Rabbit Anti-Human PAK7 (Phospho-Ser602/Ser560) polyclonal antibody

Endothelial barrier function is carefully controlled to protect tissues from edema and damage inflicted by extravasated leukocytes. RhoGTPases, in conjunction with myriad regulatory proteins, exert both positive and negative effects on the endothelial barrier integrity. Precise knowledge about the relevant mechanisms is currently fragmented and we therefore performed a comprehensive analysis of endothelial barrier regulation by RhoGTPases and their regulators. Combining RNAi with electrical impedance measurements we quantified the relevance of 270 Rho-associated genes for endothelial barrier function. Statistical analysis identified 10 targets of which six promoted-and four reduced endothelial barrier function upon downregulation. We analyzed in more detail two of these which were not previously identified as regulators of endothelial integrity. We found that the Rac1-GEF (Guanine nucleotide Exchange Factor) TIAM2 is a positive regulator and the Cdc42(Rac1)- GAP (GTPase-Activating Protein) SYDE1 is a negative regulator of the endothelial barrier function. Finally, we found that the GAP SYDE1 is part of a Cdc42-centered signaling unit, also comprising the Cdc42-GEF FARP1 and the Cdc42 effector PAK7 which controls the integrity of the endothelial barrier. In conclusion, using a siRNA-based screen, we identified new regulators of barrier function and found that Cdc42 is a dominant positive regulator of endothelial integrity.

Human immunodeficiency virus type-1 (HIV-1)-specific dendritic cell (DC) vaccines have been used in clinical trials. However, they have been found to only induce some degree of immune responses in these studies. We previously demonstrated that the HIV-1 Gag-specific Gag-Texo vaccine stimulated Gag-specific effector CD8(+) cytotoxic T lymphocyte (CTL) responses, leading to completely protective, but very limited, therapeutic immunity. In this study, we constructed a recombinant adenoviral vector, adenovirus (AdV)(4-1BBL), which expressed mouse 4-1BB ligand (4-1BBL), and generated transgenic 4-1BBL-engineered OVA-Texo(/4-1BBL) and Gag-Texo(/4-1BBL) vaccines by transfecting ovalbumin (OVA)-Texo and Gag-Texo cells with AdV(4-1BBL), respectively. We demonstrate that the OVA-specific OVA-Texo/(4-1BBL) vaccine stimulates more efficient OVA-specific CTL responses (3.26%) compared to OVA-Texo-activated responses (1.98%) in wild-type C57BL/6 mice and the control OVA-Texo(/Null) vaccine without transgenic 4-1BBL expression, leading to enhanced therapeutic immunity against 6-day established OVA-expressing B16 melanoma BL6-10(OVA) cells. OVA-Texo(/4-1BBL)-stimulated CTLs, which have a CD44(+)CD62L(high)IL-7R(+) phenotype, are likely memory CTL precursors, demonstrating prolonged survival and enhanced differentiation into memory CTLs with functional recall responses and long-term immunity against BL6-10(OVA) melanoma. In addition, we demonstrate that OVA-Texo(/4-1BBL)-stimulated CTLs up-and downregulate the expression of anti-apoptosis (Bcl2l10, Naip1, Nol3, Pak7 and Tnfrsf11b) and pro-apoptosis (Casp12, Trp63 and Trp73) genes, respectively, by RT2 Profiler PCR array analysis. Importantly, the Gag-specific Gag-Texo(/4-1BBL) vaccine also stimulates more efficient Gag-specific therapeutic and long-term immunity against HLA-A2/Gag-expressing B16 melanoma BL6-10(Gag/A2) cells than the control Gag-Texo(/Null) vaccine in transgenic HLA-A2 mice. Taken together, our novel Gag-Texo(/4-1BBL) vaccine, which is capable of stimulating potent Gag-specific therapeutic and long-term immunity, may represent a new immunotherapeutic vaccine for controlling HIV-1 infection.