Expression and characterization of the soluble form of recombinant mature HSV-2 glycoprotein G for use in anti-HSV-2 IgG serodiagnostic immunoassay.

Herpes simplex virus type-2 (HSV-2) specific glycoprotein G (gG-2) is widely used as the antigen of choice for serodiagnosis of HSV-2. In order to develop an ELISA for serodetection of HSV-2 IgG in patient sera, the soluble form of the mature gG-2 antigen (mgG-2), gG283-649, was expressed using a baculovirus expression system. gG283-649 contains the complete extracellular domain of mgG-2 including the C-terminal region, which despite homology to gG-1, does not cross-react with HSV-1 antibodies present in HSV-1 positive patient sera. gG283-649 had increased performance compared to a previously described gG-2 fragment and showed high sensitivity and specificity in a method comparison with HerpeSelect 1 & 2 Immunoblot IgG, a commercially available FDA-cleared assay for serodetection of HSV-1 and 2 antibodies. A total of 234 clinical samples consisting of 134 high risk samples, including 45 samples from pregnant subjects, and a panel of 100 mixed diagnosis samples, spanning the measurable range were tested in the method comparison. Clinical sensitivity and specificity were determined to be 94.2% and 100%, respectively. We conclude that this soluble form of mgG-2 is a novel antigen of choice for developing an ELISA for type-specific serodiagnosis of HSV-2.

Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling.

Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs, progenitors, and, ultimately, neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, although a few endogenous factors are known to regulate neuronal differentiation. Astrocytes have been implicated, but their role in juxtacrine (that is, cell-cell contact dependent) signaling in NSC niches has not been investigated. We found that ephrin-B2 presented from rodent hippocampal astrocytes regulated neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies revealed a previously unknown role for ephrin-B2 in instructing neuronal differentiation. In addition, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activated ß-catenin in vitro and in vivo independently of Wnt signaling and upregulated proneural transcription factors. Ephrin-B2(+) astrocytes therefore promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.

Understanding intracellular transport processes pertinent to synthetic gene delivery via stochastic simulations and sensitivity analyses.

A major challenge in synthetic gene delivery is to quantitatively predict the optimal design of polymer-based gene carriers (polyplexes). Here, we report a consistent, integrated, and fundamentally grounded computational methodology to address this challenge. This is achieved by accurately representing the spatio-temporal dynamics of intracellular structures and by describing the interactions between gene carriers and cellular components at a discrete, nanoscale level. This enables the applications of systems tools such as optimization and sensitivity analysis to search for the best combination of systems parameters. We validate the approach using DNA delivery by polyethylenimine as an example. We show that the cell topology (e.g., size, circularity, and dimensionality) strongly influences the spatiotemporal distribution of gene carriers, and consequently, their optimal intracellular pathways. The model shows that there exists an upper limit on polyplexes' intracellular delivery efficiency due to their inability to protect DNA until nuclear entry. The model predicts that even for optimally designed polyethylenimine vectors, only approximately 1% of total DNA is delivered to the nucleus. Based on comparison with gene delivery by viruses, the model suggests possible strategies to significantly improve transfection efficiencies of synthetic gene vectors.