The phytoplasma bacterial plant parasite depends on leafhopper insects to spread and propagate itself. This study reveals how phytoplasma subverts plant development to turn flowers into leaves and thus make its host more attractive to leafhoppers.

The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function.

Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-γ, IL-4 and TNF-α level upon stimulation. We observed diminished secretion of proinflammatory TNF-α in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.

Background: The immunotherapeutic approach, adoptive cell transfer (ACT) have in malignant melanoma studies showed clinical durable responses in more than 50% of patients. However, the expansion of tumor infiltrating lymphocytes (TILs) requires extensive ex vivo culturing often at the cost of T cell differentiation and functional capacity. Most current strategies involve non-specific expansion of bulk TILs, often providing growth preference to co-infiltrated virus specific T cells and driving an exhausted phenotype of the T cell product. Methods: It is aimed to develop a new technology to expand tumor reactive T cells, through use of Major histocompatibility complex (MHC)-loaded artificial antigenpresenting scaffolds (Ag-scaffold) to provide the T cells with specific functional stimulation to obtain phenotypic and functional properties to mediate tumor regression. These scaffolds will be build using a dextran-based polysaccharide backbone associated with streptavidin molecules where biotinylated peptide-MHC class I molecules are attached to govern the specific interaction with a specific T cell, and a combination of biotinylated cytokines and co-stimulatory molecules are co-attached to provide stimulation to the T cell to achieve increased functional properties. The Ag-scaffolds interacts specifically with T cells based on recognition of the peptide-MHC molecule and effectively expand and functionally stimulate specific T cells, while leaving all other T cell specificities untouched. Results: from in vitro experiments have showed that antigen specific CD8 T cells stimulated with these Ag-scaffolds has high CD28 expression and low PD-1 expression, associated with high proliferation potential and enhanced antitumor effect in vivo. Furthermore, this expansion strategy provides a high frequency of multifunctional antigen specific CD8 T cells expressing IFN-, TNF-a, and CD107a upon target recognition. Conclusions: This expansion technology could with great advantage be used in ACT, to increase the anti-tumor effect of the transferred T cell product, as all of the achieved T cell characteristics are of significant importance for in vivo tumor cell recognition following ACT of expanded T cell products.Legal entity responsible for the study: Sine Reker Hadrup Funding: Lundbeck foundation Disclosure: All authors have declared no conflicts of interest.

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Depletion of arginine represents an important mechanism of immunosuppression, and high plasma and tumor arginase (ARG) activity has been demonstrated in patients with a wide spectrum of cancers and correlated with a poor prognosis. Low arginine levels inhibit proliferation and activation of cytotoxic T and NK cells. Preclinical and clinical studies show that simultaneous interference with multiple mechanisms of immunosuppression results in a strongly improved antitumor efficacy. In this context, we have developed OAT-1746, a novel, potent, and selective small-molecule inhibitor of ARG1 and ARG2 with profound antitumor efficacy as a monotherapy and in combinations with gemcitabine and PD-L1 inhibitor. The IC50 of OAT-1746 was determined against the recombinant human and murine ARG1 and ARG2. Cellular activity was evaluated in M2-polarized, bone marrow-derived murine macrophages, and CHO cells transfected with human ARG1 or ARG2. The in vivo antitumor efficacy of OAT-1746 was evaluated in syngeneic mouse models and in an orthotopic C6 rat glioma model after oral administration. In the rat model, C6 glioma cells were implanted by stereotactic intracranial inoculation and the tumor size was measured by bioluminescence in vivo imaging or immunohistochemistry. Quantitative real-time PCR was used to determine inflammatory markers. The tumor arginase activity was assessed using the urea detection assay. L-Arginine and drug levels in plasma and tumor were evaluated by LC/MS method. We have developed potent, selective, orally active inhibitors of ARG1 and ARG2. The clinical candidate, OAT-1746, has IC50=32 nM and 50 nM against human ARG1 and ARG2, respectively, and a potent cellular activity (M2 macrophages IC50=32 nM, CHO-K1 hARG1 IC50=55 nM). In vivo, OAT-1746 showed good pharmacologic properties and demonstrated significant antitumor efficacy as a monotherapy in 4 tumor models: B16 (melanoma), LLC (lung carcinoma), CT26 (CRC), and C6 glioma with TGI=34-53%. The antitumor efficacy correlated with sustained pharmacodynamic (PD) effects: suppression of tumor arginase activity and 3-6 fold increases in plasma and tumor arginine concentrations. Induction of inflammatory markers in tumors confirmed reversal of immunosuppression. Combinations of OAT-1746 with PD-L1 checkpoint inhibitor and gemcitabine showed increased efficacy. Administration of OAT-1746 with anti-PD-L1 antibody resulted in a “controlled” tumor growth with 55% of tumors remaining under 500 mm3 at day 24 versus 36% for PD-L1 monotherapy. Full regression was observed in 20% of the animals treated in combination with gemcitabine in comparison to 0% for monotherapy. OAT-1746 also demonstrated efficacy and PD effects in the orthotopic model of GBM, confirming its ability to cross the blood-brain barrier. Together these results support the clinical development of OAT-1746 for cancer therapy. Citation Format: Paulina Seweryna Stanczak, Marcin Mikolaj Grzybowski, Paulina Wolska, Anna Maria Zdziarska, Marcin Mazurkiewicz, Paulina Pilanc, Anna Gieryng, Bozena Kaminska, Anna Gzik, Marek Dziegielewski, Karol Jedrzejczak, Bartlomiej Borek, Roman Blaszczyk, Adam Golebiowski, Pawel Dobrzanski, Karolina Dzwonek. Development of OAT-1746, a novel arginase 1 and 2 inhibitor for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B003.