The spatial distribution of mast cells inside the tumor stroma has been little investigated. In this study, we have evaluated tumor mast cells distribution through the analysis of the morphological features of the spatial patterns generated by these cells, including size, shape, and architecture of the cell pattern. We have compared diffuse large B cells lymphoma (DLBCL) and systemic mastocytosis in two different anatomical localizations (lymph nodes for DLBCL and, respectively, bone marrow for mastocytosis). Results have indicated that, despite the high difference in size exhibited by the mast cells patterns in the two conditions, the spatial relationship between the mast cells forming the aggregates resulted similar, characterized by a significant tendency of the mast cells to self-organize in clusters.

Murine cancer models have been extremely useful for analyzing the biology of pathways involved in cancer initiation, promotion, and progression. Interestingly, several murine cancer models also exhibit heterogeneity, genomic instability and an intact immune system. However, they do not adequately represent several features that define cancer in humans, including long periods of latency, the complex biology of cancer recurrence and metastasis and outcomes to novel therapies. Therefore, additional models that better investigate the human disease are needed. In the pet population, with special references to the dog, cancer is a spontaneous disease and dogs naturally develop cancers that share many characteristics with human malignancies More than 40 years ago, optimization of bone marrow transplantation protocols was undertaken in dogs and recently novel targeted therapies such as liposomal muramyl tripeptide phosphatidylethanolamine and several tyrosine kinase inhibitors, namely masitinib (AB1010) and toceranib phosphate (SU11654), have been developed to treat dog tumors which have then been translated to human clinical trials. In this review article, we will analyze biological data from dog tumors and comparative features with human tumors, and new therapeutic approaches translated from dog to human cancer.

Chemokines, small pro-inflammatory cytokines, are involved in migration of inflammatory cells in inflamed tissues and recent studies established their role in angiogenesis, hematopoiesis, cancer and autoimmune conditions. Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXCR2 are involved in the inflammatory processes. Since there is no previous report that supports a possible role of GRO-α/CXCR2 receptor complex during inflammation and neovascularization existing in the autoimmune disease Sjögren's syndrome (SS), in this study, we examined CXCR2 and its ligand GRO-α expression in SS tissues. Immunohistochemistry revealed that GRO-α and its receptor CXCR2 were expressed at high levels in diseased tissues compared to healthy controls. In addition, human salivary gland epithelial cells (SGEC) cultures were submitted to a pro-inflammatory microenvironment using cytokines IL-6 and TNF-α in order to demonstrate that CXCR2 may change its initial expression pattern to another under inflammatory condition. The data show an increased expression of CXCR2 depending on the inflammatory cytokine used in culture in a time-dependent manner. Furthermore, silencing of the pro-angiogenic chemokine GRO-α is proportionally correlated with decreased expression of CXCR2 in pro-inflammatory cytokine-stimulated SGEC indicating the GRO-α/CXCR2 complex as a novel therapeutic target for the chronic inflammatory disease Sjögren's syndrome

A decreased saliva production occurs in primary Sjögren’s syndrome (pSS), an autoimmune disease characterized by oral and ocular dryness due to dysfunction of the lacrimal and salivary glands (SGs). Since water movement is involved in saliva secretion, the expression, localization and function of the water channels aquaporins (AQPs) have been extensively studied in SGs. To date, the presence of AQP4 remains controversial and ambiguous in human SGs. We investigated by immunohistochemistry, high-resolution confocal microscopy and quantitative image analysis, western blot and real-time RT-PCR, the presence of the AQP4 gene and the distribution of AQP4 protein in healthy controls and pSS SGs biopsies. Through the immunohistochemical analysis we demonstrated that AQP4 presence is confined to the basal region of acini, to the lateral and apical membrane of intercalated and striated ducts in both control and pSS glands. The most striking observation was the discovery of AQP4 localization in myoepithelial cells (MECs) that surround acini lobules and intercalated ducts, and the demonstration of AQP4-down-regulated immunoreactivity in pSS MECs. Our studies suggest that the capacity for water flow across the membrane of MECs may be altered in pSS, identifying AQP4 as a promising new therapeutic agent to treat xerostomia

