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Therapeutic angiogenesis by intentional formation of blood vessels is essential for treating various ischemic diseases, including limb ischemia. Because Wnt/β-catenin and angiopoietin-1/Tie2 signaling play important roles in endothelial survival and vascular stability, coactivation of these signaling pathways can potentially achieve therapeutic angiogenesis. In this study, we developed a bifunctional antibody fusion, consisting of a Tie2-agonistic antibody and the Furin domains of R-spondin 3 (RSPO3), to simultaneously activate Tie2 and Wnt/β-catenin signaling. We identified a Tie2-agonistic antibody T11 that cross-reacted with the extracellular domain of human and mouse Tie2, and evaluated its ability to increase endothelial cell survival and tube formation. We generated a bifunctional T11–RF12 by fusing T11 with the Furin-1 and −2 domains of RSPO3. T11–RF12 could bind not only to Tie2, but also to LGR5 and ZNRF3, which are counterparts of the Furin-1 and −2 domains. T11–RF12 significantly increased Wnt/β-catenin signaling, as well as the formation of capillary-like endothelial tubes, regardless of the presence of Wnt ligands. Coactivation of Tie2 and Wnt/β-catenin signaling by T11–RF12 increased the blood flow, and thereby reduced foot necrosis in a mouse hindlimb ischemia model. In particular, T11–RF12 induced therapeutic angiogenesis by promoting vessel stabilization through pericyte coverage and retaining endothelial expression of Frizzled 10 and active β-catenin. These results indicate that the agonistic synergism of Tie2 and Wnt/β-catenin signaling achieved using T11–RF12 is a novel therapeutic option with potential for treating limb ischemia and other ischemic diseases.

IntroductionThe binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp.MethodsWe utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp.ResultsAntibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp.DiscussionThese results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

Summary: Pluripotent stem cells (PSCs) represent the most promising clinical source for regenerative medicine. However, given the cellular heterogeneity within cultivation and safety concerns, the development of specific and efficient tools to isolate a pure population and eliminate all residual undifferentiated PSCs from differentiated derivatives is a prerequisite for clinical applications. In this study, we raised a monoclonal antibody and identified its target antigen as desmoglein-2 (DSG2). DSG2 co-localized with human PSC (hPSC)-specific cell surface markers, and its expression was rapidly downregulated upon differentiation. The depletion of DSG2 markedly decreased hPSC proliferation and pluripotency marker expression. In addition, DSG2-negative population in hPSCs exhibited a notable suppression in embryonic body and teratoma formation. The actions of DSG2 in regulating the self-renewal and pluripotency of hPSCs were predominantly exerted through the regulation of β-catenin/Slug-mediated epithelial-to-mesenchymal transition. Our results demonstrate that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal. : DSG2 is a desmosomal cadherin molecule. In this article, Min and colleagues show that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal and pluripotency and the acquisition of pluripotency during somatic cell reprogramming through the regulation of β-catenin-mediated EMT signaling. keywords: desmoglein-2, cell surface marker, pluripotent stem cells, monoclonal antibody, EMT

Introduction: Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. Methods: We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Result: Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 x 10-10 M and 1.32 x 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Discussion: Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide. [ABSTRACT FROM AUTHOR]

Introduction: The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp. Methods: We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp. Results: Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp. Discussion: These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry. [ABSTRACT FROM AUTHOR]