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S U M M A R Y P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the blood-brain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.

Previous studies have shown that increased vascularity is associated with haematogenous metastasis and poor prognosis in gastric cancer. The role of mast cells in gastric cancer angiogenesis has not been clarified completely. In this study, we correlated microvascular density and tryptase- and chymase-positive mast cells with histopathological type in gastric cancer. Specimens of primary gastric adenocarcinomas obtained from 30 patients who had undergone curative gastrectomy were investigated immunohistochemically by using anti-CD31 antibody to stain endothelial cells and anti-tryptase and anti-chymase antibodies to stain mast cells. The results showed that stage IV gastric carcinoma has a higher degree of vascularization than other stages and that both tryptase- and chymase-positive mast cells increase in parallel with malignancy grade even if the density of chymase-positive mast cells was significantly lower than the density of tryptase-positive mast cells and is highly correlated with the extent of angiogenesis. This study has demonstrated that mast cell density correlates with angiogenesis and progression of patients with gastric carcinoma. Understanding the mechanisms of gastric cancer angiogenesis provides a basis for a rational approach to the development of an antiangiogenic therapy in patients with this malignancy.

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Tissue engineering applications need a continuous development of new biomaterials able to generate an ideal cellextracellular matrix interaction. The stem cell fate is regulated by several factors, such as growth factors or transcription factors. The most recent literature has reported several publications able to demonstrate that environmental factors also contribute to the regulation of stem cell behavior, leading to the opinion that the environment plays the major role in the cell differentiation.The interaction between mesenchymal stem cells (MSCs) and extracellular environment has been widely described, and it has a crucial role in regulating the cell phenotype. In our laboratory (Tecnologica Research Institute, Crotone, Italy), we have recently studied how several physical factors influence the distribution and the morphology of MSCs isolated from dental pulp, and how they are able to regulate stem cell differentiation. Mechanical and geometrical factors are only a small part of the environmental factors able to influence stem cell behavior, however, this influence should be properly known: in fact, this assumption must be clearly considered during those studies involving MSCs; furthermore, these interactions should be considered as an important bias that involves an high number of studies on the MSCs, since in worldwide laboratories the scientists mostly use tissue culture plates for their experiments.

Human mast cells (MCs) are divided in two types depending on the expression of tryptase and chymase in their granules. Literature data indicate that both tryptase and chymase are angiogenic, but there is currently no evidence of their direct angiogenic activity in vivo. In this study, we have investigated the capacity of tryptase and chymase to promote vasoproliferation in chick embryo chorioallantoic membrane (CAM), a well established in vivo assay to study angiogenesis and anti-angiogenesis. The results showed that both tryptase and chymase stimulate angiogenesis and that the response is similar to that obtained with vascular endothelial growth factor (VEGF), a well-known angiogenic cytokine, and confirm the angiogenic activity of these two proteases stored in MC granules.

'Non-traditional' large neurons of the granular layer of the cerebellar cortex include all its large neuronal types, except the Golgi neuron, which is instead one of the five 'classic' types of corticocerebellar neurons. The morphological, chemical and functional characteristics of the 'non-traditional' large neurons have not been entirely ascertained. The aim of this study was to ascertain whether morphological evidence can be provided of GABA synthesis within the 'non-traditional' large neurons of the human cerebellar cortex by means of immunocytochemistry for glutamic acid decarboxylase (GAD). Fragments of postmortem cerebellar cortex of various lobules from the hemispheres and vermis were studied. Immunoreactions revealed large neurons distributed throughout the granular layer in all lobules examined. They were discriminated by analyzing the morphological features of their bodies and processes and were identified as Golgi neurons and as some 'non-traditional' types, such as the candelabrum, Lugaro and synarmotic neurons. In addition, immunoreactive large neurons, with their bodies and processes closely adjacent to microvessels, were observed throughout the layer: these perivascular neurons could represent a new type of 'non-traditional' neuron of the cerebellar cortex. This study supplies the first indication that in the human cerebellar cortex some types of 'non-traditional' large neurons are GAD-immunoreactive, in addition to those neurons already known to be GABAergic (i.e., stellate, basket, Purkinje and Golgi neurons). These morphological data further point out possible functional roles for GABA as a neurotransmitter/neuromodulator in intrinsic, associative and projective circuits of the cerebellar cortex.

In this study, we investigated the involvement of dystrophin-associated proteins (DAPs) and their relationship with the perivascular basement membrane in the brains of mdx mice and controls at the age of 2 months. We analyzed (1) the expression of glial DAPs a-b-dystroglycan (DG), a-syntrophin, aquaporin-4 (AQP4) water channel, Kir 4.1 and dystrophin isoform (Dp71) by immunocytochemistry, laser confocal microscopy, immunogold electron microscopy, immunoblotting and RT-PCR; (2) the ultrastructure of the basement membrane and expression of laminin and agrin; and (3) the dual immunofluorescence colocalization of AQP4/a-b-DG, and of Kir 4.1/agrin. The following results were observed in mdx brain as compared with controls: (1) a significant reduction in protein content and mRNA expression of DAPs; (2) ultrastructurally, a thickened and discontinuous appearance of the basement membrane and a significant reduction in laminin and agrin; and (3) a molecular rearrangment of a-b-DG, coupled with a parallel loss of agrin and Kir 4.1 on basement membrane and glial endfeet. These data indicate that in mdx brain the deficiency in dystrophin and dystrophin isoform (Dp71) is coupled with a reduction of DAP components, coupled with an altered anchoring to the basement membrane. The blood-brain barrier (BBB) controls the selective exchanges between blood and neuropil by specific molecular and structural features of its vascular and perivascular components, including endothelial cells, pericytes, glial endfeet and basement membrane. 1-3 Glial cells have a key role in the control of BBB development and integrity. They express in a polarized way, on their perivascular endfeet, carrier membranes and channel proteins, including aquaporin-4 (AQP4) water channel and potassium channel Kir 4.1, which are responsible for the water flow rate and spatial K þ buffering control. [4][5][6][7][8][9] In the retina, AQP4 colocalizes with Kir 4.1, 9-11 suggesting a tight cooperation between the water flux and K þ siphoning generated by neuronal activity at the BBB interfaces. Moreover, AQP4 and Kir 4.1 rearrangement has been described in BBB alterations, coupled with cytotoxic edema and K þ -delayed buffering capacity. 12,13 AQP4 has a crucial role in the regulation of the interactions occurring between endothelial and glial cells and between glial cells and the extracellular matrix components. AQP4 is a component of the orthogonal aggregate particles (OAPs), demonstrated by freeze fracturing on the ependimoglial membrane and perivascular glial endfeet, 14 the development of which occurs in parallel with AQP4 glial endfeet expression and BBB maturation. 7 Moreover, OAPs contain AQP4 associated with Kir 4.1 12 and their polarized distribution on the glial endfeet seems to be dependent by the presence of the perivascular basement membrane. 15 AQP4 controls BBB functioning, modifying