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Globoid cell leukodystrophy (GLD), or Krabbe disease, is a neurodegenerative sphingolipidosis caused by genetic deficiency of lysosomal β-galactosylceramidase (GALC), characterized by neuroinflammation and demyelination of the central (CNS) and peripheral nervous system. The acute phase protein long pentraxin-3 (PTX3) is a soluble pattern recognition receptor and a regulator of innate immunity. Growing evidence points to the involvement of PTX3 in neurodegeneration. However, the expression and role of PTX3 in the neurodegenerative/neuroinflammatory processes that characterize GLD remain unexplored. Here, immunohistochemical analysis of brain samples from Krabbe patients showed that macrophages and globoid cells are intensely immunoreactive for PTX3. Accordingly, Ptx3 expression increases throughout the course of the disease in the cerebrum, cerebellum, and spinal cord of GALC-deficient twitcher (Galctwi/twi) mice, an authentic animal model of GLD. This was paralleled by the upregulation of proinflammatory genes and M1-polarized macrophage/microglia markers and of the levels of PTX3 protein in CNS and plasma of twitcher animals. Crossing of Galctwi/twi mice with transgenic PTX3 overexpressing animals (hPTX3 mice) demonstrated that constitutive PTX3 overexpression reduced the severity of clinical signs and the upregulation of proinflammatory genes in the spinal cord of P35 hPTX3/Galctwi/twi mice when compared to Galctwi/twi littermates, leading to a limited increase of their life span. However, this occurred in the absence of a significant impact on the histopathological findings and on the accumulation of the neurotoxic metabolite psychosine when evaluated at this late time point of the disease. In conclusion, our results provide the first evidence that PTX3 is produced in the CNS of GALC-deficient Krabbe patients and twitcher mice. PTX3 may exert a protective role by reducing the neuroinflammatory response that occurs in the spinal cord of GALC-deficient animals. [ABSTRACT FROM AUTHOR]

In the present study, we report the analysis of NK cells derived from patients suffering from a rare ovarian cancer histotype of clear cell carcinoma (OCCC) resistant to conventional chemotherapies. We analyzed the phenotype of NK cells derived from peripheral blood (PB) and peritoneal fluid (PF) and evaluated cytotoxic interactions between NK cells and autologous tumor cells (ATC) derived from patients. We provided evidence of impaired degranulation capacity of NK cells derived from patients' PF in the presence of ATC. Analyzing tumor cell ligands recognized by NK cell receptors, we found that ATC are characterized by an HLA class I+ phenotype (although the level of HLA‐I expression varies among all patients) and by a heterogeneous expression of ligands for activating NK receptors (from normal to decreased expression of some markers). Furthermore, we observed a down‐regulation of crucial NK cell activating receptors, primarily DNAX Accessory Molecule‐1 (DNAM‐1), on tumor‐associated NK cells. Based on these results, we propose that this severe lysis defect may be due to both negative interactions between HLA‐I‐specific inhibitory NK cell receptors/HLA‐I molecules and to defective interactions between activating NK receptors and cognate ligands. In conclusion, for the first time, the phenotypic and functional properties of tumor‐associated NK cells and their ATC derived from PF of patients with advanced stage of OCCC were characterized. Taken together results indicate altered interactions between NK cells and ATC and shed light on the aggressive mechanisms of this cancer histotype. Further studies on this rare tumor will be helpful to improve and define more effective therapies. [ABSTRACT FROM AUTHOR]

Alessandro Moretta was Professor of Histology at University of Brescia from 1994 to 1997. It was in that period that we met and started a collaboration that continued in the years to follow. He immediately involved us in the production of monoclonal antibodies (mAbs) that allowed the identification and fine characterization of novel receptor molecules that were able to activate or inhibit human Natural Killer cell function, including several antibodies specific for Natural Cytotoxicity Receptor (NCR) and Killer-cell Immunoglobulin-like Receptor (KIR) molecules. These reagents, generated in our laboratory in Brescia, contributed to complete the studies aimed to characterize innate lymphoid NK cells, that had been initiated by Alessandro and his brother Lorenzo in Genoa. Soon, we identified an anti-KIR3DL2 that was subsequently shown to be helpful for the diagnosis and treatment of various forms of cutaneous T cell lymphoma. While in Brescia, Alessandro established a partnership with those of us who were working in the Department of Pediatrics; together, in short time we tackled the goal of studying the role of NK cells in patients with primary immunodeficiencies. This collaboration led to novel discoveries that shed light on the critical role played by NK cells in the immune response against virus and tumors in humans, as best exemplified by our characterization of the molecular mechanisms of impaired control of Epstein-Barr Virus (EBV) infection in patients with X-linked lymphoproliferative (XLP) disease. After Alessandro left Brescia to return to Genoa, our collaboration continued with the same enthusiasm, and even from a distance he remained an extraordinary example of an inspirational and generous mentor. This review is a sign of our gratitude to a mentor and a friend whom we deeply miss. [ABSTRACT FROM AUTHOR]

