α-Synuclein is a chaperone-like protein implicated in Parkinson's disease (PD). Among α-synuclein's normal functions is an ability to bind to and stimulate the activity of the protein phosphatase 2A (PP2A) catalytic subunit in vitro and in vivo. PP2A activity is impaired in PD and in dementia with Lewy Bodies in brain regions harboring α-synuclein aggregates. Using PP2A as the readout, we measured PP2A activity in response to α-synuclein, ceramides, and FTY720, and then on the basis of those results, we created new FTY720 compounds. We then measured the effects of those compounds in dopaminergic cells. In addition to stimulating PP2A, all three compounds stimulated the expression of brain derived neurotrophic factor and protected MN9D cells against tumor-necrosis-factor-α-associated cell death. FTY720-C2 appears to be more potent while FTY720-Mitoxy targets mitochondria. Importantly, FTY720 is already FDA approved for treating multiple sclerosis and is used clinically worldwide. Our findings suggest that FTY720 and our new FTY720-based compounds have considerable potential for treating synucleinopathies such as PD.

The goal of this study is to develop an integrated, mathematical-experimental approach for understanding the interactions between the immune system and the effects of trastuzumab on breast cancer that overexpresses the human epidermal growth factor receptor 2 (HER2+). A system of coupled, ordinary differential equations was constructed to describe the temporal changes in tumour growth, along with intratumoural changes in the immune response, vascularity, necrosis and hypoxia. The mathematical model is calibrated with serially acquired experimental data of tumour volume, vascularity, necrosis and hypoxia obtained from either imaging or histology from a murine model of HER2+ breast cancer. Sensitivity analysis shows that model components are sensitive for 12 of 13 parameters, but accounting for uncertainty in the parameter values, model simulations still agree with the experimental data. Given theinitial conditions, the mathematical model predicts an increase in the immune infiltrates over time in the treated animals. Immunofluorescent staining results are presented that validate this prediction by showing an increased co-staining of CD11c and F4/80 (proteins expressed by dendritic cells and/or macrophages) in the total tissue for the treated tumours compared to the controls ($p < 0.03$). We posit that the proposed mathematical-experimental approach can be used to elucidate driving interactions between the trastuzumab-induced responses in the tumour and the immune system that drive the stabilization of vasculature while simultaneously decreasing tumour growth-conclusions revealed by the mathematical model that were not deducible from the experimental data alone.

Despite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells (DCs), from the thymic tumor microenvironment (TME) support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in two distinct mouse models of T-ALL, and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of anti-tumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.

α-Synuclein (aSyn), β-Synuclein (bSyn), and γ-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo.

Immune cells undergo dramatic metabolic reprogramming in response to external stimuli. These metabolic pathways, long considered as simple housekeeping functions, are increasingly understood to critically regulate the immune response, determining the activation, differentiation, and downstream effector functions of both lymphoid and myeloid cells. Within the complex metabolic networks associated with immune activation, several enzymes play key roles in regulating inflammation and represent potential therapeutic targets in human disease. In some cases, these enzymes control flux through pathways required to meet specific energetic or metabolic demands of the immune response. In other cases, key enzymes control the concentrations of immunoactive metabolites with direct roles in signaling. Finally, and perhaps most interestingly, several metabolic enzymes have evolved moonlighting functions, with roles in the immune response that are entirely independent of their conventional enzyme activities. Here, we review key metabolic enzymes that critically regulate inflammation, highlighting mechanistic insights and opportunities for clinical intervention.