Background: Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXC chemokine receptor 2 (CXCR2) are involved in the inflammatory processes. In many models of acute and chronic inflammatory diseases, blockade of CXCR2 substantially reduces leukocyte recruitment, tissue damage and mortality. There are evidences that the CXCR2 expression can also be regulated, by enzymatic cleavage via metalloproteinases. Actually, the ADAM17 inhibitors are under development for the treatment of a variety of inflammatory autoimmune disorders. In addition, the ADAM17 activation was recently associated with the chronic inflammatory condition observed in Sjögren's syndrome (SS). Objectives: To better understand the molecular mechanisms by which the GRO-α /CXCR2 system is involved in the SS inflammatory condition and clarify the role of ADAM17 activation in the modulation of the GRO-α/CXCR2 chemokine system in epithelial cells (SGEC) from SS salivary glands. Methods: RT-PCR, Real Time-PCR, western blot, ELISA, flow cytometry techniques were employed to examine the expression of GRO-α /CXCR2 system in presence or not of the ADAM17 inhibitor TAPI-1 in salivary gland epithelial cells cultures from SS patients. Results: The CXCR2 overexpression observed in SS SGEC resulted dramatically decreased by ADAM17 inhibitor TAPI-1. In addition, comparing the expression levels of ADAM17 in healthy SGEC in presence or not of GRO-α treatment, we observed that GRO-α, dose-dependently, influences ADAM17 activation, effect that was inhibited by blocking the interaction of GRO-α with its CXCR2 receptor. Conclusions: Our data show for the first time that ADAM17 has an important role in GRO-α/CXCR2 system activity regulation, suggesting that regulating CXCR2/ADAM17 interaction could be an attractive therapeutic target in SS.

Plaque rupture is more frequent in symptomatic than asymptomatic patients and in symptomatic patients in which the fibrous cap is thinner with greater inflammatory infiltrate. The aim of this study was to evaluate the role of major cardiovascular risk factors and histomorphological characteristics, in particular of macrophages and mast cells and of microvascular density, in atherosclerotic plaques collected from 33 consecutive symptomatic and asymptomatic patients undergoing carotid endoarterectomy for carotid disease. Patients were divided in two groups: symptomatic patients [defined according to the North American Symptomatic Carotid Endoarterectomy Trial Classification presenting with cerebrovascular TIAs or stroke within the last three months, and asymptomatic patients, exhibiting progressive ipsilateral internal carotid artery stenosis. Morphological analysis revealed an higher neovascularization in plaques from symptomatic patients, as compared to asymptomatic patients. New-formed blood vessels were located in the intima, interfacing the necrotic core, and at the shoulder area. In parallel, we found a significant increased number of CD68-positive macrophages and tryptase-positive mast cells in plaques from symptomatic patients, as compared to asymptomatic patients. Morphological evidence concerning microvascular density, CD68-positive macrophages and tryptase-postive mast cells have been confirmed by morphometric analysis. Increased plaque vascularity may contribute to lesion progression; being a source of nutrient, inflammatory cell recruitment, and intraplaque hemorrhage, which tends to weaken the plaque and predispose to plaque rupture with ensuing clinical sequelae, such as stroke.

Angiogenesis, the formation of new vessels, is found in Multiple Sclerosis (MS) demyelinating lesions following Vascular Endothelial Growth Factor (VEGF) release and the production of several other angiogenic molecules. The increased energy demand of inflammatory cuffs and damaged neural cells explains the strong angiogenic response in plaques and surrounding white matter. An angiogenic response has also been documented in an experimental model of MS, experimental allergic encephalomyelitis (EAE), where blood – brain barrier disruption and vascular remodelling appeared in a pre-symptomatic disease phase. In both MS and EAE, VEGF acts as a pro-inflammatory factor in the early phase but its reduced responsivity in the late phase can disrupt neuroregenerative attempts, since VEGF naturally enhances neuron resistance to injury and regulates neural progenitor proliferation, migration, differentiation and oligodendrocyte precursor cell (OPC) survival and migrati on to demyelinated lesions. An giogenesis, neurogenesis and oligodendroglia maturation are closely intertwined in the neurovascular niches of the subventricular zone, one of the preferential locations of inflammatory lesions in MS, and in all the other temporary vascular niches where the mutual fostering of angiogenesis and OPC maturation occurs. Angiogenesis, induced either by CNS inflammation or by hypoxic stimuli related to neurovascular uncoupling, appears to be ineffective in chronic MS due to a counterbalancing effect of vasoconstrictive mechanisms determined by the reduced axonal activity, astrocyte dysfunction, microglia secretion of free radical species and mitochondrial abnormalities. Thus, angiogenesis, that supplies several trophic factors, should be promoted in therapeutic neuroregeneration efforts to combat the progressive, degenerative phase of MS.

