Comparison of RNA extracts from in vitro shoot tip cultures of leafroll-affected and leafroll-free grapevine cultivars

The RNA content of in vitro shoot tip cultures from grapevine leafroll (GLR) disease-affected grapevines was analyzed and compared to that of similar cultures from GLR-free grapevines. A previously unreported low-molecular-weight single-stranded RNA (LMWssRNA) was detected in in vitro shoot tip cultures of 65 % (11 out of 17) of GLR-affected cultivars. This LMWssRNA was absent from disease-free cultivars and may be associated with a virus or a strain of a virus responsible for GLR. Numerous high-molecular-weight (HMW) dsRNA bands were also detected in GLR-affected grapevine cultivars. The intensities and mobilities (apparent molecular weights) of these dsRNA bands varied considerably from one GLR-affected cultivar to the next, but were reproducible for each cultivar. The detection of multiple distinctive RNA banding patterns is consistent with the possibility that more than one agent can cause grapevine leafroll disease

Double-stranded RNA from rupestris stem pitting-affected grapevines

Nucleic acids were extracted from in vitro shoot tip cultures of 31 grapevine cultivars affected with rupestris stem pitting (RSP) disease and from cultures of 11 RSPfree cultivars, 4 of which were disease-free, 2 of which were fleck-affected and 5 of which were grapevine leafroll disease-affected. Analysis of the extracts by polyacrylamide slab gel electrophoresis showed that 21 of the 31 RSP-affected cultivars contained a previously unreported nucleic acid which was absent from the RSP-free controls. Nuclease digestions showed that the nucleic acid was double-stranded RNA (dsRNA). The apparent size of the dsRNA was inconsistent with that expected for either a viroid or a closterovirus. The observation that 10 of the 31 RSP-affected cultivars lacked this dsRNA is consistent with the view that there may be more than one 'RSP-like' disease. The dsRNA detected in this investigation may be associated with one of these diseases

Mechanical sap transmission of a closterovirus from in vitro shoot tip cultures of a leafroll-affected grapevine to Nicotiana benthamiana

Transmission manuelle d'un clostérovirus au Nicotiana benthamiana à partir de cultures in vitro d'une vigne atteinte de l'enroulementDes cultures in vitro d'une vigne de Vitis vinifera Limberger atteinte de l'enroulement ont été broyées dans une solution tampon contenant de la nicotine. Les extraits ont été inoculés sur des feuilles de Nicotiana benthamiana et de 6 autres espèces de plantes herbacées. 3 semaines plus tard, seuls les plants de N. benthamiana ont démontré des symptômes. Ceux-ci consistaient d'un nanissement systémique accompagné d'un éclaircissement des nervures qui se transformait en chlorose interveinale. Les feuilles de ces N. benthamiana contenaient le virus A de la vigne (GVA), démontré par la méthode ISEM

Retinol improves bovine embryonic development in vitro

Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7%) and under atmospheric oxygen conditions (20%). In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF) in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM) tended (p < 0.07) to increase blastocyst formation (blastocyst/putative zygote; 26.1% +/- 2.2%) compared to the controls (21.9% +/- 1.9%). Further analysis revealed when blastocyst development rates fell below 20% in the control groups, 5 micromolar retinol treatment dramatically improved embryonic development, measured by blastocyst/putative zygote rate (14.4 +/- 2.1 vs 23.7 +/- 2.5; p < 0.02). The 5 micomolar retinol treatment also enhanced the blastocyst/cleaved rate by nearly 10% (23.7% vs 34.6%; p < 0.02). In the second and third experiments addition of 5 micromolar retinol to the embryo culture medium (IVC) under low oxygen conditions did not significantly improve cleavage or blastocyst rates, but 5 micromolar retinol significantly increased blastocyst development under 20% O2 conditions (p < 0.001). These studies demonstrate that supplementation of 5 micromolar retinol to the maturation medium may improve embryonic development of bovine oocytes indicated by their increased blastocyst rate. A significant improvement in the blastocyst development with the 5 micromolar retinol treatment under atmospheric conditions suggests a beneficial antioxidant effect during embryo culture

Phospholipase A2 regulation of bovine endometrial (BEND) cell prostaglandin production

Background Prostaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production. Methods Cells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates. Results BEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity. Conclusion Results from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production

Distinct phospholipase A2 enzymes regulate prostaglandin E2 and F2alpha production by bovine endometrial epithelial cells

