Predicting the risk to develop preeclampsia in the first trimester combining promoter variant -98A/C of LGALS13 (placental protein 13), Black ethnicity, previous preeclampsia, obesity, and maternal age

BACKGROUND: We studied LGALS13 [Placental Protein 13 (PP13)] promoter DNA polymorphisms in preeclampsia (PE) prediction, given PP13’s effects on hypotension, angiogenesis and immunotolerance. METHODS: We retrieved 67 PE (49 term, 18 preterm) cases and 196 matched controls from first trimester plasma samples prospectively collected at King's College Hospital, London. Cell-free DNA was extracted and the four LGALS13 exons were sequenced after PCR amplification. Expression of LGALS13 promoter reporter constructs were determined in BeWo trophoblast-like cells with luciferase assays. RESULTS: A/C genotype in –98 position was the lowest in term PE compared to controls (p<0.032), similar to a South African cohort. Control but not all PE allele frequencies were in Hardy-Weinberg equilibrium (p=0.036). The Odds ratio for term PE calculated from prior risk, the A/A genotype and black ethnicity was 14 (p<0.001). In luciferase assays, the LGALS13 promoter “-98A" variant had 13% (p=0.04) and 26% (p<0.001) lower expression than the "-98C" variant in non-differentiated and differentiated BeWo cells, respectively. After 48-hour differentiation, there was 4.55- fold increase in expression of "-98C" variant versus 3.85-fold of "-98A" variant (p<0.001). CONCLUSION: Lower LGALS13 (PP13) expression by the "-98A/A" genotype appears to impose higher risk to develop PE and could aid in PE prediction

Liver-Targeting of Interferon-Alpha with Tissue-Specific Domain Antibodies

PMCID: PMC3581439This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Hepatitis C Virus Core-Derived Peptides Inhibit Genotype 1b Viral Genome Replication via Interaction with DDX3X

The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16–36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent protein (GFP) fusions to HCV core protein residues 16–36, but not by GFP fusions to core protein residues 16–35 or 16–34. Notably, the inhibition of HCV replication due to expression of the GFP fusion to HCV core protein residues 16–36 can be reversed by overexpression of DDX3X. These results suggest that the protein interface on DDX3X that binds the HCV core protein is important for replicon maintenance. However, infection of HuH-7 cells by HCV viruses of genotype 2a (JFH1) was not affected by expression of the GFP fusion protein. These results suggest that the role of DDX3X in HCV infection involves aspects of the viral life cycle that vary in importance between HCV genotypes

Removal of Hepatitis C Virus-Infected Cells by a Zymogenized Bacterial Toxin

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named “zymoxins”. These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases

Rise and Fall of an Anti-MUC1 Specific Antibody

So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology

Developing a new algorithm for first and second trimester preeclampsia screening in twin pregnancies

Objectives: Construct a new preeclampsia predicting algorithm in twins. Methods: Twins sampled at 10–13 and 16–20 gestational weeks and their marker values were log transformed into multiples of the gestation-specific medians (MoMs) for singletons and entered into a new logistic regression model with/without prior risk factors. Results: The cohort included 9 PE (18 samples) and 96 unaffected cases (175 samples) twin pregnant women. The algorithm constructed of PlGF, PAPP-A, PP13, Doppler UTPI, and MAP with prior risk factors generated an area under the curve of 0.918, 75% detection rate for 10% false-positive rate. Conclusions: The algorithm effectively forecasted twin risk to develop PE