Angiogenesis is a constant hallmark of multiple myeloma progression and has prognostic potential. Multiple myeloma cells interact with surrounding host cells and extracellular matrix, this crosstalk affecting the most important aspects of the malignant phenotype, both at primary and secondary tumor sites. The pathophysiology of multiple myeloma-induced angiogenesis involves both direct production of angiogenic cytokines by plasma cells and their induction within the bone marrow microenvironment cells. A direct involvement of bone marrow macrophages and mast cells in vasculogenic mimicry has been demonstrated, thus contributing together with circulating endothelial cells and endothelial precursor cells to the multiple myeloma neovascularization. The role of host cells or the niche microenvironment and extracellular matrix represents an intense area of research, finalized at a better understanding of the pathophysiological modifications of the complete tumor entity, i.e. malignant cells and microenvironment

Calbindin-D28k (CB) is a calcium-binding protein largely distributed in the cerebellum of various species of vertebrates. As regards the human cerebellar cortex, precise data on the distribution of CB have not yet been reported. Aim of the present work was to analyze the distribution of CB in postmortem samples of human cerebellar cortex using light microscopy immunohistochemical techniques. Immunoreactivity to CB was detected within neuronal bodies and processes distributed in all cortex layers. In the molecular layer, the immunoreactivity was observed in subpopulations of stellate and basket neurons. In the Purkinje neuron layer, the immunoreactivity was observed in practically all the Purkinje neurons. In the granular layer, the immunoreactivity was observed in subpopulations of granules, of Golgi neurons, and also of other types of large neurons (candelabrum, Lugaro neurons, etc.). Immunoreactivity to CB was also observed in axon terminals distributed throughout the cortex according to layer-specific patterns of distribution. The qualitative and quantitative patterns of distribution of CB showed no difference among the different lobes of the cerebellar cortex. This study reports that CB is expressed by different neuron types, both inhibitory (GABAergic) and excitatory (glutamatergic), involved in both intrinsic and extrinsic circuits of the human cerebellar cortex. The study provides further insights on the functional role of CB and on the neuronal types of the cerebellar cortex in which it is expressed.

Tumor microenvironment plays an important role in cancer initiation and progression. In hematological malignancies, the bone marrow represents the paradigmatic anatomical site in which tumor microenvironment expresses its morphofunctional features. Among the cells participating in the composition of this microenvironment, cancer associated fibrobasts (CAFs) have received less attention in hematopoietic tumors compared to solid cancers. In this review article, we discuss the involvement of CAFs in progression of hematological malignancies and the potential targeting of CAFs in a therapeutic perspective.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune exocrine disease associated with variable lymphocytic infiltration of the affected organs (primarily salivary and lachrymal glands). To investigate the potential implication of nerve growth factor-β (NGF-β) and its high affinity receptor tyrosine kinase receptor A (TrkA) in the regulation of pSS inflammatory responses, we studied their expression in the human salivary gland epithelial cells (SGEC) cultures from pSS minor salivary glands (MSG) biopsies and their relationship with histopathological disease parameters. Here, we demonstrated an increased expression of the NGF-β/TrkA system in pSS SGEC, correlated with the MSG inflammation grade. The results demonstrate that the pro-inflammatory cytokines TNF-α and IL-6 enhance NGF-β production; on the contrary, NGF-β production was reduced in the presence of both Raf-1 kinase and MEK inhibitors. Furthermore, TNF-α/IL-6 treatment increased ERK1/2 phosphorylation. Inhibition of the EGF/EGFR system also decreased NGF-β release by pSS SGEC, indicating that the chronic inflammatory condition characteristic of pSS enhances NGF-β production via EGFR/Raf-1/MEK/ERK pathway activation

Canine cutaneous mast cell tumour (CMCT) is a c-Kit driven tumour sharing similar c-Kit aberrations found in human gastrointestinal stromal tumour. CMCT is classified into three forms: well-(G1), intermediately (G2) (more benign diseases), and poorly (G3) differentiated (malignant) forms. We assess a correlation between c-Kit status, grading, and angiogenesis in CMCTs to explore their potential significance in humans. C-Kit receptor (c-KitR) expression, microvascular density (MVD), and mast cell granulated and degranulated status density (MCGD and MCDD, resp.) were analyzed in 97 CMCTs, by means of histochemistry, immunohistochemistry double staining, and image analysis system. Data showed that predominantly diffuse cytoplasmic-and predominantly focal paranuclear-(Golgi-like) c-Kit protein (PDC-c-Kit and PFP-c-Kit, resp.) expression correlate with high MVD, G3 histopathological grade, and MCDD. Moreover, predominant cellmembrane-c-KitR (PCM-c-KitR) expression status correlates with low MVD, G1-G2 histopathological grade, and MCGD. These findings underline the key role of c-Kit in the biopathology of canine MCTs, indicating a link between aberrant c-Kit expression, increased angiogenesis, and higher histopathological grade. CMCT seems to be a model to study contributions of c-Kit activated MCs in tumour angiogenesis and to evaluate the inhibition of MCs activation by means of c-Kit tyrosine kinase inhibitors, currently translated in humans.

