The endoskeleton of elasmobranchs (sharks and rays) is comprised largely of unmineralized cartilage, differing fundamentally from the bony skeletons of other vertebrates. Elasmobranch skeletons are further distinguished by a tessellated surface mineralization, a layer of minute, polygonal, mineralized tiles called tesserae. This 'tessellation' has defined the elasmobranch group for more than 400 million years, yet the limited data on development and ultrastructure of elasmobranch skeletons (e.g. how tesserae change in shape and mineral density with age) have restricted our abilities to develop hypotheses for tessellated cartilage growth. Using high-resolution, two-dimensional and three-dimensional materials and structural characterization techniques, we investigate an ontogenetic series of tessellated cartilage from round stingray Urobatis halleri, allowing us to define a series of distinct phases for skeletal mineralization and previously unrecognized features of tesseral anatomy. We show that the distinct tiled morphology of elasmobranch calcified cartilage is established early in U. halleri development, with tesserae forming first in histotroph embryos as isolated, globular islets of mineralized tissue. By the sub-adult stage, tesserae have increased in size and grown into contact with one another. The intertesseral contact results in the formation of more geometric (straight-edged) tesseral shapes and the development of two important features of tesseral anatomy, which we describe here for the first time. The first, the intertesseral joint, where neighboring tesserae abut without appreciable overlapping or interlocking, is far more complex than previously realized, comprised of a convoluted bearing surface surrounded by areas of fibrous attachment. The second, tesseral spokes, are lamellated, high-mineral density features radiating outward, like spokes on a wheel, from the center of each tessera to its joints with its neighbors, likely acting as structural reinforcements of the articulations between tesserae. As tesserae increase in size during ontogeny, spokes are lengthened via the addition of new lamellae, resulting in a visually striking mineralization pattern in the larger tesserae of older adult skeletons when viewed with scanning electron microscopy (SEM) in backscatter mode. Backscatter SEM also revealed that the cell lacunae in the center of larger tesserae are often filled with high mineral density material, suggesting that when intratesseral cells die, cell-regulated inhibition of mineralization is interrupted. Many of the defining ultrastructural details we describe relate to local variation in tissue mineral density and support previously proposed accretive growth mechanisms for tesserae. High-resolution micro-computed tomography data indicate that some tesseral anatomical features we describe for U. halleri are common among species of all major elasmobranch groups despite large variation in tesseral shape and size. We discuss hypotheses about how these features develop, and compare them with oth...

Abstract-Membrane vesicles (MVs) released from activated cells and blebs from apoptotic cells are increased in patients with vascular disease and in those with atherosclerotic lesions, and their contribution to inflammatory reactions has been suggested. At sites of inflammation, MVs could serve as rapidly available substrates for peroxidation, carry oxidized compounds to activate other cells, and amplify inflammation. Here, we show that MVs released from tert-butyl hydroperoxide-treated endothelial cells (ECs) and apoptotic blebs, but not MVs from Ca 2ϩ ionophore-treated ECs, stimulate monocyte adhesion to ECs, an important step in atherogenesis. We show that oxidized phospholipids, such as the previously identified 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), are responsible for biological activity in MVs and apoptotic blebs. Natural antibodies from apolipoprotein E-null mice that recognize POVPC also recognize oxidized MVs, and pretreatment of MVs with these antibodies inhibits their ability to activate ECs. Furthermore, the biological activity of oxidized MVs is inhibited by platelet-activating factor receptor antagonists, which have been shown to inhibit the action of POVPC. Taken together, we show that oxidized MVs and apoptotic blebs stimulate ECs to specifically bind monocytes, with oxidized phospholipids (POVPC) being the active principle. In addition to oxidized lipoproteins, oxidized MVs and apoptotic blebs may play an important role in chronic inflammatory diseases, such as atherosclerosis.

To provide a tool to investigate the mechanisms inducing and maintaining cancer-related pain and hyperalgesia, a soft tissue tumor/ metastasis model was developed that is applicable in C57BL/6J wild-type and transgenic mice. We show that the experimental tumorinduced heat hyperalgesia and nociceptor sensitization were prevented by systemic treatment with the tumor necrosis factor ␣ (TNF␣) antagonist etanercept. In naive mice, exogenous TNF␣ evoked heat hyperalgesia in vivo and sensitized nociceptive nerve fibers to heat in vitro. TNF␣ enhanced the expression of the nociceptor-specific heat transducer ion channel transient receptor potential vanilloid 1 (TRPV1) and increased the amplitudes of capsaicin and heat-activated ionic currents via p38/MAP (mitogen-activated protein) kinase and PKC (protein kinase C). Deletion of the tumor necrosis factor receptor type 2 (TNFR2) gene attenuated heat hyperalgesia and prevented TRPV1 upregulation in tumor-bearing mice, whereas TNFR1 gene deletion played a minor role. We propose endogenous TNF␣ as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2.

IntroductionWe describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model.MethodsAutomation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC.ResultsLower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype.ConclusionWe demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.