Sara Paccosi

Characterization of circulating and monocyte-derived dendritic cells in obese and diabetic patients

Dendritic cells (DCs) are suspected to be involved in the development of atherogenesis, but their role is still unclear. The aim of this study was to characterize circulating DCs and monocyte-derived DCs (Mo-DCs) of obese and diabetic patients (T2D), and to study their interaction with human coronary smooth muscle cells (CASMCs). Obese post-menopausal women with or without insulin resistance were enrolled and were compared to age-matched healthy women. Myeloid circulating DCs significantly increased in obese T2D patients compared to healthy donors and a smaller increase was observed for plasmacytoid one. Mature Mo-DCs from obese T2D patients significantly decreased when compared to control, but they were significantly more capable of adhering to CASMCs compared to that from healthy controls and from not-T2D obese subjects. Altogether these data suggest that in conditions of insulin-resistance and obesity there is an up-regulation of myeloid DCs that might contribute to pathological vascular remodeling.

Pharmacologically active microcarriers for endothelial progenitor cell support and survival

The regenerative potential of endothelial progenitor cell (EPC)-based therapies is limited due to poor cell viability and minimal retention following application. Neovascularization can be improved by means of scaffolds supporting EPCs. The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured on pharmacologically active microcarriers (PAMs), made with poly(d,l-lactic-coglycolic acid) and coated with adhesion/extracellular matrix molecules. They may serve as a support for stem cells and may be used as cell carriers providing a controlled delivery of active protein such as the angiogenic factor, vascular endothelial growth factor-A (VEGF-A). eEPC adhesion to fibronectin-coated PAMs (FN-PAMs) was assessed by means of microscopic evaluation and by means of Alamar blue assay. Phospho ERK(1/2) and PARP-1 expression was measured by means of Western blot to assess the survival effects of FN-PAMs releasing VEGF-A (FN-VEGF-PAMs). The Alamar blue assay or a modified Boyden chamber assay was employed to assess proliferative or migratory capacity, respectively. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-VEGF-PAMs increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation. These data strongly support the use of PAMs for supporting eEPC growth and survival and for stimulating resident mature human endothelial cells.

Nutritionally relevant concentrations of resveratrol and hydroxytyrosol mitigate oxidative burst of human granulocytes and monocytes and the production of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages

&NA; The health benefits of bio‐active phenolic compounds have been largely investigated in vitro at concentrations which exceed those reachable in vivo. We investigated and compared the anti‐inflammatory effects of resveratrol, hydroxytyrosol and oleuropein at physiologically relevant concentrations by using in vitro models of inflammation. Human granulocytes and monocytes were stimulated with phorbol myristate acetate (PMA) and the ability of resveratrol, hydroxytyrosol and oleuropein to inhibit the oxidative burst and CD11b expression was measured. Nitric oxide (NO), prostaglandin E2 (PGE2) levels, COX‐2, iNOS, TNF&agr;, IL‐1&bgr; and miR‐146a expression and activation of the transcription factor Nrf2 were evaluated in macrophages RAW 264.7 stimulated with LPS (1 &mgr;g/ml) for 18 h, exposed to resveratrol, hydroxytyrosol and oleuropein (5 and 10 &mgr;M). Synergistic effects were explored as well, together with the levels of PGE2, COX‐2 and IL‐1&bgr; expression in macrophages after 6 h of LPS stimulation. PGE2 and COX‐2 expression were also assessed on human monocytes. All the tested compounds inhibited granulocytes oxidative burst in a concentration dependent manner and CD11b expression was also significantly counteracted by resveratrol and hydroxytyrosol. The measurement of oxidative burst in human monocytes produced similar effects being resveratrol more active. Hydroxytyrosol and resveratrol inhibited the production of NO and PGE2 but did not reduce iNOS, TNF&agr; or IL‐1&bgr; gene expression in LPS‐stimulated RAW 264.7 for 18 h. Resveratrol slightly decreased COX‐2 expression after 18 h but not after 6 h, but reduced PGE2 levels after 6 h. Resveratrol and hydroxytyrosol 10 &mgr;M induced NRf2 nuclear translocation and reduced miR‐146a expression in LPS treated RAW 264.7. Overall, we reported an anti‐inflammatory effect of resveratrol and hydroxytyrosol at low, nutritionally relevant concentrations, involving the inhibition of granulocytes and monocytes activation, the modulation of miR‐146a expression and the activation of Nrf2. A regular dietary intake of resveratrol and hydroxytyrosol may be a useful complementary strategy to control inflammatory diseases. HighlightsLow concentrations of resveratrol and hydroxytyrosol attenuate inflammation.Resveratrol and hydroxytyrosol mitigate oxidative stress.Resveratrol and hydroxytyrosol decrease PGE2 levels.miR‐146a and Nrf2 are involved in the observed anti‐inflammatory effects.

The monocyte chemotactic protein synthesis inhibitor bindarit prevents mesangial cell proliferation and extracellular matrix remodeling.

