bulls. Strikingly, our results do not support the hypothesis that these markers distinguish European aurochs from domesticated cattle.

Conclusions

The model of a rapid introduction of domestic cattle into Central Europe without significant crossbreeding with local wild cattle remains unchallenged.     

A Phase 1 Trial of MSP2-C1, a Blood-Stage Malaria Vaccine Containing 2 Isoforms of MSP2 Formulated with Montanide? ISA 720

Background

In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naïve adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion.

Methodology/Principal Findings

The trial was designed to include three dose cohorts (10, 40, and 80 µg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 µg dose; no subjects received the 80 µg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 µg and 40 µg dose cohorts, with antibody levels by ELISA higher in the 40 µg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth.

Conclusions/Significance

As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant.

Trial Registration

Australian New Zealand Clinical Trials Registry 12607000552482