The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.

Opposite effects of nuclear factor-jB (NF-jB) on neuron survival rely on activation of diverse NF-jB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1b. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-b (Ab), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Ab toxicity depends on cRel activation. Ab peptide induced NF-jB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X L . Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X L prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TATBcl-X L addition rescued neurons from Ab toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Reldependent antiapoptotic pathway responsible for neuroprotection against Ab peptide.

The occurrence of emerging organic micropollutants (OMPs) in sewage sludge has been widely reported; nevertheless, their fate during sludge treatment remains unclear. The objective of this work was to study the fate of OMPs during mesophilic and thermophilic anaerobic digestion (AD), the most common processes used for sludge stabilization, by using raw sewage sludge without spiking OMPs. Moreover, the results of analytical chemistry were complemented with biological assays in order to verify the possible adverse effects (estrogenic and genotoxic) on the environment and human health in view of an agricultural (re)use of digested sludge. Musk fragrances (AHTN, HHCB), ibuprofen (IBP) and triclosan (TCS) were the most abundant compounds detected in sewage sludge. In general, the efficiency of the AD process was not dependent on operational parameters but compound-specific: some OMPs were highly biotransformed (e.g. sulfamethoxazole and naproxen), while others were only slightly affected (e.g. IBP and TCS) or even unaltered (e.g. AHTN and HHCB). The MCF-7 assay evidenced that estrogenicity removal was driven by temperature. The Ames test did not show point mutation in Salmonella typhimurium while the Comet test exhibited a genotoxic effect on human leukocytes attenuated by AD. This study highlights the importance of combining chemical analysis and biological activities in order to establish appropriate operational strategies for a safer disposal of sewage sludge. Actually, it was demonstrated that temperature has an insignificant effect on the disappearance of the parent compounds while it is crucial to decrease estrogenicity.

Gap‐junctional intercellular communication (GJIC) in 20 primary human liver tumors with different degrees of malignancy has been studied at the functional and molecular levels. When GJIC capacity was determined by dye‐transfer assay performed directly with freshly removed tumor tissue, significant reduction was found in all samples, regardless of their morphology. In addition, a selective lack of GJIC between tumor and surrounding non‐tumorous cells was observed in some cases, probably due to the physical separation between them resulting from encapsulation of tumors. There was, however, no essential change in the level of expression of the major liver gap‐junction protein, connexin (ex) 32, in liver tumors as measured by Northern and Western blot analyses. Immunohistochemical study revealed aberrant localization of ex 32 in the majority of malignant liver tumors. Instead of cytoplasmic membrane localization at intercellular contacts, ex 32 was detected mainly either intracytoplasmically or in plasma membrane free from contact with other cells. We did not detect any mutation in the coding sequence of the ex 32 gene from any of the human liver tumors we tested. Thus it is likely that the aberrant localization of ex 32 in tumor cells is due to disruption of the mechanisms for establishment of this protein into gap‐junction plaques, rather than to structural abnormality of the ex 32 protein itself. Another member of the connexin family, ex 43, not detectable in non‐tumorigenic hepatocytes, was expressed in several tumors, especially in invasive areas, but was detected in only a few tumor cells and was localized intracytoplasmically, suggesting that ex 43 protein is not involved in GJIC in the tumors. © 1994 Wiley‐Liss, Inc.

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo.Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.