Background:HER-2 targeted vaccine approaches appear promising for the treatment of breast cancer. HER-2 is an oncogenic growth factor receptor, is immunogenic, is expressed in stem cell subset of many breast cancers, and is overexpressed in 20–30% of human breast cancers. MVA-BN®-HER2 is a candidate immunotherapy product which utilizes a poxviral vector that encodes a modified form of the HER-2 protein. The MVA-BN® vector is a highly attenuated vaccinia virus, non-replicating in humans, which is an immunogenic vaccine vehicle. The vector induces minimal neutralizing antibodies and is capable of multiple immunizations. Preclinical data with mouse models demonstrates that immunization with MVA-BN®-HER2 can break tolerance in a transgenic model and has anti tumor activity against HER-2 expressing tumors.Methods: MVA-BN®-HER2 was evaluated in two fixed-dose, single arm studies. The first study, based in Poland and Serbia (7 sites), evaluated 21 patients (pts) either following first or second-line chemotherapy (10 pts), or in a separate cohort, in combination with single agent taxane chemotherapy (11 pts). The second study, based in the US (2 sites), evaluated 9 patients following first- or second-line chemotherapy. Patients were vaccinated with 1x108 TCID50 subcutaneously, 3 times, at 3 week intervals. Eligible patients had metastatic disease, with stable disease or better after chemotherapy. Patients with brain metastasis were excluded. The endpoints were safety and immunogenicity.Results: Twenty-eight patients received all three vaccinations. Vaccinations were well tolerated with typically only local injection site reactions as side effects. No dose-limiting toxicities were recorded, and no patient discontinued due to an adverse event. Two patients discontinued after 2 vaccinations due to disease progression. Two patients had asymptomatic declines in LV ejection fraction, possibly but unlikely related to vaccination. Both were heavily pretreated with anthracyclines and Herceptin, and one was on concurrent Herceptin. Both resolved on follow-up ECHO within 1 and 2 months. In the group where vaccine was combined with chemotherapy, one complete response and one partial response were observed. With 6 months of follow-up, after completion of treatment, 15 out of 28 patients have not progressed. Immune evaluation of samples from patients treated with MVA BN®-HER2 revealed that treatment was able to break tolerance against HER-2 in a metastatic setting, inducing either a humoral and/or T-cell response in greater than 66% of the patients. Specifically, HER-2 specific antibodies were detected in more than 50% of patients tested. And although modest (50-100 spots per 106 PBMC), T-cell responses were boosted post treatment (with at least one HER-2 reagent peptide pools or recombinant protein) in five out of eight patients upon stimulation.Conclusions: These preliminary data show that MVA-BN®-based, HER2-directed vaccination is a biologically active treatment for patients with HER-2- positive breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5089.

Bulk sampling of aerosols is often needed for the determination of physical properties, chemical composition and toxicity assessments of airborne particulate matter. Conventional aerosol samplers have several limitations for use as bulk aerosol collectors including cost, noise levels, power requirements associated with the use of a pump, limited flow rate, and a relatively long sampling time needed to collect sufficient mass to achieve gravimetric or other method limits of detection. In this study, a low-cost ionic charging device (ICD) was evaluated that addresses many of the drawbacks of conventional aerosol samplers. Different types of particles including incense fume, Arizona Road Dust (ARD) powders and Polystyrene Latex (PSL) spheres of different sizes were aerosolized then sampled using three ICDs and compared to conventional inhalable and PM 2.5 (particulate matter with aerodynamic diameter less than 100 µm and 2.5 µm, respectively) aerosol samplers in a controlled laboratory chamber at varying concentrations. The device was also evaluated in indoor environments. ICDs operate at almost 18.5 times higher flow rate than conventional personal samplers and provided up to 9 times greater total collected mass compared to the conventional samplers over the same time frame. Using a regression analysis, aerosol-specific linear equations with slopes (C PM2.5 /C ICD ) from 1.21 to 7.10 and R 2 from 0.74 to 0.99 for estimating the inhalable and PM 2.5 mass concentrations using the ICD were derived. This study suggests that the ICD provides a less accurate estimate of size-selective PM mass concentrations than conventional personal aerosol samplers; however, it collects coarse particles efficiently and increases total sampled mass per time at a lower cost and without noise associated with traditional sampling methods. Therefore, the ICD can be used as a bulk aerosol collector for composition analyses and in-vitro toxicology tests of coarse PM.

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