Diffuse large B cell lymphoma (DLBCL) is recognized as the most common non-Hodgkin lymphoma subtype. Advanced high-resolution digital scans of pathology slides have enabled the development of computer-based image analysis algorithms that may assist pathologists in quantifying immunohistochemical stains. In this retrospective study, we reviewed data from 29 patients affected by DLBCL. In order to evaluate the number of tumor cells and microenvironment T cells, we performed an analysis of CD20, Ki67, and CD3 counts, assessed with the Positive Pixel Count algorithm embedded in the Aperio ImageScope software. A lower tumor cell count was observed in patients with a non-germinal center immunophenotype, high LDH, splenomegaly and an IPI ≥ 3. A lower number of CD3 was observed in patients with bulky disease, an IPI ≥ 3 and disease stage 3-4. Overall, these data confirm that quantitative analysis of the tumor cells and of the tumor microenvironment by means of computer-driven quantitative image analysis may add new information in DLBCL diagnosis.

PURPOSE: In clear cell renal cell carcinoma tissue samples we identified and characterized a population of renal cell carcinoma derived CD133+/CD24+ cancer cells. We studied differences between these cells and their nonneoplastic counterpart, tubular adult renal progenitor cells. MATERIALS AND METHODS: CD133+/CD24+ renal cell carcinoma derived cells were isolated from 40 patients. The mesenchymal phenotype and stemness proteomic profile of these renal cell carcinoma derived cells were characterized. Colony forming efficiency and self-renewal ability were tested by limiting dilution. Tumorigenic properties were evaluated in vitro by soft agar assay. The angiogenic response was evaluated in vivo by the chorioallantoic membrane angiogenic assay. Microarray analysis was performed on 6 tubular adult renal progenitor cell and 6 renal cell carcinoma derived cell clones. Membrane protein expression was evaluated by flow cytometry and immunofluorescence staining. RESULTS: CD133+/CD24+ cells were isolated from normal and tumor kidney tissue. Fluorescence activated cell sorting revealed that renal cell carcinoma derived cells did not express mesenchymal stem cell markers. CD133+/CD24+ tumor cells were more undifferentiated than tubular adult renal progenitor cells. Renal cell carcinoma derived cells were clonigenic and could differentiate into adipocytes, epithelial and osteogenic cells. They could also regenerate tumor cells in vitro and induce angiogenesis in vivo. Gene expression profile identified CTR2 as a membrane marker for this neoplastic population. CTR2 was involved in renal cell carcinoma derived cell cisplatin resistance. CONCLUSIONS: Our results indicate the presence of a CD133+/CD24+/CTR2+ cancer cell population in clear cell renal cell carcinoma. These cells have some stem cell-like features, including in vitro self-maintenance and differentiating capabilities, and they can induce an angiogenic response in vivo.

The mdx mouse, the most widely used animal model of Duchenne muscular dystrophy (DMD), develops a seriously impaired blood-brain barrier (BBB). As glucocorticoids are used clinically to delay the progression of DMD, we evaluated the effects of chronic treatment with α-methyl-prednisolone (PDN) on the expression of structural proteins and markers in the brain and skeletal muscle of the mdx mouse. We analyzed the immunocytochemical and biochemical expression of four BBB markers, including endothelial ZO-1 and occludin, desmin in pericytes, and glial fibrillary acidic protein (GFAP) in glial cells, and the expression of the short dystrophin isoform Dp 71, the dystrophin-associated proteins (DAPs), and aquaporin-4 (AQP4) and α-β dystroglycan (DG) in the brain. We evaluated the BBB integrity of mdx and PDN-treated mdx mice by means of intravascular injection of horseradish peroxidase (HRP). The expression of DAPs was also assessed in gastrocnemius muscles and correlated with utrophin expression, and laminin content was measured in the muscle and brain. PDN treatment induced a significant increase in the mRNA and protein content of the BBB markers; a reduction in the phosphorylation of occludin in the brain and of AQP4/β DG in both tissues; an increase of Dp71 protein content; and an increase of both mRNA and protein levels of the AQP4/α-β DG complex. The latter was associated with enhanced laminin and utrophin in the muscle. The HRP assay demonstrated functional restoration of the BBB in the PDN-treated mdx mice. Specifically, mdx mice showed extensive perivascular labeling due to escape of the marker, while HRP was exclusively intravascular in the PDN-treated mice and the controls. These data illustrate for the first time that PDN reverses the BBB alterations in the mdx mouse and re-establishes the proper expression and phosphorylation of β-DG in both the BBB and skeletal muscle. Further, PDN partially protects against muscle damage. The reduction in AQP4 and occludin phosphorylation, coupled with their anchoring to glial and endothelial membranes in PDN-treated mice, suggests that the drug may target the glial and endothelial cells. Our results suggest a novel mechanism for PDN action on cerebral and muscular function, restoring the link between DAPs and the extracellular matrix, most likely through protein kinase inactivation.

