Latent KSHV Infection of Endothelial Cells Induces Integrin Beta3 to Activate Angiogenic Phenotypes

Kaposi's Sarcoma (KS), the most common tumor of AIDS patients, is a highly vascularized tumor supporting large amounts of angiogenesis. The main cell type of KS tumors is the spindle cell, a cell of endothelial origin, the primary cell type involved in angiogenesis. Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of KS and is likely involved in both tumor formation and the induction of angiogenesis. Integrins, and specifically integrin αVβ3, have known roles in both tumor induction and angiogenesis. αVβ3 is also important for KSHV infection as it has been shown to be involved in KSHV entry into cells. We found that during latent infection of endothelial cells KSHV induces the expression of integrin β3 leading to increased surface levels of αVβ3. Signaling molecules downstream of integrins, including FAK and Src, are activated during viral latency. Integrin activation by KSHV is necessary for the KSHV-associated upregulation of a number of angiogenic phenotypes during latent infection including adhesion and motility. Additionally, KSHV-infected cells become more reliant on αVβ3 for capillary like formation in three dimensional culture. KSHV induction of integrin β3, leading to induction of angiogenic and cancer cell phenotypes during latency, is likely to be important for KS tumor formation and potentially provides a novel target for treating KS tumors

Kaposi's Sarcoma Associated Herpes Virus (KSHV) Induced COX-2: A Key Factor in Latency, Inflammation, Angiogenesis, Cell Survival and Invasion

Kaposi's sarcoma (KS), an enigmatic endothelial cell vascular neoplasm, is characterized by the proliferation of spindle shaped endothelial cells, inflammatory cytokines (ICs), growth factors (GFs) and angiogenic factors. KSHV is etiologically linked to KS and expresses its latent genes in KS lesion endothelial cells. Primary infection of human micro vascular endothelial cells (HMVEC-d) results in the establishment of latent infection and reprogramming of host genes, and cyclooxygenase-2 (COX-2) is one of the highly up-regulated genes. Our previous study suggested a role for COX-2 in the establishment and maintenance of KSHV latency. Here, we examined the role of COX-2 in the induction of ICs, GFs, angiogenesis and invasive events occurring during KSHV de novo infection of endothelial cells. A significant amount of COX-2 was detected in KS tissue sections. Telomerase-immortalized human umbilical vein endothelial cells supporting KSHV stable latency (TIVE-LTC) expressed elevated levels of functional COX-2 and microsomal PGE2 synthase (m-PGES), and secreted the predominant eicosanoid inflammatory metabolite PGE2. Infected HMVEC-d and TIVE-LTC cells secreted a variety of ICs, GFs, angiogenic factors and matrix metalloproteinases (MMPs), which were significantly abrogated by COX-2 inhibition either by chemical inhibitors or by siRNA. The ability of these factors to induce tube formation of uninfected endothelial cells was also inhibited. PGE2, secreted early during KSHV infection, profoundly increased the adhesion of uninfected endothelial cells to fibronectin by activating the small G protein Rac1. COX-2 inhibition considerably reduced KSHV latent ORF73 gene expression and survival of TIVE-LTC cells. Collectively, these studies underscore the pivotal role of KSHV induced COX-2/PGE2 in creating KS lesion like microenvironment during de novo infection. Since COX-2 plays multiple roles in KSHV latent gene expression, which themselves are powerful mediators of cytokine induction, anti-apoptosis, cell survival and viral genome maintainence, effective inhibition of COX-2 via well-characterized clinically approved COX-2 inhibitors could potentially be used in treatment to control latent KSHV infection and ameliorate KS

Electrochemiluminescent arrays utilizing layer-by-layer films of cytochrome P450/DNA/ruthenuim polymer for drug toxicity screening & unraveling direct electrochemistry of cytochrome P450s