The subcutaneous Matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic molecules. However, quantification of the angiogenic response in the plug remains a problematic task. Here we report a simple, rapid, unbiased and reverse transcription-quantitative PCR (RT-qPCR) method to investigate the angiogenic process occurring in the Matrigel plug in response to fibroblast growth factor-2 (FGF2). To this purpose, a fixed amount of human cells were added to harvested plugs at the end of the in vivo experimentation as an external cell tracer. Then, mRNA levels of the pan-endothelial cell markers murine CD31 and vascular endothelial- cadherin were measured by species-specific RT-qPCR analysis of the total RNA and data were normalized for human GAPDH or β- actin mRNA levels. RT-qPCR was used also to measure the levels of expression in the plug of various angiogenesis/inflammation-related genes. The procedure allows the simultaneous, quantitative evaluation of the newly-formed endothelium and of non-endothelial/inflammatory components of the cellular infiltrate in the Matrigel implant, as well as the expression of genes involved in the modulation of the angiogenesis process. Also, the method consents the quantitative assessment of the effect of local or systemic administration of anti-angiogenic compounds on the neovascular response triggered by FGF2. [ABSTRACT FROM AUTHOR]

Fibronectin (FN) is an extracellular matrix cell-adhesive glycoprotein. The alternative spliced isoform EDB-FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro-environments on EDB-FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet-FGF2 cells overexpressing fibroblast growth factor-2 (FGF2) driven by the tetracycline-responsive promoter were further transfected with a VEGF antisense cDNA, generating AS-VEGF/Tet-FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast-growing, highly vascularized Tet-FGF2 tumours. Down-regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS-VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro-environment. Quantitative RT-PCR analysis using human EDB-FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up-regulation of EDB-FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki-1 cells. However, in vivo down-regulation of VEGF expression, as occurs in AS-VEGF/Tet-FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline-treated Tet-FGF2 tumour-bearing animals, causes significant inhibition of EDB-FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT-PCR analysis. Accordingly, treatment of Tet-FGF2 tumour-bearing animals with the neutralizing anti-murine VEGF receptor-2 antibody DC101, or of Caki-1 tumour-bearing animals with the anti-VEGF antibody bevacizumab, inhibited EDB-FN expression in tumour grafts. EDB-FN down-regulation was paralleled by a decrease in vascularity, thus confirming EDB-FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro-environment, and in particular the VEGF/VEGFR-2 system, plays a key role in modulating EDB-FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB-FN in combination with anti-angiogenic and/or cytotoxic drugs. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor-2 (FGF2) is a potent angiogenic growth factor. Here, gene expression profiling of FGF2-stimulated microvascular endothelial cells revealed, together with a prominent pro-angiogenic profile, a pro-inflammatory signature characterized by the up-regulation of pro-inflammatory cytokine/chemokines and their receptors, endothelial cell adhesion molecules and members of the eicosanoid pathway. Real-time quantitative PCR demonstrated early induction of most of the FGF2-induced, inflammation-related genes. Accordingly, chick embryo chorioallantoic membrane (CAM) and murine Matrigel plug angiogenesis assays demonstrated a significant monocyte/macrophage infiltrate in the areas of FGF2-driven neovascularization. Similar results were obtained when the conditioned medium (CM) of FGF2-stimulated endothelial cells was delivered onto the CAM, suggesting that FGF2-upregulated chemoattractants mediate the inflammatory response. Importantly, FGF2-triggered new blood vessel formation was significantly reduced in phosphatidylinositol 3-kinase-γ null mice exhibiting defective leucocyte migration or in clodronate liposome-treated, macrophage-depleted mice. Furthermore, the viral pan-chemokine antagonist M3 inhibited the angiogenic and inflammatory responses induced by the CM of FGF2-stimulated endothelial cells and impaired FGF2-driven neovascularization in the CAM assay. These findings point to inflammatory chemokines as early mediators of FGF2-driven angiogenesis and indicate a non-redundant role for inflammatory cells in the neovascularization process elicited by the growth factor. [ABSTRACT FROM AUTHOR]