Sjögren's syndrome (SS) is an autoimmune disease characterized by an inflammatory mononuclear infiltration and the destruction of epithelial cells of the lachrymal and salivary glands. The aetiology is unknown. The expression "autoimmune epithelitis" has been proposed as an alternative to SS, in view of the emerging central role of the epithelial cells in the disease pathogenesis. At the biomolecular level, the epithelial cells play an important role in triggering the autoimmune condition via antigen presentation, apoptosis, and chemokine and cytokines release. Inflammation and angiogenesis are frequently coupled in the pathological conditions associated to autoimmune diseases, and an angiogenic imbalance contributes to the pathogenesis of a number of inflammatory disorders. This work reviews the current knowledge of the molecular and cellular mechanisms underlying the pathogenesis of the inflammatory reactions that characterize SS. The literature and our data on the role of angiogenesis in the pathophysiology of the disease are discussed.

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 α-β-dystroglycan (DG), α-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/α-β-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 α-β-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 chemokine GRO-α and its receptor CXCR2 are associated with the chronic inflammation in Sjögren's syndrome (SS). To better understand the molecular mechanisms by which the GRO-α/CXCR2 system is involved in the SS inflammatory condition, our studies were designed to clarify the role of ADAM17 activation in the modulation of the GRO-α/CXCR2 chemokine system in epithelial cells (SGEC) from SS salivary glands. The CXCR2 overexpression observed in SS SGEC was dramatically decreased by ADAM17 inhibitor TAPI-1. In addition, comparing the expression levels of ADAM17 in healthy SGEC in presence or not of GRO-α treatment, we observed that GRO-α dose-dependently influences ADAM17 activation, an effect that was inhibited by blocking the interaction of GRO-α with its CXCR2 receptor. Our data show for the first time that ADAM17 has an important role in GRO-α/CXCR2 system activity regulation, suggesting that regulating CXCR2/ADAM17 interaction could be an attractive therapeutic target in SS.

Background: The transcription factor nuclear factor-κB (NF-κB) pathway has long been considered a prototypical pro-inflammatory signalling pathway, largely based on the role of NF-κB in the expression of pro-inflammatory genes including cytokines, chemokines, and adhesion molecules. The NF-κB pathway does indeed regulate pro-inflammatory cytokine production, leukocyte recruitment, or cell survival, which are important contributors to the inflammatory response during the development of autoimmune diseases. Although aberrant regulation in the NF-κB signal transduction pathway involving the inhibitory IκBa was observed in several autoimmune disorders, thereis presently nopublication investigating the alteration of IκBα expression in human salivary gland epithelial cells (SGEC) from SS patients. Objectives: In this study we examine the expression of the NF-κB inhibitory protein termed IκBα in SGEC comparing it with SGEC from healthy controls, to test the hypothesis that an altered expression of IκBα occurs in SGEC from SS biopsies. Methods: RT-PCR, Real Time-PCR, western blot, immunohistochemistry and flow cytometry were employed to examine the expression IκBα in SS biopsies in comparison with salivary gland biopsies from healthy subjects. Results: Changes in the levels of IκBαin SS SGEC in comparison with healthy subjects were demonstrated, suggesting that the attenuated expression of IκBα could contribute to the deregulation of NF-κB pathways in SS. Given its key function in the fine-tuning of NF-κB signalling, it was to be expected that defects in IκBα expression or function could lead to chronic inflammation and tissue damage. In line with gene and protein analysis results, we delineated the changes in IκBα both at gene and protein expression levels, finding a clear reduction of IκBα in tissue samples from active Sjögren's syndrome patients in comparison with healthy subjects. In biopsy specimens, we found a moderate positive staining for the IκBα protein in healthy controls examined in theimmunohistochemical study, located in the cytoplasm of acini and ductal cells. Conversely, SS salivary gland biopsies elicited a weak cytoplasmic positivity for IκBα. Conclusions: The analysis of IκBα expression at salivary gland epithelial cell level could be a potential new hallmark of SS progression and sustain a rationale to more deeply investigate the therapeutic potential of specific NF-κB inhibitors in SS