Glomerular expression of chemotactic protein-1/chemokine (C-C motif) ligand-2 (MCP-1/CCL2) correlates with the degree of renal damage, suggesting a role of this chemokine in the pathogenesis of renal diseases. Bindarit is an original indazolic derivative able to inhibit MCPs synthesis and to significantly decrease MCP-1/CCL2 urinary excretion in patients with Lupus Nephritis, in correlation with reduction in albuminuria. Aim of the present work was to elucidate the effect of MCP-1/CCL2 synthesis inhibition on in vitro models of mesangial cell dysfunction. ET1 (10nM) and AngII (10nM) significantly stimulated MCP-1/CCL2 release by human renal mesangial cells (HRMCs) after 3-12h stimulation. Bindarit (10-300 μM) significantly inhibited MCP-1/CCL2 release in response to both stimuli within 12h. Bindarit also inhibited mRNA MCP-1/CCL2 expression, confirming an effect of the drug at transcriptional level. Bindarit significantly and concentration-dependently inhibited HRMC proliferation, measured as either cell duplication or total DNA/well, and impaired mRNA collagen IV expression, collagen deposition and fibronectin expression induced by AngII and ET1. Exposure of HRMCs to bindarit also impaired MMP2 activation in response to both stimuli, measured by means of gelatin zymography. These data confirm the important role of MCP-1/CCL2 synthesis in mesangial cell dysfunction and support the potential of therapeutic intervention targeting this chemokine in kidney disease.

Development and characterization of an in vitro model of colorectal adenocarcinoma with MDR phenotype

The major cause of failure in cancer chemotherapy is the development of multidrug resistance (MDR), and the characterization of biological factors involved in this response to therapy is particularly needed. A doxorubicin‐resistant HCT‐8/R clone was selected from sensitive parental cells and characterized analyzing several parameters (cell cycle phase distribution, apoptotic activity, expression, distribution and functionality of the P‐gp efflux pump, the response to other chemotherapy agents, its ultrastructural features, invasiveness, and transcriptomic profile). HCT‐8/R cells showed a peculiar S phase distribution, characterized by a single pulse of proliferation, resistance to drug‐mediated apoptosis, increased expression and functionality of P‐gp and overexpression of stem cell markers (CD44 and aldehyde dehydrogenase 1A2). At the ultrastructural level, HCT‐8/R presented a greater cell volume and several intracytoplasmic vesicles respect to HCT‐8. Moreover, the resistant clone was characterized by cross resistance to other cytotoxic drugs and a greater capacity for migration and invasion, compared to parental cells. Our data reinforce the concept that the MDR phenotype in HCT‐8/R cells is multifactorial and involves multiple mechanisms, representing an interesting tool to understand the biological basis of MDR and to test strategies that overcome resistance to chemotherapy.

Stimulatory interactions between human coronary smooth muscle cells and dendritic cells.

Despite inflammatory and immune mechanisms participating to atherogenesis and dendritic cells (DCs) driving immune and non-immune tissue injury response, the interactions between DCs and vascular smooth muscle cells (VSMCs) possibly relevant to vascular pathology including atherogenesis are still unclear. To address this issue, immature DCs (iDCs) generated from CD14+ cells isolated from healthy donors were matured either with cytokines (mDCs), or co-cultured (ccDCs) with human coronary artery VSMCs (CASMCs) using transwell chambers. Co-culture induced DC immunophenotypical and functional maturation similar to cytokines, as demonstrated by flow cytometry and mixed lymphocyte reaction. In turn, factors from mDCs and ccDCs induced CASMC migration. MCP-1 and TNFα, secreted from DCs, and IL-6 and MCP-1, secreted from CASMCs, were primarily involved. mDCs adhesion to CASMCs was enhanced by CASMC pre-treatment with IFNγ and TNFα ICAM-1 and VCAM-1 were involved, since the expression of specific mRNAs for these molecules increased and adhesion was inhibited by neutralizing antibodies to the counter-receptors CD11c and CD18. Adhesion was also inhibited by CASMC pre-treatment with the HMG-CoA-reductase inhibitor atorvastatin and the PPARγ agonist rosiglitazone, which suggests a further mechanism for the anti-inflammatory action of these drugs. Adhesion of DCs to VSMCs was shown also in vivo in rat carotid 7 to 21 days after crush and incision injury. The findings indicate that DCs and VSMCs can interact with reciprocal stimulation, possibly leading to perpetuate inflammation and vascular wall remodelling, and that the interaction is enhanced by a cytokine-rich inflammatory environment and down-regulated by HMGCoA-reductase inhibitors and PPARγ agonists.

