Cultural landscape and plant use at the Phoenician site of Motya (Western Sicily,Italy) inferred from a disposal pit

Vegetation History and Archaeobotany - The present study concerns the Phoenician-Punic site of Motya, a small island set in Western Sicily (Italy), in the Marsala Lagoon (Stagnone di Marsala),...     

Cytotoxic effectors in human peripheral blood mononuclear cells induced by a mannoprotein complex of Candida albicans: a comparison with interleukin 2-activated killer cells

In this study, the major histocompatibility complex-unrestricted cytotoxic effectors elicited in human peripheral blood mononuclear cells (PBMC) by a mannoprotein (MP) component from the cell wall of the human indigenous microorganism Candida albicans have been compared with those obtained by stimulation with interleukin 2. (Interleukin 2-activated killer cells: LAK). It has been found that MP-induced lytic effectors were substantially similar to LAK in potency, target specificity, and type of precursor/effector cells. In both cases, natural killer (NK)-susceptible and NK-resistant targets as well as fresh tumor (glioma) cells were efficiently killed by a population of effectors showing a predominant CD3-, CD16+ phenotype. However, the precursors of MP-induced killers were highly sensitive to the lysosomotropic toxic drug L-leucine methyl ester (Leu-OME) whereas the generation of LAK cells was unaffected by this drug. The Leu-OME sensitivity of MP-induced cytotoxicity generation was not due to a nonspecific effect on antigen-presenting cells or inhibition of cell proliferation. In addition, the generation of MP-induced killer cells was totally abrogated by treatment with CD16 antibodies and complement, whereas a minor but significant fraction of LAK precursors was not susceptible to the above treatment. These results indicate that a defined component(s) of the cell wall of C. albicans has some properties of biological response modifiers in cultures of human PBMC in vitro.     

Generation and characterization of cytotoxic activity against tumor cell lines in human peripheral blood mononuclear cells stimulated "in vitro" by a glucomannan-protein preparation of Candida albicans

Human peripheral blood mononuclear cells (PBMC) proliferated and generated non-specific cell-mediated cytotoxicity (CMC) after stimulation with a cell-wall glucomannan-protein (GMP) fraction of Candida albicans or chemically-inactivated intact microrganism. No effects were observed using other fungal cell wall components such as glucan or alkali-acid treated glucomannan. The highest CMC level was detected after 7-10 days of PBMC incubation in the presence of 50 micrograms/ml of whole Candida cells and the cytotoxic immunoeffectors elicited by these antigenic stimulations equally affected NK-susceptible (K562) and NK-resistant (Raji, Daudi and Jurkat) tumor cell lines. Both Interleukin-2 (IL-2) and gamma interferon (IFN-gamma) were produced by GMP-stimulated PBMC, the IL-2 peak production constantly preceding that of IFN production. GMP-induced generation of natural CMC was potentiated by the addition of IFN-gamma and a monospecific anti IFN-gamma serum totally abrogated both IFN activity and CMC generation. The cytolytic effectors were shown to be OKT3-, OKT8- and HLA-DR-. They did not possess typical NK markers (e.g. Leu-7 and AB8.28) but were partially recognized by A10, a IgG2a monoclonal antibody reacting to PBMC-NK lymphocytes and activated T cells. These results suggest that the antitumor cytolytic effectors generated in PBMC cultures exposed to Candida material belong either to a discrete subset of natural effectors lacking classical NK markers or to other lymphokine-activated cells. This study also suggests that the human indigenous microrganisms C.albicans may play a role in raising nonspecific antitumor effects in normal host.     

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.     

Degradation of the tumor antigen epitope gp100(280-288) by fibroblast-associated enzymes abolishes specific immunorecognition

Degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) was investigated in the presence of cultured human fibroblasts, and acellular supernatants obtained from these cells; the possible effect of substrate degradation on in vitro immunorecognition was also addressed. In the presence of fibroblasts, gp100(280-288) was degraded to free amino acids with a half-life of less than 4 min; hydrolysis data support the hypothesis that substrate degradation was mainly caused by the activity of cell-expressed amino- and carboxypeptidases. Gp100(280-288) was also degraded in the presence of acellular supernatants: under these conditions, the hydrolysis pattern was similar to that observed in the presence of whole cells, but degradation kinetics was slower. As a result of these phenomena, immunorecognition of gp100(280-288)-specific cytotoxic T lymphocyte (CTL) clones was severely hampered, and was totally suppressed after 90 min. In conclusion, the high activity of fibroblast-expressed proteases, and the presence of wide-scope soluble enzymes, may explain, at least in part, the low activity of peptide-based antineoplastic vaccines, as well as the transient effectiveness of subcutaneously administered peptides in general.     

Stimulation via CD3-Ti but not CD2 induces rapid tyrosine phosphorylation of a 68-kDa protein in the human Jurkat T cell line.

Tyrosine phosphorylation is an early biochemical event associated with surface receptor triggering in many cellular systems. In T lymphocytes, Ag receptor (CD3-Ti) stimulation results in tyrosine phosphorylation of the CD3 zeta subunit. The tyrosine kinase responsible for this modification after CD3-Ti triggering has not been identified. Here we reported that a 68-kDa T cell membrane-associated protein (pp68) in human Jurkat T cells is phosphorylated on tyrosine residues within 1 min after anti-CD3 mAb addition. This induced tyrosine phosphorylation is detected either by in vivo [32P]orthophosphate labeling of the Jurkat T cells or by in vitro [32P]ATP labeling after immunoprecipitation by antiphosphotyrosine antibody. In contrast, mAb stimulation via CD2 and CD4 structures does not induce phosphorylation of pp68. These data are among the first to provide evidence that CD3-Ti and CD2 activation pathways are distinct. Furthermore, they imply that pp68 is itself a tyrosine kinase and/or is a rapidly phosphorylated substrate of a tyrosine kinase.     

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels¹. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation². A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo^3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ^2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing β-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium^7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.     

MHC-peptide specificity and T-cell epitope mapping: where immunotherapy starts

The evaluation and characterization of epitope-specific human leukocyte antigen (HLA)-restricted memory T-cell reactivity is an important step for the development of preventive vaccines and peptide-based immunotherapies for viral and tumor diseases. The past decade has witnessed the use of HLA-restricted peptides as tools to activate strong immune responses of na?ve or memory T cells specifically. This has fuelled an active search for methodological approaches focusing on HLA and peptide associations. Here, we outline new perspective on the emerging opportunity of evaluating HLA and peptide restriction by using novel approaches, such as quantitative real-time PCR, that can identify epitope specificities that are potentially useful in clinical settings.     

Guiding embryonic stem cells towards differentiation: lessons from molecular embryology

Embryonic stem cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types, including germ cells. These qualities have made mouse embryonic stem cells a valuable resource for genetic manipulation of the mouse genome. In addition, they present a powerful system for the in vitro dissection of mammalian embryonic development. The recent isolation of human embryonic stem cells has raised a lot of interest for the potential of transposing our knowledge of lineage-specific differentiation of embryonic stem cells to cell-based therapy of human disease. Recent reports have provided insights into the specific differentiation of embryonic stem cells to different cell types of the embryo. However, progress in this direction seems to depend on the knowledge of the mechanisms controlling lineage decisions during embryogenesis.