Propanil (3,4-DCPA)-induced alterations of macrophage function

The pesticide 3,4-dichloropropionanilide (propanil or alternatively, DCPA) is a post-emergent herbicide predominantly used for the control of weeds on commercial rice crops worldwide. The US Environmental Protection Agency report on Pesticides Industry Sales and Usage in 2001 ranked propanil as the 17th most used herbicide in the US. It is applied on the fields via groundboom sprayers or aerial equipment suppressing photosynthesis and carbon fixation of a growing plant. The primary target of herbicide exposure is personnel working in the manufacturing and application who are required to wear personal protective equipment to minimize the exposure. The common routes of this exposure are inhalational and dermal, and to a lesser extent, oral. A number of toxic side effects are induced by propanil include hemolytic anemia, methemoglobinemia, and myelotoxicity. The majority of current knowledge about propanil\u27s toxic effects comes from animal studies. Liver and spleen were found to be the primary target organs, with many cell populations of the immune system affected, including T and B lymphocytes, NK cells and macrophages.;Macrophages play essential roles in both innate and adaptive immune responses. They recognize and destroy pathogens through phagocytosis and generation of reactive oxygen and nitrogen species (ROS and RNS). Macrophages can also function as antigen presenting cells in the initiation of an adaptive immune response and secrete a number of cytokines and inflammatory mediators thereby modulating the immune reaction at the inflamed site.;Previous studies have demonstrated that propanil was able to suppress interleukin (IL)-1beta, IL-6 and TNF-alpha cytokine production by LPS-activated murine macrophages. The analysis of nuclear factor-kappaB (NF-kappaB), responsible for TNF-alpha production, detected a decreased nuclear localization of NF-kappaB accompanied by a reduced, but not entirely abrogated binding to DNA activity.;The first study of this dissertation demonstrated the ability of propanil to inhibit TNF-alpha production by LPS-stimulated human monocytic cells line and suppress phagocytosis of fluorescent beads and Listeria monocytogenes . The ability to directly kill this bacterium and produce ROS and RNS was also assessed. The results demonstrate that DCPA has profound effects on macrophage function, and provide insight into the potential mechanisms of immunosuppression by DCPA. The second study focused on the mechanism of dramatic suppression of an inflammatory mediator secreted by activated macrophages, prostaglandin E2 (PGE2). Enzymatic activity and expression level of the proteins involved in PGE2 production were studied. However, the exact mechanism of propanil\u27s action is still unknown. The third study describes a novel behavior on NF-kappaB, called oscillations, in continuously LPS-stimulated macrophages. Binding activity of NF-kappaB was studied by two independent methods, and a mathematical model describing the oscillatory behavior was created based on experimental data. It demonstrated that propanil exposure potentiated the NF-kappaB activation process changing the oscillation behavior which could influence the transcription activity of the NF-kappaB-inducible genes.;Taken together our findings indicate that propanil exposure affects a number of important macrophage functions, such as phagocytosis, ROS and RNS production and inflammatory mediator\u27s secretion. Our studies also demonstrated a potential mechanism for the inhibition of a number of inflammatory cytokines through changes in NF-kappaB binding activity and oscillatory behavior

Application of replication-defective West Nile virus vector to non-flavivirus vaccine targets

The RepliVax vaccine platform(RV) is based on flavivirus genomes that are rationally attenuated by deletion. The self-limiting infection provided by RV has been demonstrated to be safe, highly immunogenic and efficacious for several vaccine candidates against flaviviruses. Here respiratory syncytial virus (RSV) F, influenza virus HA, and simian immunodeficiency virus (SIV) Env proteins were expressed in place of either prM-E or C-prM-E gene deletions of the West Nile (WN) virus genome. The resulting RV-RSV, -influenza and -SIV vaccine prototypes replicated efficiently in complementing helper cells expressing the WN structural proteins in trans. Expressed antigens exhibited correct post-translational processing and the RV recombinants were shown to be highly attenuated and immunogenic in mice, eliciting strong antigen-specific antibodies as well as detectable T-cell responses. These data support the utility of RV vectors for development of vaccines against non-flavivirus targets including rabies and HIV

Modulating Temporal Control of NF-κB Activation: Implications for Therapeutic and Assay Selection

The activation of transcription factor NF-κB (nuclear factor-κB) plays a central role in the induction of many inflammatory response genes. This process is characterized by either oscillations or stable induction of NF-κB nuclear binding. Changes in dynamics of binding result in the expression of distinct subsets of genes leading to different physiological outcomes. We examined NF-κB DNA binding activity in lipopolysaccharide (LPS)-stimulated IC-21 cells by electromobility shift assay and nonradioactive transcription factor assay and interpreted the results using a kinetic model of NF-κB activation. Both assays detected damped oscillatory behavior of NF-κB with differences in sensitivity and reproducibility. 3,4-Dichloropropionaniline (DCPA) was used to modulate the oscillatory behavior of NF-κB after LPS stimulation. DCPA is known to inhibit the production of two NF-κB-inducible cytokines, IL-6 and tumor necrosis factor α, by reducing but not completely abrogating NF-κB-induced transcription. DCPA treatment resulted in a potentiation of early LPS-induced NF-κB activation. The nonradioactive transcription factor assay, which has a higher signal/noise ratio than the electromobility shift assay, combined with in silico modeling, produced results that revealed changes in NF-κB dynamics which, to the best of our knowledge, have never been previously reported. These results highlight the importance of cell type and stimulus specificity in transcription factor activity assessment. In addition, assay selection has important implications for network inference and drug discovery