An altered metabolism is involved in the development of clear cell - renal cell carcinoma (ccRCC), and in this tumor many altered genes play a fundamental role in controlling cell metabolic activities. We delineated a large-scale metabolomic profile of human ccRCC, and integrated it with transcriptomic data to connect the variations in cancer metabolism with gene expression changes. Moreover, to better analyze the specific contribution of metabolic gene alterations potentially associated with tumorigenesis and tumor progression, we evaluated the transcription profile of primary renal tumor cells. Untargeted metabolomic analysis revealed a signature of an increased glucose uptake and utilization in ccRCC. In addition, metabolites related to pentose phosphate pathway were also altered in the tumor samples in association with changes in Krebs cycle intermediates and related metabolites. We identified NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) as the most highly expressed gene in renal cancer cells and evaluated its role in sustaining angiogenesis, chemoresistance, and mitochondrial dysfunction. Finally, we showed that silencing of NDUFA4L2 affects cell viability, increases mitochondrial mass, and induces ROS generation in hypoxia.

The aim of this study was to evaluate the role of macrophages and mast cells and of microvascular density in atherosclerotic plaques collected from 63 consecutive symptomatic and asymptomatic patients undergoing carotid endarterectomy for carotid disease. Results have shown no statistically significant differences between the two groups as concerns: (i) the degree of stenosis; (ii) the extention of the lipidic core; (iii) the thickness of the fibrous cup; (iv) the inflammatory infiltrate; (v) the degree of calcification; (vi) the intraplaque hemorrhage. Otherwise, statistically significant difference was found in microvascular density, in the number of CD68-positive macrophages and tryptase-positive mast cells in plaques from symptomatic patients, as compared to asymptomatic patients. Overall, this study indicate that although advanced symptomatic and asymptomatic carotid plaques present similar histomorphological characteristics, the degree of macrophage and mast cell infiltration and differences in microvascular density could help to discriminate between symptomatic and asymptomatic patients.

Vasculitides are characterized by inflammation and necrosis of blood vessels leading to vessel occlusion and ischemic damages of tissues. Among the inflammatory cells involved in vasculitides, neutrophils, T cells, and macrophages have been identified as the predominant cell type. This review article is focused on the role of mast cells in these chronic inflammatory processes. Mast cells are characterized by their complex plasticity. Increasing evidences document that mast cells exert both pro- and anti-inflammatory functions depending on the cell types and the microenvironment they reside in. In this context, mast cell mediators able to modulate progression of vasculitides at different levels and the anatomic localization of mast cells in different vasculitides will be described. Finally, therapeutic approach including inhibition of recruitment of mast cells to the inflammatory infiltrate and blockade of their proinflammatory effects and proangiogenic functions as potential new targets for the treatment of these diseases will be discussed.

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.

Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of Non-Hodgkin lymphoma characterized by clinical and biological heterogeneity attributable both to the tumor cells and the complex tumor-microenvironment surrounding them. Tumor-associated macrophages (TAMs) and mast cells are two major components of the tumor inflammatory infiltrate with a definite role in enhancing tumor angiogenesis. In this study, we have investigated CD68 and tryptase expression and their relationship with microvascular density (MVD) in chemo-resistant and chemosensitive patients affected by DLBCL. CD68 and tryptase expression as well as MVD were increased in chemo-resistant patients when compared with chemosensitive patients. Tryptase expression showed a positive correlation with MVD, supporting a role for mast cell in DLBCL tumor angiogenesis, while CD68 correlation with MVD was not significant, indicating a different role for TAMs than angiogenesis in DLBCL.

Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in Sjögren's syndrome (SS) remains to be elucidated. Previous SS studies have demonstrated an increase in VEGF-A/VEGFR-2 system expression in minor salivary gland (MSG) biopsies from patients with SS, but differences in the new blood vessel formation between the different grades of disease severity have not been reported. Therefore, experiments were performed to demonstrate angiogenesis during different phases of primary SS (pSS) and to define the relationship between the microvessel density (MVD), macrophage infiltration and histiocyte distribution in SS MSG inflammatory lesions. In this series of experiments, immunohistochemistry was used to examine angiogenesis in serial sections of pSS MSG. Patients with pSS were classified accordingly with the grade of inflammatory lesions as I = low-grade (low focus score of 1 or 2), II = intermediate-grade (focus score of 3–6) and III = extensive inflammation in the MSG (high focus score of 12). Histological examination demonstrated that the MVD increased with the severity of the inflammatory lesions, and in addition, we found an increased infiltration of inflammatory and pro-angiogenic cells.These findings reveal that angiogenesis is intimately involved in the progression of pSS, may be central to the propagation of the chronic immune response observed in pSS and could represent a novel potential biomarker of pSS disease activity.

Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of Sjögren's syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sjögren's syndrome.

