Direct Detection of Isolevuglandins in Tissues using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence.

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive.

Sodium activates human monocytes via the NADPH oxidase and isolevuglandin formation.

Aims: Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro.

Methods And Results: To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments.

A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity.

Enzyme-catalyzed chemical reactions produce heat. We developed an enclosed, capillary-perfused nanocalorimeter platform for thermometric enzyme-linked immunosorbent assay (TELISA). We used catalase as enzymes to model the thermal characteristics of the micromachined calorimeter.

Agonistic anti-CD148 monoclonal antibody attenuates diabetic nephropathy in mice.

CD148 is a transmembrane protein tyrosine phosphatase (PTP) that is expressed in the renal vasculature, including the glomerulus. Previous studies have shown that CD148 plays a role in the negative regulation of growth factor signals (including epidermal growth factor and vascular endothelial growth factor), suppressing cell proliferation and transformation. However, the role of CD148 in kidney disease remains unknown.

Arachidonic Acid Kills Staphylococcus aureus through a Lipid Peroxidation Mechanism.

infects every niche of the human host. In response to microbial infection, vertebrates have an arsenal of antimicrobial compounds that inhibit bacterial growth or kill bacterial cells. One class of antimicrobial compounds consists of polyunsaturated fatty acids, which are highly abundant in eukaryotes and encountered by at the host-pathogen interface.

Microfabricated calorimeters for thermometric enzyme linked immunosorbent assay in one-Nanoliter droplets.

Advances in microfabrication allow for highly sensitive calorimeters with dramatically reduced volume, decreased response time and increased energy resolution. These calorimeters hold the potential for designs of ELISA platforms competitive with fluorescent and chemiluminescent technologies. We have developed a new assay platform using conventional ELISA reagents to produce a thermal signal quantifiable using calorimetry.

Isolevuglandin scavenger attenuates pressure overload-induced cardiac oxidative stress, cardiac hypertrophy, heart failure and lung remodeling.

Increased levels of reactive isolevuglandins (IsoLGs) are associated with vascular inflammation and hypertension, two important factors affect heart failure (HF) development. The role of IsoLGs in HF development is unknown. Here we studied the role of IsoLG scavenger 2-hydroxybenzylamine (2-HOBA) in transverse aortic constriction (TAC) induced heart failure.

Dendritic Cell Amiloride-Sensitive Channels Mediate Sodium-Induced Inflammation and Hypertension.

Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47, and association of p47 with gp91.

Isolevuglandins as a gauge of lipid peroxidation in human tumors.

The cellular production of free radicals or reactive oxygen species (ROS) can lead to protein, lipid or DNA modifications and tumor formation. The cellular lipids undergo structural changes through the actions of enzymes (e.g.

Comparative Kinetic Analysis of Closed-Ended and Open-Ended Porous Sensors.

Efficient mass transport through porous networks is essential for achieving rapid response times in sensing applications utilizing porous materials. In this work, we show that open-ended porous membranes can overcome diffusion challenges experienced by closed-ended porous materials in a microfluidic environment. A theoretical model including both transport and reaction kinetics is employed to study the influence of flow velocity, bulk analyte concentration, analyte diffusivity, and adsorption rate on the performance of open-ended and closed-ended porous sensors integrated with flow cells.

Determination of the CD148-Interacting Region in Thrombospondin-1.

CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148.

Accumulation of isolevuglandin-modified protein in normal and fibrotic lung.

Protein lysine modification by γ-ketoaldehyde isomers derived from arachidonic acid, termed isolevuglandins (IsoLGs), is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by IsoLGs are subject to genetic regulation and the identity of IsoLG-modified proteins remain unclear. Herein we show that Nrf2 and Nox2 are key regulators of IsoLG modification in pulmonary tissue and report on the identity of proteins analyzed by LC-MS following immunoaffinity purification of IsoLG-modified proteins.

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.

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.

Phenotype-Driven Plasma Biobanking Strategies and Methods.

Biobank development and integration with clinical data from electronic medical record (EMR) databases have enabled recent strides in genomic research and personalized medicine. BioVU, Vanderbilt's DNA biorepository linked to de-identified clinical EMRs, has proven fruitful in its capacity to extensively appeal to numerous areas of biomedical and clinical research, supporting the discovery of genotype-phenotype interactions. Expanding on experiences in BioVU creation and development, we have recently embarked on a parallel effort to collect plasma in addition to DNA from blood specimens leftover after routine clinical testing at Vanderbilt.

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.

Porous silicon Bloch surface and sub-surface wave structure for simultaneous detection of small and large molecules.

A porous silicon (PSi) Bloch surface wave (BSW) and Bloch sub-surface wave (BSSW) composite biosensor is designed and used for the size-selective detection of both small and large molecules. The BSW/BSSW structure consists of a periodic stack of high and low refractive index PSi layers and a reduced optical thickness surface layer that gives rise to a BSW with an evanescent tail that extends above the surface to enable the detection of large surface-bound molecules. Small molecules were detected in the sensor by the BSSW, which is a large electric field intensity spatially localized to a desired region of the Bragg mirror and is generated by the implementation of a step or gradient refractive index profile within the Bragg mirror.

Biosensor design based on Marangoni flow in an evaporating drop.

Effective point-of-care diagnostics require a biomarker detection strategy that is low-cost and simple-to-use while achieving a clinically relevant limit of detection. Here we report a biosensor that uses secondary flows arising from surface Marangoni stresses in an evaporating drop to concentrate target-mediated particle aggregates in a visually detectable spot. The spot size increases with increasing target concentration within the dynamic range of the assay.

Early detection of NSCLC with scFv selected against IgM autoantibody.

Survival of patients with lung cancer could be significantly prolonged should the disease be diagnosed early. Growing evidence indicates that the immune response in the form of autoantibodies to developing cancer is present before clinical presentation. We used a phage-displayed antibody library to select for recombinant scFvs that specifically bind to lung cancer-associated IgM autoantibodies.

Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin.

Targeted multiplex imaging mass spectrometry with single chain fragment variable (scfv) recombinant antibodies.

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e.

Thrombospondin-1 acts as a ligand for CD148 tyrosine phosphatase.

CD148 is a receptor-type protein tyrosine phosphatase that is expressed in several cell types, including vascular endothelial cells and duct epithelial cells. Growing evidence demonstrates a prominent role for CD148 in negative regulation of growth factor signals, suppressing cell proliferation and transformation. However, its extracellular ligand(s) remain unknown.

Recombinant antibodies and their use in biosensors.

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically.