Enzyme-Linked Immunosorbent Assay Using Henipavirus-Receptor EphrinB2 and Monoclonal Antibodies for Detecting Nipah and Hendra Viruses.

The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.

Cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein.

Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiVB) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape.

The protective role of Ephrin-B2/EphB4 signaling in osteogenic differentiation under inflammatory environment.

Inflammation and alveolar bone destruction constitute the main pathological process of periodontitis. However, the molecular mechanisms of bone destruction under the inflammation environment remain unclear. This study aims to explore the role of Ephrin-B2/EphB4 signaling in osteogenic differentiation under the inflammation environment. Mouse pre-osteoblasts MC3T3-E1 were pretreated with lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS). The Ephrin-B2/EphB4 signaling was activated, and the osteogenic differentiation of cells was examined. The results showed that activation of Ephrin-B2/EphB4 signaling promoted the expression levels of osteogenic differentiation-related genes, and also relieved the inhibitory effect of Pg-LPS on osteogenesis. Noticeably, the effect of Ephrin-B2/EphB4 signaling might be related to the mitogen-activated protein kinase (MAPK) pathway. While applying Ephrin-B2-Fc and EphB4-Fc to periodontitis mice, we observed the reduction of alveolar crest destruction. The current study revealed the possible role of Ephrin-B2/EphB4 signaling in reducing bone destruction in periodontitis and suggested its potential values for further research.

Single Nucleotide Polymorphism Analysis in HIV and Kaposi's Sarcoma Disease by Microarray Technique.

BACKGROUND: Emergence of Kaposi's Sarcoma in the cases other than HIV, following the use of immunosuppressant drugs, demonstrates that it is related to weak immunity. The fact that this malignancy does not occur in every HIV-positive patient suggests that genetic predisposition may also be effective. Replacement of one of the base pairs of adenine, guanine, cytosine, and thymine that constitute the DNA sequence in the human genome with another base pair can affect susceptibility to disease, response to treatment, and immunity. OBJECTIVE: The purpose of this study is to analyze the Single Nucleotide Polymorphism that could predispose to Kaposi's sarcoma of an HIV-infected patient and to identify which nucleotides such SNPs correspond to, using the microarray technology. MATERIALS AND METHODS: The blood samples of individuals, one of whom was diagnosed with Kaposi's Sarcoma HIV (+) visiting the outpatient clinic of infectious diseases polyclinic of Harran University Research and Practice Hospital and of a healthy individual with no Kaposi's Sarcoma, were used in the study. Following the DNA isolation of the blood samples taken from the respective individuals, a SNP analysis was conducted on the microarray device. 204,000 SNPs obtained were scanned later on in the databases in an attempt to identify the SNPs related to Kaposi's Sarcoma. RESULTS: In the 204,000 SNP screenings, we scrutinized the SNPs that differ in the case of Kaposi's Sarcoma [KS (+) and HIV (+)] on the basis of Control [KS(-) and HIV(-)] and HIV+ [KS(-)], and two SNPs of the ENDRA gene, three SNPs of the ADRA1A gene, six SNPs of the STIM1 gene, four SNPs of the EFNB2 gene, and one SNP of the CD209 gene were found to be different. However, when it comes to all SNPs (all the 204.000 SNPs) screened in terms of allele, it was observed that the AA and BB alleles were lower in the patient with Kaposi's Sarcoma [KS (+) and HIV (+)] compared to other groups and AB alleles were found to be higher than others in the patient with Kaposi's sarcoma [KS] (+) and HIV (+)]. CONCLUSION: In the microarray study we have conducted, 204,000 SNPs were screened for Control (HIV-) HIV (+) and HIV (+) patient with Kaposi's Sarcoma. It was found that 32,362 of those SNPs had different alleles in the Kaposi's Sarcoma [KS + HIV (+)] patient, while they had the same ones in the control [KS (-) and HIV (-)] and HIV + [KS (-)] group. 16 of the 32,362 SNPs took place among the genes related to Kaposi's Sarcoma. In the cases of Kaposi's Sarcoma with suspected diagnosis, it can be used as a beneficial laboratory test.