Glucocorticoids are clinically used in Duchenne muscular dystrophy although their mechanism of action is largely unclear. Part of their effect in dystrophic muscle can be mediated by the enhancement of utrophin expression; however the impact of this increase on dystrophin–glycoprotein complex (DGC) is unknown. In the present work we assessed the effect of a chronic treatment with α-methyl-prednisolone (PDN) (4–6 weeks at 1 mg/kg day) in the exercised mdx mouse model, on the expression of utrophin and key proteins of the DGC in skeletal muscle. We found a significant increase in expression of utrophin, α and β dystroglycan and DGC-bound aquaporin (AQP)-4 at both mRNA and protein level in PDN-treated muscles. Interestingly, this was paralleled by a normalization of phosphorylation state of β-dystroglycan and an increase in laminin. Immunohistochemistry supported the correct sarcolemmal localization of the proteins. On pathology-related signs, PDN increased mdx mouse force in vivo, while ex vivo it ameliorated calcium homeostasis and reduced signs of myofiber degeneration; however plasma creatine kinase and susceptibility to eccentric contraction were not ameliorated. In parallel we also assessed the impact of PDN treatment on blood–brain barrier (BBB), based on its serious impairment in mdx mice and on the clinical use of glucocorticoids in disorders characterized by leaky BBB. Interestingly, we found a restoration of key BBB markers at level of endothelium (ZO-1 and occludin), pericytes (desmin) and glial cells (glial fibrillary acidic protein, GFAP) in PDN-treated mdx mice. In parallel, an increase of mRNA and protein content of DGC (α–β dystroglycan complex Dp 71, AQP4), and a rescue of the phosphorylation state of AQP4 and β dystroglycan were found. Then PDN exerts a positive action on DGC in both muscle and brain, disclosing a novel mechanism of action whose impact on therapeutic effect deserves to be better investigated (Supported by Duchenne Parent Project NL).

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor that binds with high affinity and selectivity to fibroblast growth factor-2 (FGF2), thus inhibiting its pro-angiogenic activity. Here we investigated the effects of PTX3 on monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patient-derived bone marrow (BM) plasma cells (PCs), endothelial cells (ECs), and fibroblasts (FBs), and assessed whether PTX3 can modulate the cross-talk between PCs and those microenvironment cells. PTX3 and FGF2 expression was evaluated by ELISA. Functional studies, including cell viability, wound healing, chemotaxis, and Matrigel(®) assays, were performed on MGUS and MM ECs and FBs upon the PTX3 treatment. Through western blot PTX3-induced modulation in FGF2/FGF receptor signalling pathways was evaluated in MGUS and MM ECs and FBs through western blot. Co-cultures between MM ECs/FBs and human PC lines were used to evaluate possible PTX3 indirect effects on MM PCs. Adhesion molecules were studied by flow cytometry. PTX3 provides a direct time- and dose-dependent apoptotic effect on MM ECs and FBs, but not on either MM primary PCs or human PC lines. PTX3 inhibits migration of MM ECs and FBs in a dose-dependent manner, and impacts in vitro and in vivo FGF2-mediated MM angiogenesis. Co-cultures of PCs and ECs/FBs show that PTX3 treatment indirectly impairs PC viability and adhesion. We conclude that PTX3 is an anti-angiogenic factor in MM and behaves as a cytotoxic molecule on MM cells by inhibiting the cross-talk between PCs and ECs/FBs

We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren's syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

With the aim of elucidating the relationship between Stat3 expression and tumor vessels abnormalities in the PCNLs, in this study we evaluated Stat3 and pStat3 expression by Real-time PCR and by immunohistochemistry in biopsy sections from PCNSL patients. Correlations of the expression levels with the presence of aberrant vessels were analyzed by confocal laser microscopy analysis, using FVIII as endothelial cell marker, CD133 and nestin as cancer stem cell (CSC) marker, CD20 as tumor cell marker, and Stat3. In addition, we investigated Stat3 mutations in lymphoma cells to clarify the role of the constitutive expression of Stat3 and of its phosphorylated forms. Results showed that in PCNSL, putative endothelial cells lining the vessels are heterogeneous, expressing FVIII/ pStat3/CD133 (presumably originally they are vascular progenitor cells), as well as FVIII/CD20/CD133 (presumably originally they are tumor cells). Finally, we detected a fraction of the FVIII+ endothelial cell that co-expressed Stat3 bearing a tetraploid karyotype, while no amplification signal for the Stat3 gene was detected.

