Identification of Novel Proteins in Neospora caninum Using an Organelle Purification and Monoclonal Antibody Approach

Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

<p>Abstract</p> <p>Background</p> <p>Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD). One hypothesis is that amyloid beta (Aβ) peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD.</p> <p>Methods</p> <p>Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis.</p> <p>Results</p> <p>Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals.</p> <p>Conclusions</p> <p>These data suggest that amyloid dependent microgliosis may be Src kinase dependent <it>in vitro</it> and <it>in vivo.</it> This study defines a role for Src kinase in the microgliosis characteristic of diseased brains and suggests that particular tyrosine kinase inhibition may be a valid anti-inflammatory approach to disease. Dasatinib is an FDA-approved drug for treating chronic myeloid leukemia cancer with a reported ability to cross the blood-brain barrier. Therefore, this suggests a novel use for this drug as well as similar acting molecules.</p

Tentative and transient natural killer cell polarization balances the requirements for discriminatory recognition and cytolytic efficacy

Natural killer (NK) cells are immune cells that lyse virally infected and tumor cells. Initially, their cytolytic capability is induced by cytokines. Subsequently, in their decision whether to kill a potential target cell, NK cells have to distinguish between small differences in the expression of ligands that report on the viral infection or transformation of the target. NK killing requires tight coupling to the target cell and extensive NK cell polarization. Here we discuss, often in contrast to the second cytolytic immune cell type, cytotoxic T cells, how NK cell polarization is shaped by three constraints of their activation. First, NK cell have to respond to cytokines: Different priming cytokines yield dramatically divergent NK cell polarization. Second, NK cells have to distinguish small differences in ligand expression: NK cell polarization is tentative, likely to allow discriminatory recognition close to the NK cell activation threshold. A critical contributor to the tentative nature of NK cell polarization may be poorly developed spatiotemporal organization of NK cell signaling. Third, NK cells have to kill effectively: NK cell polarization is transient, allowing for efficient killing by sequential interactions of a single NK cell with numerous target cells