EPH receptor B2 stimulates human monocyte adhesion and migration independently of its EphrinB ligands.

The molecular basis of atherosclerosis is not fully understood and mice studies have shown that Ephrins and EPH receptors play a role in the atherosclerotic process. We set out to assess the role for monocytic EPHB2 and its Ephrin ligands in human atherosclerosis and show a role for EPHB2 in monocyte functions independently of its EphrinB ligands. Immunohistochemical staining of human aortic sections at different stages of atherosclerosis showed that EPHB2 and its ligand EphrinB are expressed in atherosclerotic plaques and that expression proportionally increases with plaque severity. Functionally, stimulation with EPHB2 did not affect endothelial barrier function, nor did stimulation with EphrinB1 or EphrinB2 affect monocyte-endothelial interactions. In contrast, reduced expression of EPHB2 in monocytes resulted in decreased monocyte adhesion to endothelial cells and a decrease in monocyte transmigration, mediated by an altered morphology and a decreased ability to phosphorylate FAK. Our results suggest that EPHB2 expression in monocytes results in monocyte accumulation by virtue of an increase of transendothelial migration, which can subsequently contribute to atherosclerotic plaque progression.

The heparan sulfate editing enzyme Sulf1 plays a novel role in zebrafish VegfA mediated arterial venous identity.

Arterial and venous specification is critical for establishing and maintaining a functioning vascular system, and defects in key arteriovenous signaling pathways including VEGF (vascular endothelial growth factor) lead to congenital arteriopathies. The activities of VEGF, are in part controlled by heparan sulfate (HS) proteoglycans, significant components of the endothelial glycocalyx. The level of 6-O sulfation on HS polysaccharide chains, that mediate the interaction between HS and VEGFA, is edited at the cell surface by the enzyme SULF1. We investigated the role of sulf1 in vascular development. In zebrafish sulf1 is expressed in the head and tail vasculature, corresponding spatially and temporally with vascular development. Targeted knockdown of sulf1 by antisense morpholinos resulted in severe vascular patterning and maturation defects. 93 % of sulf1 morphants show dysmorphogenesis in arterial development leading to occlusion of the distal aorta and lack of axial and cranial circulation. Co-injection of vegfa165 mRNA rescued circulatory defects. While the genes affecting haematopoiesis are unchanged, expression of several arterial markers downstream of VegfA signalling such as notch and ephrinB2 are severely reduced in the dorsal aorta, with a concomitant increase in expression of the venous markers flt4 in the dorsal aorta of the morphants. Furthermore, in vitro, lack of SULF1 expression downregulates VEGFA-mediated arterial marker expression, confirming that Sulf1 mediates arterial specification by regulating VegfA165 activity. This study provides the first in vivo evidence for the integral role of the endothelial glycocalyx in specifying arterial-venous identity, vascular patterning and arterial integrity, and will help to better understand congenital arteriopathies.

Direct inhibition of cell surface ephrin-B2 by recombinant ephrin-B2/FC.

First messengers and viral transfection are the two most common ways to stimulate cells for signal output, although their applications are limited. We investigated mechanisms of inducing neural stem cell differentiation using recombinant ephrin-B2/Fc and found that it acted as a ligand and inhibited endogenous ephrin-B2, which maintenance of the neural progenitor cell state, by direct interference. Our results showed the movement of ephrin-B2/Fc within the cell and indicated that it recycled to the plasma membrane surface, revealing a possible pattern of ephrin trafficking. Our results also serve as proof of concept for the reconstruction of the intracellular domain of ephrin using an artificial receptor to direct input signals in future studies.

T cell-specific deletion of EFNB2 minimally affects T cell development and function.