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na+/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Uveal melanoma is a highly metastatic tumor, representing the most common primary intraocular malignancy in adults. Tumor cell xenografts in zebrafish embryos may provide the opportunity to study in vivo different aspects of the neoplastic disease and its response to therapy. Here, we established an orthotopic model of uveal melanoma in zebrafish by injecting highly metastatic murine B16-BL6 and B16-LS9 melanoma cells, human A375M melanoma cells, and human 92.1 uveal melanoma cells into the eye of zebrafish embryos in the proximity of the developing choroidal vasculature. Immunohistochemical and immunofluorescence analyses showed that melanoma cells proliferate during the first four days after injection and move towards the eye surface. Moreover, bioluminescence analysis of luciferase-expressing human 92.1 uveal melanoma cells allowed the quantitative assessment of the antitumor activity exerted by the canonical chemotherapeutic drugs paclitaxel, panobinostat, and everolimus after their injection into the grafted eye. Altogether, our data demonstrate that the zebrafish embryo eye is a permissive environment for the growth of invasive cutaneous and uveal melanoma cells. In addition, we have established a new luciferase-based in vivo orthotopic model that allows the quantification of human uveal melanoma cells engrafted in the zebrafish embryo eye, and which may represent a suitable tool for the screening of novel drug candidates for uveal melanoma therapy. [ABSTRACT FROM AUTHOR]

In the present study we have attempted a characterization of the biochemical bases of the interaction of human basic fibroblast growth factor (bFGF) with glycosaminoglycans (GAGs) in solution. This interaction has been evidenced as the capacity of different GAGs and various sulfated compounds to protect bFGF and different bFGF mutants from tryptic cleavage. Heparin protects bFGF from trypsin digestion in a dose-dependent fashion, Substitution by sitedirected mutagenesis of two or more basic residues with neutral glutamine residues in the amino, terminal region bFGF(27-32) or in the carboxyl4erminai region bFGF(118-129) does not significantly affect the protective effect exerted by heparin. In contrast, heparin protection is abolished when the full region bFGF(27-32) is deleted. The capacity of different GAGs to protect bFGF from proteolytic cleavage decreases in the following order: heparin > heparan sulfate > dermatan sulfate = chondroitin sulfates A and C > hyaluronic acid = K5 polysaccharide, indicating that both the degree of sulfation and the backbone structure of GAG modulate its interaction with bFGE This is confirmed by the different capacity of various sulfated compounds (including dextran sulfates, suramin, trypan blue, and sulfate ion) to protect bFGF from tryptic digestion. Moreover, tryptic digestion studies performed with various heparin molecules and dextran sulfates of different size, ranging from 2.0 kDa to 500 kDa, indicate that the number of bFGF molecules which interact with a single molecule of polysaccharide is related to the molecular mass of the GAG and that six hexose residues are sufficient to protect 1 2 molecules bFGE In conclusion, our findings indicate that the capacity of GAGs to protect bFGF from tryptic cleavage depends upon their size, sulfation, distribution of the anionic sites along the chain, and structural requirements of the bFGF molecule, These studies will help to design synthetic oligosaccharides endowed with different bFGF agonist and/or antagonist activities. [ABSTRACT FROM AUTHOR]

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase β-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31−/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease. [ABSTRACT FROM AUTHOR]

In this study the phenotype and function of tumor-associated NK cells from peritoneal fluids of a selected cohort of patients with seropapillary ovarian carcinoma were analyzed. In > 50% of these patients, the expression of the activating receptor NKp30 in tumor-associated NK cells was substantially reduced as compared to autologous peripheral blood (PB) NK cells. The impaired expression of this receptor was associated with the presence of one of its cellular ligands (B7-H6), which was detectable as a surface/cytosolic molecule in tumor cells and as a soluble molecule in the peritoneal fluid. NK cells from patients expressing this NKp30low phenotype displayed an impaired interferon-gamma (IFNγ) production and cytolytic function when tested against target cells expressing surface B7-H6. Our data also suggest that in these patients, the defective expression and function of NKp30 may be induced by the chronic engagement of this receptor by soluble B7-H6 or by tumor cells expressing this ligand. The impairment of NK cell functions described herein could represent a novel mechanism by which the tumor microenvironment may contribute to the escape from immune surveillance. [ABSTRACT FROM PUBLISHER]