ENDOGLIN (ENG) is a co-receptor for transforming growth factor-β (TGF-β) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis.

HSP gp96-based vaccines have been trialled in rodent models and, more recently, in humans. Better understanding of gp96's immunomodulatory role will help with the design of more effective strategies for treatment of cancer and infectious diseases. In this study, we monitored the activities of T cells and activation of Treg in BABL/c mice after immunization using different doses of gp96 as adjuvant. We found that co-injection of gp96 simultaneously stimulated both CTL and Treg activity. Activation of CTL at low dose was far more pronounced than Treg activation. Treg population and suppression increased with gp96 dose, eventually abrogating the T-cell response induced by immunization. Lowdose cyclophosphamide treatment could restore the T-cell responses lost after high-dose gp96 adjuvant injection by suppression of Treg activation. We further examined the effect of different doses of gp96 or N355 peptide administration on tumor rejection. Our results provide new insights into the mechanisms of gp96-mediated balance between regulatory and responder T cells, which may facilitate future development of an effective gp96-based therapeutic vaccine.Key words: CTL . gp96 . Hepatitis B virus . Treg Introduction HSP gp96, a counterpart of the cytosolic HSP90 family residing in the endoplasmic reticulum, plays important roles in the activation of innate and adaptive immunity. This molecule has the unique ability to associate with antigenic peptides from tumor, virus and intracellular bacteria. Peptides bound by gp96 are taken up by APC through cell surface receptor CD91 and can be presented by both MHC class I and MHC class II molecules [1][2][3]. In mouse models, gp96-peptide complexes have been shown to prime and induce MHC class I-restricted CTL responses by crosspresentation of peptides to MHC class I molecules. Additionally, gp96 may act as co-stimulator and activator of CD4 1 and CD8 1T cells [4], increasing cell proliferation and secretion of cytokines. The gp96 protein also induces innate immune responses. Interaction of gp96 with APC (e.g. dendritic cells) leads to maturation or enhanced function of dendritic cells [5]. Recently gp96 was demonstrated to function in the expression of both intracellular and cell surface TLR [6]. In rodent models, autologous tumor-derived gp96-peptide complexes are highly effective in the elimination of tumor burden [7,8]. Immunization of mice with gp96 complexed with specific CTL epitopes derived from various viruses has been shown to elicit virus-specific CTL or protective immunity [1,9,10]. Since gp96 has been demonstrated to act as a powerful adjuvant, clinical trials of gp96-based therapeutic vaccines have been initiated for treatment of cancer [8,11].Previous study in our lab found that gp96 and its N-terminal fragment, N355, have the ability to augment CTL responses against to hepatitis B virus (HBV). However, higher amounts of à These authors contributed equally to this work. 3110gp96 were detrimental to the adjuvant effect and decreased the capability of mice to generate an i...

Preparation of agents that can successfully traverse the blood-brain-barrier (BBB) is a key challenge in brain cancer therapeutics. In this study, angiopep-2 was used as a brain-targeting peptide for preparing multifunctional Angiopep-2-modified poly nanoparticles, angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles. In vitro experiments showed a greater uptake of Angiopep-2 modified nanoparticles, also angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles by bEnd.3 cells versus nanoparticles and nanoparticles modified by IP10-EGFRvIIIscFv. Angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles accumulated in brain tissue after intravenous injection and recruited activated CD8+ T lymphocytes to location of glioblastoma cells. In vivo experiments to assess anti-glioblastoma effect of angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles showed significantly reduced tumor volume in angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles+ CD8+ cytotoxic T lymphocytes group versus in NPs modified by IP10-EGFRvIIIscFv+ CD8+ cytotoxic T lymphocytes, CD8+ cytotoxic T lymphocytes, Angiopep-2 modified nanoparticles+ CD8+ cytotoxic T lymphocytes, angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles and PBS groups. Leukocytes infiltrated in brain tissues showed strong anti-glioblastoma activity in angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles+ CD8+ cytotoxic T lymphocytes treated mice. Thus, angiopep-2 and IP10-EGFRvIIIscFv fusion protein modified nanoparticles may be useful for brain-targeted delivery and recruitment of activated CD8+ T lymphocytes to glioblastoma cells.