Diffuse large B cell lymphoma (DLBCL) is recognized as the most common form of non-Hodgkin lymphoma (NHL), accounting for about 40 % of all cases of NHL. Among the cellular components of the tumor inflammatory infiltrate, T cells and mast cells have been demonstrated to be correlated with tumor angiogenesis. In this report, we have investigated CD3 and tryptase expression and their relationship with microvascular density (MVD) in DLBCL patients. Moreover, we determined the significance of CD3 expression in bulky and non-bulky disease. CD3 expression was significantly lower in bulky disease patients when compared to non-bulky ones. CD3 showed a positive correlation with tryptase and MVD, while multiple regression analysis efficaciously predicted MVD depending on CD3 and tryptase as predictors, supporting a complex interplay between these cells in sustaining tumor angiogenesis in DLBCL patients.

The growth, survival and proliferation of cancer cells are guaranteed by a crosstalk between cancer cells themselves and surrounding host cells and extracellular matrix. An intense area of research has contributed to a better understanding of the pathophysiological modification of tumor progression, e.g., the role of microenvironment. Multiple Myeloma (MM) is a malignancy of immunoglobulin-synthesizing plasma cells with symptoms mainly related to imbalance of bone homeostasis, kidney damage, anemia, impaired humoral immunity, and sometimes nervous system dysfunctions. Plasma cells home and expand in the bone marrow where cause an unbalanced bone remodelling with increased bone resorption and low bone formation that represent the typical feature in the majority of patients. MM plasma cells are thought to be responsible for the osteolytic bone lesions, which occur by increased osteoclast formation/activity and inhibition of osteoblast formation/differentiation. In physiological conditions, this process is critically regulated by the transcription factor Runx2 and by the Wnt signaling pathway. Moreover, MM plasma cells accelerate the differentiation of resident macrophages to osteoclasts. Finally, plasma cells themselves can transdifferentiate to functional osteoclasts. Another relevant aspect of the interactions of MM plasma cells with stromal cells in the bone marrow microenvironment is neovascularization, a constant hallmark of disease progression. MM plasma cells induce angiogenesis both directly, via their own factors (vascular endothelial growth factor [VEGF], fibroblast growth factor-2 [FGF-2], hepatocyte growth factor [HGF] and metalloproteinases), and indirectly via recruitment and activation of stromal inflammatory cells to secrete their own angiogenic factors. Macrophages and mast cells play an important role in this sense. They are recruited and activated by tumor plasma cells through the secretion of FGF-2, interleukin-8 (IL-8) and chemokines, such as ITAC, Mig, IP-10. When macrophages and mast cells are activated they secrete potent angiogenic factors (FGF-2, VEGF, granulocyte-colony stimulating factor [G-CSF], granulocyte macrophage-colony stimulating factor [GM-CSF]), which contribute to the tumor neovascularization. Recent evidence demonstrates the vasculogenic ability of active MM macrophages exposed to VEGF and FGF-2, the major angiogenic cytokines secreted by plasma cells, and present in the bone marrow microenvironment at four-to five-fold higher levels than in peripheral blood. Under these stimuli, bone marrow macrophages acquire endothelial cell (EC) markers and transform into cells functionally and phenotypically similar to paired bone marrow ECs (MM patient-derived endothelial cells, MMECs). So they generate capillary-like networks mimicking those of MMECs. Thus, MM macrophages contribute to build the neovessel wall via a “vasculogenic mimicry”, hence helping MM progression by this way.

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.

Background The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1. Results The examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2) and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively). The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons. Conclusion The present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins. --------------------------------------------------------------------------------

Gastric cancer is the fifth most common cancer and third leading cause of cancer-related death worldwide. Several studies on angiogenic blocking agents in gastric cancer revealing promising results by the use of monoclonal antibodies against VEGFA or its receptor VEGFR2 or against VEGFA activating pathway. The validation of biomarkers useful to better organize the clinical trials involving anti-angiogenic therapies is crucial. Molecular markers such as RNA are increasingly used for cancer diagnosis, prognosis, and therapy guidance as in the case of the targeted therapies concerning the inhibition of angiogenesis. The aim of this study is to set the conditions for evaluating the expression of VEGFA and VEGFR2 in gastric cancer specimens and in healthy gastric mucosa by the use of RNAscope, a novel RNA in situ hybridization (ISH) method that allows the visualization of a specific gene expression in individual cells. We found the increased expression of VEGFA in the tubular glands and VEGFR2 in the endothelium of gastric cancer samples mainly in the T2, T3 and T4 stages of tumor progression as compared to the healthy controls. These results obtained by the application of this highly sensitive method for oligonucleotide detection the role of angiogenesis in gastric cancer progression already highlighted by conventional immunohistochemical methods, and offer significant promise as a new platform for developing and implementing RNA-based molecular diagnostics also in the conditions in which immunohistochemistry is not applicable.