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Tracking the kinetics of intrahepatic immune responses by repeated fine needle aspiration of the liver

Liver disease is an increasing global health burden. The final sequalae of cirrhosis, liver failure and hepatocellular carcinoma are often the result of inflammation driven by intrahepatic lymphocytes. Accurate assessment of organ-specific diseases ideally employs tissue sampling though this is rarely performed. Here we report our experiences of utilising repeated fine needle aspirations (FNAs) to assess liver-derived leukocytes. In 88 patient samples, we obtained a mean of 36,959 lymphocytes from each FNA-derived biopsy (SD 22,319 cells, range 5034–91,242 cells) measured by flow cytometry. This quick technique required minimal analgesia compared to liver biopsy (p = 0.03); was well tolerated and safe, and hence repeated sampling up to 3 times within a week was feasible. We detail the technique to rapidly derive a single cell suspension suitable for multiparameter flow cytometry analysis. Finally we illustrate the importance of organ-derived sampling by showing that natural killer (NK) cells from FNA samples have a markedly altered phenotype compared to those assessed in peripheral blood. In combination these data validate FNA as a powerful and well-tolerated method of sampling intrahepatic lymphocytes to study the immunology of acute and chronic liver diseases

Re-directing CD4+ T cell responses with the flanking residues of MHC class II-bound peptides: The core is not enough

Recombinant αβ T cell receptors, expressed on T cell membranes, recognize short peptides presented at the cell surface in complex with MHC molecules. There are two main subsets of αβ T cells: CD8(+) T cells that recognize mainly cytosol-derived peptides in the context of MHC class I (pMHC-I), and CD4(+) T cells that recognize peptides usually derived from exogenous proteins presented by MHC class II (pMHC-II). Unlike the more uniform peptide lengths (usually 8–13mers) bound in the MHC-I closed groove, MHC-II presented peptides are of a highly variable length. The bound peptides consist of a core bound 9mer (reflecting the binding motif for the particular MHC-II type) but with variable peptide flanking residues (PFRs) that can extend from both the N- and C-terminus of the MHC-II binding groove. Although pMHC-I and pMHC-II play a virtually identical role during T cell responses (T cell antigen presentation) and are very similar in overall conformation, there exist a number of subtle but important differences that may govern the functional dichotomy observed between CD8(+) and CD4(+) T cells. Here, we provide an overview of the impact of structural differences between pMHC-I and pMHC-II and the molecular interactions with the T cell receptor including the functional importance of MHC-II PFRs. We consider how factors such as anatomical location, inflammatory milieu, and particular types of antigen presenting cell might, in theory, contribute to the quantitative (i.e., pMHC ligand frequency) as well as qualitative (i.e., variable PFR) nature of peptide epitopes, and hence offer a means of control and influence of a CD4(+) T cell response. Lastly, we review our recent findings showing how modifications to MHC-II PFRs can modify CD4(+) T cell antigen recognition. These findings may have novel applications for the development of CD4(+) T cell peptide vaccines and diagnostics

Home sweet home: The tumor microenvironment as a haven for regulatory T cells

CD4+Foxp3+ regulatory T cells (Tregs) have a fundamental role in maintaining immune balance by preventing autoreactivity and immune-mediated pathology. However this role of Tregs extends to suppression of anti-tumor immune responses and remains a major obstacle in the development of anti-cancer vaccines and immunotherapies. This feature of Treg activity is exacerbated by the discovery that Treg frequencies are not only elevated in the blood of cancer patients, but are also significantly enriched within tumors in comparison to other sites. These observations have sparked off the quest to understand the processes through which Tregs become elevated in cancer-bearing hosts and to identify the specific mechanisms leading to their accumulation within the tumor microenvironment. This manuscript reviews the evidence for specific mechanisms of intra-tumoral Treg enrichment and will discuss how this information may be utilized for the purpose of manipulating the balance of tumor-infiltrating T cells in favor of anti-tumor effector cells

Eat Walk Sleep Discuss: Building a Multi-Dimensional Participatory Relationship

A multi-faceted relationship has developed between UMass Worcester and the Worcester Refugee Assistance Project (WRAP). The relationship has its roots in student engagement, and has grown to include faculty, students and community members in a range of community-based participatory activities, which can be shaped in response to needs as they are identified and defined. This poster describes the different ways student engagement and community partnerships worked together in a research project