This thesis describes development and fundamental studies related to simple, inexpensive, high throughput electrochemiluminescent arrays made of layer-by-layer (LbL) films of cytochrome (cyt) P450, DNA and redox ruthenium polymer ([Ru(bpy)2PVP10]2+, RuPVP) for drug/chemical toxicity screening. A major focus includes unraveling direct electron transfer and biocatalytic kinetics of drug metabolizing cytochrome P450s films, and mimicking natural cytochrome P450 catalytic cycle on electrodes with high catalytic efficiency. ^ Chapter one describes the background and significance of direct electrochemistry of metabolic cyt P450 enzymes with some of the key issues addressed, as well as the need for developing simple electrochemiluminescent arrays for drug/chemical toxicity screening that can complement the existing bioassays in drug development. ^ Chapter two discusses the first report on the use of voltammetric sensors/ECL arrays applied to predict genotoxicity of model N-nitroso compound, N-nitrosopyrrolidine (NPYR), bioactivated by human cyt P450 2E1 in LbL films with DNA and RuPVP. ^ Chapter three reports the application of ECL arrays containing films of DNA, RuPVP and selected human cyt P450 enzymes to elucidate cyt P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). ^ Chapter four describes the successful application of cheap and commercially available liver microsomes as cyt P450 source on the electrochemical sensors to detect comparative toxicity between nitrosamine and styrene compounds, and their evaluation to in vivo toxicity data and reactive metabolite formation. ^ Chapter five reports the first implementation of liver microsomes as LbL films with DNA and RuPVP on ECL arrays to test toxicity of several model nitroso compounds and styrene. Correlation of the toxicity array responses to in vivo toxicity data and to reactive metabolite-DNA adducts formation in the sensor films was observed. ^ Chapter six is on the direct electrochemistry study of cyt P450s films on electrodes that discusses for the first time about the influences of iron heme spin state and amino acids nature in the heme distal region on the direct electron transfer and active ferryloxy formation kinetics of cyt P450s in polyion films. ^ Chapter seven discusses the first ever demonstrated natural cyt P450 catalytic cycle mechanism mimicked electrochemically using LbL films of purified human cytochrome P450 and microsomal cyt P450-reducase on electrodes having high catalytic efficiency.

Magnetic Particle Bioconjugates: A Versatile Sensor Approach

Nanomaterial biosensors have revolutionized the entire scientific, technology, biomedical, materials science, and engineering fields. Among all nanomaterials, magnetic nanoparticles, microparticles, and beads are unique in offering facile conjugation of biorecognition probes for selective capturing of any desired analytes from complex real sample matrices (e.g., biofluids such as whole blood, serum, urine and saliva, tissues, food, and environmental samples). In addition, rapid separation of the particle-captured analytes by the simple use of a magnet for subsequent detection on a sensor unit makes the magnetic particle sensor approach very attractive. The easy magnetic isolation feature of target analytes is not possible with other inorganic particles, both metallic (e.g., gold) and non-metallic (e.g., silica), which require difficult centrifugation and separation steps. Magnetic particle biosensors have thus enabled ultra-low detection with ultra-high sensitivity that has traditionally been achieved only by radioactive assays and other tedious optical sources. Moreover, when traditional approaches failed to selectively detect low-concentration analytes in complex matrices (e.g., colorimetric, electrochemistry, and optical methods), magnetic particle-incorporated sensing strategies enabled sample concentration into a defined microvolume of large surface area particles for a straightforward detection. The objective of this article is to highlight the ever-growing applications of magnetic materials for the detection of analytes present in various real sample matrices. The central idea of this paper was to show the versatility and advantages of using magnetic particles for a variety of sample matrices and analyte types and the adaptability of different transducers with the magnetic particle approaches

Voltammetric Immunosensor Assembled on Carbon-Pyrenyl Nanostructures for Clinical Diagnosis of Type of Diabetes

Herein we report the first serum insulin voltammetric immunosensor for diagnosis of type 1 and type 2 diabetic disorders. The sensor is composed of multiwalled carbon nanotube-pyrenebutyric acid frameworks on edge plane pyrolytic graphite electrodes (PGE/MWNT/Py) to which an anti-insulin antibody was covalently attached. The detection of picomolar levels of serum insulin binding to the surface antibody was achieved by monitoring the decrease in voltammetric current signals of a redox probe taken in the electrolyte solution. This method offered a detection limit of 15 pM for free insulin present in serum. This detection limit was further lowered to 5 pM by designing serum insulin conjugates with poly­(acrylic acid)-functionalized magnetite nanoparticles (100 nm hydrodynamic diameter) and detecting the binding of MNP-serum insulin conjugate to the surface insulin-antibody on PGE/MWNT/Py electrodes. When tested on real patient serum samples, the sensor accurately measured insulin levels. To our knowledge, this is the first report of a voltammetric immunosensor capable of both diagnosing and distinguishing the type of diabetes based on serum insulin levels in diabetic patients

Microsome Biocolloids for Rapid Drug Metabolism and Inhibition Assessment by LC-MS

Rat liver microsomes attached to nanoparticles were used for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC50 values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were estimated