BACKGROUND: Eph kinases and their ephrin ligands (EFN) are all cell surface molecules, capable of transmitting signals in both directions (1, 2). Such bidirectional signaling is called forward (from EFNs to Ephs) and reverse (from Ephs to EFNs) signaling. Eph family kinases have 15 members, divided into A and B subfamilies. Ephrin ligands have 9 members, also classified into A and B families. Ephs and ephrins interact promiscuously, but EphAs mainly interact with EFNAs, and EphBs with EFNBs. EphB family kinases and their ephrin ligands (EFN) are expressed in the T cell compartment. RESULTS: In this study, using mice with T cell-specific EFNB2 gene knockout (EFNB2 KO mice), we investigated T cell development and function after EFNB2 deletion. EFNB2 KO mice presented normal thymus weight and cellularity. Their thymocyte subpopulations, such as CD4CD8 double positive cells and CD4 and CD8 single positive cells, were normally distributed, but there was a significant relative increase of CD4CD8 double negative cells. Flow cytometry analysis revealed that there was a moderate increase in the DN3 subpopulation. This augmented percentage of DN cells was further confirmed in competitive repopulation chimeras, suggesting that EFNB2 is involved in thymocyte development. The EFNB2 KO mice had normal T cell numbers and percentages in the spleen, and the T cells were able to be activated and to proliferate normally upon TCR ligation. Further, EFNB2 KO naïve CD4 cells were capable of differentiating into Th1, Th2, Th17 and Treg cells similar to WT naïve CD4 cells. CONCLUSIONS: Our results suggest the involvement of EFNB2 in thymocyte development. However, heavy redundancy among Eph/EFN family members prevents the occurrence of detrimental phenotypes in the T cell compartment caused by T cell-specific EFNB2 gene null mutation.

Blocking ephrinB2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth.

Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor-independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.

Novel strategy for selection of monoclonal antibodies against highly conserved antigens: phage library panning against ephrin-B2 displayed on yeast.

Ephrin-B2 is predominately expressed in endothelium of arterial origin, involved in developmental angiogenesis and neovasculature formation through its interaction with EphB4. Despite its importance in physiology and pathological conditions, it has been challenging to produce monoclonal antibodies against ephrin-B2 due to its high conservation in sequence throughout human and rodents. Using a novel approach for antibody selection by panning a phage library of human antibody against antigens displayed in yeast, we have isolated high affinity antibodies against ephrin-B2. The function of one high affinity binder (named as 'EC8') was manifested in its ability to inhibit ephrin-B2 interaction with EphB4, to cross-react with murine ephrin-B2, and to induce internalization into ephrin-B2 expressing cells. EC8 was also compatible with immunoprecipitation and detection of ephrin-B2 expression in the tissue after standard chemical fixation procedure. Consistent with previous reports on ephrin-B2 induction in some epithelial tumors and tumor-associated vasculatures, EC8 specifically detected ephrin-B2 in tumors as well as the vasculature within and outside of the tumors. We envision that monoclonal antibody developed in this study may be used as a reagent to probe ephrin-B2 distribution in normal as well as in pathological conditions and to antagonize ephrin-B2 interaction with EphB4 for basic science and therapeutic applications.

Ephrinb1 and Ephrinb2 are associated with interleukin-7 receptor α and retard its internalization from the cell surface.

IL-7 plays vital roles in thymocyte development, T cell homeostasis, and the survival of these cells. IL-7 receptor α (IL-7Rα) on thymocytes and T cells is rapidly internalized upon IL-7 ligation. Ephrins (Efns) are cell surface molecules and ligands of the largest receptor kinase family, Eph kinases. We discovered that T cell-specific double gene knock-out (dKO) of Efnb1 and Efnb2 in mice led to reduced IL-7Rα expression in thymocytes and T cells, and that IL-7Rα down-regulation was accelerated in dKO CD4 cells upon IL-7 treatment. On the other hand, Efnb1 and Efnb2 overexpression on T cell lymphoma EL4 cells retarded IL-7Rα down-regulation. dKO T cells manifested compromised STAT5 activation and homeostatic proliferation, an IL-7-dependent process. Fluorescence resonance energy transfer and immunoprecipitation demonstrated that Efnb1 and Efnb2 interacted physically with IL-7Rα. Such interaction likely retarded IL-7Rα internalization, as Efnb1 and Efnb2 were not internalized. Therefore, we revealed a novel function of Efnb1 and Efnb2 in stabilizing IL-7Rα expression at the post-translational level, and a previously unknown modus operandi of Efnbs in the regulation of expression of other vital cell surface receptors.

Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells.

PURPOSE: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. METHODS: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 microL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). RESULTS: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2+/-VEGF compared to IgG control+/-VEGF (P<0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. CONCLUSIONS: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.

Neutralization assays for differential henipavirus serology using Bio-Plex protein array systems.

Hendra virus (HeV) and Nipah virus (NiV) are related emerging paramyxoviruses classified in the genus Henipavirus. Both cause fatal disease in animals and humans and are classified as biosafety level 4 pathogens. Here we detail two new multiplexed microsphere assays, one for antibody detection and differentiation and another designed as a surrogate for virus neutralization. Both assays utilize recombinant soluble attachment glycoproteins (sG) whereas the latter incorporates the cellular receptor, recombinant ephrin-B2. Spectrally distinct sG(HeV)- and sG(NiV)-coupled microspheres preferentially bound antibodies from HeV- and NiV-seropositive animals, demonstrating a simple procedure to differentiate antibodies to these closely related viruses. Soluble ephrin-B2 bound sG-coupled microspheres in a dose-dependent fashion. Specificity of binding was further evaluated with henipavirus G-specific sera and MAbs. Sera from henipavirus-seropositive animals differentially blocked ephrin-B2 binding, suggesting that detection and differentiation of HeV and NiV neutralizing antibodies can be done simultaneously in the absence of live virus.

Clinical pulmonary autograft valves: pathologic evidence of adaptive remodeling in the aortic site.

OBJECTIVE: We studied the pathologic features, cellular phenotypes, and matrix remodeling of clinical pulmonary-to-aortic valve transplants functioning up to 6 years. METHODS: Nine autografts and associated vascular walls early (2-10 weeks) and late (3-6 years) postoperatively were examined by using routine morphologic methods and immunohistochemistry. In 4 cases autograft and homograft cusps were obtained from the same patients. RESULTS: Autografts had near-normal trilaminar cuspal structure and collagen architecture and viable valvular interstitial and endothelial cells throughout the time course. In contrast, cusps of homografts used to replace the pulmonary valves in the same patients were devitalized. In early autograft explants, 19.3% +/- 2.4% of cuspal interstitial cells were myofibroblasts expressing alpha-actin. In contrast, myofibroblasts comprised only 6.0% +/- 1.1% of cells in late explants and 2.5% +/- 0.4% and 4.6% +/- 0.8% of cells in normal pulmonary and aortic valves, respectively (P <.05). In early autografts only 12.0% +/- 4.6% of endothelial cells expressed the systemic arterial endothelial cell marker EphrinB2, whereas later explants had 85.6% +/- 5.4% of endothelial cells expressing EphrinB2 (P <.05). In early autografts 43.8% +/- 8.8% of interstitial cells expressed metalloproteinase 13, whereas late autografts had 11.4% +/- 2.7% of interstitial cells expressing matrix metalloproteinase 13 (P <.05). Collagen content in autografts was comparable with that of normal valves and was higher than that seen in homograft valves (P <.005). However, autograft walls were damaged, with granulation tissue (early) and scarring, with focal loss of normal smooth muscle cells, elastin, and collagen (late). CONCLUSIONS: The structure of pulmonary valves transplanted to the systemic circulation evolved toward that of normal aortic valves. Key processes in this remodeling included onset of a systemic endothelial cell phenotype and reversible plasticity of fibroblast-like valvular interstitial cells to myofibroblasts.