Proteomic Approach to Evaluate Mechanisms That Contribute to Food Allergenicity: Comparative 2D-DIGE Analysis of Radioallergosorbent Test Positive and Negative Patients

Proteomic profiles of RAST+ subjects with severe food allergies and RAST− subjects were compared using 2D-DIGE analysis to obtain candidate biomarkers specific to food allergies. Our analysis highlighted 52 proteins that were differentially expressed between the RAST+ and RAST− groups of which 37 were successfully identified that include chondroitin sulfates, zinc finger proteins, C-type lectins, retinoic acid binding proteins, heat shock proteins, myosin, cytokines, mast cell expressed proteins, and MAP kinases. Biological network analysis tool Metacore revealed that most of these regulated proteins play a role in immune tolerance, hypersensitivity and modulate cytokine patterns inducing a Th2 response that typically results in IgE-mediated allergic response which has a direct or indirect biological link to food allergy. Identifying unique biomarkers associated with certain allergic phenotypes and potentially cross-reactive proteins through bioinformatics analyses will provide enormous insight into the mechanisms that underlie allergic response in patients with food allergies

Genomic Analysis Highlights the Role of the JAK-STAT Signaling in the Anti-proliferative Effects of Dietary Flavonoid—‘Ashwagandha’ in Prostate Cancer Cells

Phytochemicals are dietary phytoestrogens that may play a role in prostate cancer prevention. Forty percent of Americans use complementary and alternative medicines (CAM) for disease prevention and therapy. Ashwagandha (Withania somnifera) contains flavonoids and active ingredients like alkaloids and steroidal lactones which are called ‘Withanolides’. We hypothesize that the immunomodulatory and anti-inflammatory properties of Ashwagandha might contribute to its overall effectiveness as an anti-carcinogenic agent. The goal of our study was gain insight into the general biological and molecular functions and immunomodulatory processes that are altered as a result of Ashwagandha treatment in prostate cancer cells, and to identify the key signaling mechanisms that are involved in the regulation of these physiological effects using genomic microarray analysis in conjunction with quantitative real-time PCR and western blot analysis. Ashwagandha treatment significantly downregulated the gene and protein expression of proinflammatory cytokines IL-6, IL-1β, chemokine IL-8, Hsp70 and STAT-2, while a reciprocal upregulation was observed in gene and protein expression of p38 MAPK, PI3K, caspase 6, Cyclin D and c-myc. Furthermore, Ashwagandha treatment significantly modulated the JAK-STAT pathway which regulates both the apoptosis process as well as the MAP kinase signaling. These studies outline several functionally important classes of genes, which are associated with immune response, signal transduction, cell signaling, transcriptional regulation, apoptosis and cell cycle regulation and provide insight into the molecular signaling mechanisms that are modulated by Ashwagandha, thereby highlighting the use of this bioflavanoid as effective chemopreventive agent relevant to prostate cancer progression

Prostate-Specific Antigen Modulates the Expression of Genes Involved in Prostate Tumor Growth

Prostate-specific antigen (PSA) is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA) was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent mono-layers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 µM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA), VEGF, and Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-γ, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-γ gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03) in tumor load when f-PSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors

Nanoparticle based galectin-1 gene silencing, implications in methamphetamine regulation of HIV-1 infection in monocyte derived macrophages

Galectin-1, an adhesion molecule, is expressed in macrophages and implicated in human immunodeficiency virus (HIV-1) viral adsorption. In this study, we investigated the effects of methamphetamine on galectin-1 production in human monocyte derived macrophages (MDM) and the role of galectin-1 in methamphetamine potentiation of HIV-1 infection. Herein we show that levels of galectin-1 gene and protein expression are significantly increased by methamphetamine. Furthermore, concomitant incubation of MDM with galectin-1 and methamphetamine facilitates HIV-1 infection compared to galectin-1 alone or methamphetamine alone. We utilized a nanotechnology approach that uses gold nanorod (GNR)-galectin-1 siRNA complexes (nanoplexes) to inhibit gene expression for galectin-1. Nanoplexes significantly silenced gene expression for galectin-1 and reversed the effects of methamphetamine on galectin-1 gene expression. Moreover, the effects of methamphetamine on HIV-1 infection were attenuated in the presence of the nanoplex in MDM

Anti-HIV-1 nanotherapeutics : promises and challenges for the future

The advent of highly active antiretroviral therapy (HAART) has significantly improved the prognosis for human immunodeficiency virus (HIV)-infected patients, however the adverse side effects associated with prolonged HAART therapy use continue. Although systemic viral load can be undetectable, the virus remains sequestered in anatomically privileged sites within the body. Nanotechnology-based delivery systems are being developed to target the virus within different tissue compartments and are being evaluated for their safety and efficacy. The current review outlines the various nanomaterials that are becoming increasingly used in biomedical applications by virtue of their robustness, safety, multimodality, and multifunctionality. Nanotechnology can revolutionize the field of HIV medicine by not only improving diagnosis, but also by improving delivery of antiretrovirals to targeted regions in the body and by significantly enhancing the efficacy of the currently available antiretroviral medications.Published Versio

New Insights into the Disease Progression Control Mechanisms by Comparing Long-Term-Nonprogressors versus Normal-Progressors among HIV-1-Positive Patients Using an Ion Current-Based MS1 Proteomic Profiling

For decades, epidemiological studies have found significant differences in the susceptibility to disease progression among HIV-carrying patients. One unique group of HIV-1-positive patients, the long-term-nonprogressors (LTNP), exhibits far superior ability in virus control compared with normal-progressors (NP), which proceed to Acquired Immune Deficiency Syndrome (AIDS) much more rapidly. Nonetheless, elucidation of the underlying mechanisms of virus control in LTNP is highly valuable in disease management and treatment but remains poorly understood. Peripheral blood mononuclear cells (PBMC) have been known to play important roles in innate immune responses and thereby would be of great interest for the investigation of the mechanisms of virus defense in LTNP. Here, we described the first comparative proteome analysis of PBMC from LTNP (<i>n</i> = 10) and NP (<i>n</i> = 10) patients using a reproducible ion-current-based MS1 approach, which includes efficient and reproducible sample preparation and chromatographic separation followed by an optimized pipeline for protein identification and quantification. This strategy enables analysis of many biological samples in one set with high quantitative precision and extremely low missing data. In total, 925 unique proteins were quantified under stringent criteria without missing value in any of the 20 subjects, and 87 proteins showed altered expressions between the two patient groups. These proteins are implicated in key processes such as cytoskeleton organization, defense response, apoptosis regulation, intracellular transport, etc., which provided novel insights into the control of disease progressions in LTNP versus NP, and the expression and phosphorylation states of key regulators were further validated by immunoassay. For instance, (1) SAMH1, a potent and “hot” molecule facilitating HIV-1 defense, was for the first time found elevated in LTNP compared with NP or healthy controls; elevated proteins from IFN-α response pathway may also contribute to viral control in LTNP; (2) decreased proapoptotic protein ASC along with the elevation of antiapoptotic proteins may contribute to the less apoptotic profile in PBMC of LTNP; and (3) elevated actin polymerization and less microtubule assembly that impede viral protein transport were first observed in LTNP. These results not only enhanced the understanding of the mechanisms for nonprogression of LTNP, but also may afford highly valuable clues to direct therapeutic efforts. Moreover, this work also demonstrated the ion-current-based MS1 approach as a reliable tool for large-scale clinical research