Raymond L. Mernaugh

DC isoketal-modified proteins activate T cells and promote hypertension

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1β, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-γ and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.

The Role of Individual SH2 Domains in Mediating Association of Phospholipase C-γ1 with the Activated EGF Receptor

The two SH2 (Src homology domain 2) domains present in phospholipase C-γ1 (PLC-γ1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 ≫ pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 ≫ pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-γ1 molecule with the activated epidermal growth factor (EGF) receptor were assessedin vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-γ1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-γ1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-γ1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Label-free porous silicon membrane waveguide for DNA sensing

We report a label-free porous silicon membrane waveguide biosensor based on a 1μm thick freestanding porous silicon film with 100nm diameter pores. The sensor operates in the Kretschmann configuration. A formvar polymer film provides robust adhesion of the porous silicon membrane to a rutile prism and enables confinement of guided modes in the porous silicon membrane. Attenuated total reflectance measurements are performed, along with theoretical calculations, to fully characterize the waveguide. The sensitivity of the sensor is investigated through DNA hybridization in the porous silicon membrane. A detection limit of 42nM was demonstrated for 24-base pair DNA oligonucleotides.

Single chain fragment variable recombinant antibody functionalized gold nanoparticles for a highly sensitive colorimetric immunoassay

In this report, the peptide linker connecting scFv V(H) and V(L) domains were genetically modified to contain different amino acids (i.e. cysteine (scFv-cys) or histidines (scFv-his)) to enable the scFv to adsorb or self-assemble onto the gold nanoparticles (NPs). The scFv-cys stabilized gold NPs were used to develop a highly sensitive colorimetric immunosensor. The scFv-cys stabilized gold NPs were characterized by UV-vis spectra, transmission electron microscope (TEM) and FTIR. After adding the antigen rabbit IgG, the solution of scFv-cys stabilized gold NPs shows obvious visible color change from deep red to light purple due to the aggregation of the gold nanoparticles. Based on the colorimetric aggregation of scFv-cys stabilized gold NPs, the immunosensor exhibits high sensitivity with a detection limit of 1.7 nM and good specificity. The good properties of the colorimetric aggregation immunosensor can be attributed to the small size of scFv and the covalent link between the scFv and gold NPs that improve the better orientation and enhance the probe density. With the advantages of speed, simplicity and specificity, the colorimetric immunoassay based on the functionalized scFv stabilized gold NPs represents a promising approach for protein analysis and clinical diagnostics.

Renal Denervation Prevents Immune Cell Activation and Renal Inflammation in Angiotensin II–Induced Hypertension

RATIONALE Inflammation and adaptive immunity play a crucial role in the development of hypertension. Angiotensin II and probably other hypertensive stimuli activate the central nervous system and promote T-cell activation and end-organ damage in peripheral tissues. OBJECTIVE To determine if renal sympathetic nerves mediate renal inflammation and T-cell activation in hypertension. METHODS AND RESULTS Bilateral renal denervation using phenol application to the renal arteries reduced renal norepinephrine levels and blunted angiotensin II-induced hypertension. Bilateral renal denervation also reduced inflammation, as reflected by decreased accumulation of total leukocytes, T cells, and both CD4+ and CD8+ T cells in the kidney. This was associated with a marked reduction in renal fibrosis, albuminuria, and nephrinuria. Unilateral renal denervation, which partly attenuated blood pressure, only reduced inflammation in the denervated kidney, suggesting that this effect is pressure independent. Angiotensin II also increased immunogenic isoketal-protein adducts in renal dendritic cells (DCs) and increased surface expression of costimulation markers and production of interleukin (IL)-1α, IL-1β, and IL-6 from splenic DCs. Norepinephrine also dose dependently stimulated isoketal formation in cultured DCs. Adoptive transfer of splenic DCs from angiotensin II-treated mice primed T-cell activation and hypertension in recipient mice. Renal denervation prevented these effects of hypertension on DCs. In contrast to these beneficial effects of ablating all renal nerves, renal afferent disruption with capsaicin had no effect on blood pressure or renal inflammation. CONCLUSIONS Renal sympathetic nerves contribute to DC activation, subsequent T-cell infiltration and end-organ damage in the kidney in the development of hypertension.

VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis

Chemotaxis regulates the recruitment of leukocytes, which is integral for a number of biological processes and is mediated through the interaction of chemokines with seven transmembrane G-protein-coupled receptors. Several studies have indicated that chemotactic signaling pathways might be activated via G-protein-independent mechanisms, perhaps through novel receptor-interacting proteins. CXCR2 is a major chemokine receptor expressed on neutrophils. We used a proteomics approach to identify unique ligand-dependent CXCR2-interacting proteins in differentiated neutrophil-like HL-60 cells. Using this approach, vasodilator-stimulated phosphoprotein (VASP) was identified as a CXCR2-interacting protein. The interaction between CXCR2 and VASP is direct and enhanced by CXCL8 stimulation, which triggers VASP phosphorylation via PKA- and PKCδ-mediated pathways. The interaction between CXCR2 and VASP requires free F-actin barbed ends to recruit VASP to the leading edge. Finally, knockdown of VASP in HL-60 cells results in severely impaired CXCR2-mediated chemotaxis and polarization. These data provide the first demonstration that direct interaction of VASP with CXCR2 is essential for proper CXCR2 function and demonstrate a crucial role for VASP in mediating chemotaxis in leukocytes.

Immune activation caused by vascular oxidation promotes fibrosis and hypertension

Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg(sm/p22phox) mice, which overexpress the NADPH oxidase subunit p22(phox) in smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg(sm/p22phox) mice produced high levels of IL-17A and IFN-γ. Crossing tg(sm/p22phox) mice with lymphocyte-deficient Rag1(-/-) mice eliminated vascular inflammation, aortic stiffening, renal dysfunction, and hypertension; however, adoptive transfer of T cells restored these processes. Isoketal-protein adducts, which are immunogenic, were increased in aortas, DCs, and macrophages of tg(sm/p22phox) mice. Autologous pulsing with tg(sm/p22phox) aortic homogenates promoted DCs of tg(sm/p22phox) mice to stimulate T cell proliferation and production of IFN-γ, IL-17A, and TNF-α. Treatment with the superoxide scavenger tempol or the isoketal scavenger 2-hydroxybenzylamine (2-HOBA) normalized blood pressure; prevented vascular inflammation, aortic stiffening, and hypertension; and prevented DC and T cell activation. Moreover, in human aortas, the aortic content of isoketal adducts correlated with fibrosis and inflammation severity. Together, these results define a pathway linking vascular oxidant stress to immune activation and aortic stiffening and provide insight into the systemic inflammation encountered in common vascular diseases.

Engineering Peptide Linkers for scFv Immunosensors

Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors.

Proapoptotic activity of cell-permeable anti-Akt single-chain antibodies.

We developed anti-Akt1 single-chain antibodies (scFv) by panning a mouse phage-displayed scFv recombinant antibody library. Recombinant scFv that bound glutathione S-transferase (GST)-Akt1 were screened for their ability to inhibit Akt activity in vitro in a kinase reaction containing human recombinant Akt1 and an Akt/serum glucocorticoid-inducible kinase (SGK) substrate. Michaelis-Menten analysis of kinase inhibition by a selected scFv was consistent with scFv-mediated competition with enzymes substrate for the catalytic site of Akt. To generate a membrane-permeable version of the anti-Akt1 scFv, the scFv gene was subcloned into a GST expression vector carrying a membrane-translocating sequence (MTS) from Kaposi fibroblast growth factor. A purified GST-anti-Akt1-MTS fusion protein accumulated intracellularly in 293T, BT-474, and PyVmT cells in a dose- and time-dependent fashion. Intracellular accumulation correlated temporally with inhibition of p-Ser(473) Akt and GSK-3alpha/beta phosphorylation, suggesting that Ser(473) is an Akt autophosphorylation site. Phosphorylated (activated) phosphoinositide-dependent kinase 1, mitogen-activated protein kinase, p38, and HER2 (erbB2) were not affected, supporting Akt kinase specificity for the inhibitory scFv. Exogenously expressed constitutively active Akt2 and Akt3 were also inhibited in vitro by the anti-Akt1 fusion protein. Furthermore, GST-anti-Akt1-MTS induced apoptosis in three cancer cell lines that express constitutively active Akt. Finally, systemic treatment with the anti-Akt scFv reduced tumor volume and neovascularization and increased apoptosis in PyVmT-expressing transgenic tumors implanted in mouse dorsal window chambers. Thus, GST-anti-Akt1-MTS is a novel cell-permeable inhibitor of Akt, which selectively inhibits Akt-mediated survival in intact cells both in vitro and in vivo.

Si-based sensor for virus detection

This paper describes the development of a sensor for the detection of viruses. The detection scheme uses sense and dummy capacitors and a sense amplifier circuit to compare capacitance values. The presence of sufficient biological material within the sense capacitor alters the sense amplifier output. This promising approach has the potential to more rapidly test for the presence of virus using an IC platform.