The chemokine (C-C motif) ligand protein synthesis inhibitor bindarit prevents cytoskeletal rearrangement and contraction of human mesangial cells

Intraglomerular mesangial cells (MCs) maintain structural and functional integrity of renal glomerular microcirculation and homeostasis of mesangial matrix. Following different types of injury, MCs change their phenotype upregulating the expression of α-smooth muscle actin (α-SMA), changing contractile abilities and increasing the production of matrix proteins, chemokines and cytokines. CCL2 is a chemokine known to be involved in the pathogenesis of renal diseases. Its glomerular upregulation correlates with the extent of renal damage. Bindarit is an indazolic derivative endowed with anti-inflammatory activity when tested in experimental diseases. It selectively inhibits the synthesis of inflammatory C-C chemokines including CCL2, CCL7 and CCL8. This work aims to analyse bindarit effects on ET1-, AngII- and TGFβ-induced mesangial cell dysfunction. Bindarit significantly reduced AngII-, ET1- and TGFβ-induced α-SMA upregulation. In a collagen contraction assay, bindarit reduced AngII-, ET1- and TGFβ-induced HRMC contraction. Within 3-6h stimulation, vinculin organization and phosphorylation was significantly impaired by bindarit in AngII-, ET1- and TGFβ-stimulated cells without any effect on F-actin distribution. Conversely, p38 phosphorylation was not significantly inhibited by bindarit. Our data strengthen the importance of CCL2 on ET-1, AngII- and TGFβ-induced mesangial cell dysfunction, adding new insights into the cellular mechanisms responsible of bindarit protective effects in human MC dysfunction.

Differences in Inflammatory Response Induced by Two Representatives of Clades of the Pandemic ST258 Klebsiella pneumoniae Clonal Lineage Producing KPC-Type Carbapenemases

ST258-K. pneumoniae (ST258-KP) strains, the most widespread multidrug-resistant hospital-acquired pathogens, belong to at least two clades differing in a 215 Kb genomic region that includes the cluster of capsule genes. To investigate the effects of the different capsular phenotype on host-pathogen interactions, we studied representatives of ST258-KP clades, KKBO-1 and KK207-1, for their ability to activate monocytes and myeloid dendritic cells from human immune competent hosts. The two ST258-KP strains strongly induced the production of inflammatory cytokines. Significant differences between the strains were found in their ability to induce the production of IL-1β: KK207-1/clade I was much less effective than KKBO-1/clade II in inducing IL-1β production by monocytes and dendritic cells. The activation of NLRP3 inflammasome pathway by live cells and/or purified capsular polysaccharides was studied in monocytes and dendritic cells. We found that glibenclamide, a NLRP3 inhibitor, inhibits more than 90% of the production of mature IL-1β induced by KKBO1 and KK207-1. KK207-1 was always less efficient compared to KKBO-1 in: a) inducing NLRP3 and pro-IL-1β gene and protein expression; b) in inducing caspase-1 activation and pro-IL-1β cleavage. Capsular composition may play a role in the differential inflammatory response induced by the ST258-KP strains since capsular polysaccharides purified from bacterial cells affect NLRP3 and pro-IL-1β gene expression through p38MAPK- and NF-κB-mediated pathways. In each of these functions, capsular polysaccharides from KK207-1 were significantly less efficient compared to those purified from KKBO-1. On the whole, our data suggest that the change in capsular phenotype may help bacterial cells of clade I to partially escape innate immune recognition and IL-1β-mediated inflammation.

Endothelial Soluble Factors Mediate Differentiation of Circulating Endothelial Precursors.

Abstract: Although endothelial progenitor cells have been used in clinical trials with promising preliminary results, the mechanism by which these cells interact with vascular wall cells and ischemic tissues remains unclear. We have previously reported that human coronary artery endothelial cells cocultured with peripheral blood mononuclear cell (PBMC) can stimulate their early differentiation toward a pre-endothelial phenotype. This study was aimed to assess possible soluble factors, released from the coculture, and involved in endothelial progenitor cell differentiation. Among cytokines and chemokines measured by means of Milliplex assay, interleukin (IL)-6, IL-8, endothelial growth factor, and CCL-2 were released in cocultures, and those levels were significantly higher than that found in human coronary artery endothelial cells or in PBMCs alone. To check their involvement in PBMC differentiation, blocking experiments with neutralizing antibodies were performed. Flow cytometry analysis confirmed an impairment of PBMC differentiation toward a pre-endothelial phenotype when IL-6, IL-8 and with a lesser extent CCL-2 were blocked. These data add a new insight into the mechanisms by which endothelial precursors interact with vascular wall, thus suggesting future directions in understanding and treating ischemic injury.

T-cell immunoglobulin mucin-3 and galectin-9 levels in peripheral blood mononuclear cells predict fetal acidemia in twin pregnancies

Abstract Purposes: The pH in the umbilical artery at delivery provides information on the fetal environment and is related to postnatal outcomes. The ability to predict fetal acidemia at delivery would improve clinical management and neonatal well-being. We hypothesized that an alteration in maternal immunity would accompany placental changes that precede a decrease in pH in the fetal circulation in twin gestations. Methods: Peripheral blood mononuclear cells (PBMCs), obtained from 86 women with twin pregnancies, were lysed and assayed for concentrations of T-cell immunoglobulin mucin domain 3 (Tim-3) and galectin-9 (gal-9) by ELISA. Tim-3–gal-9 interaction is a primary mechanism promoting immune suppression. At delivery, the pH of arterial cord blood was determined. Results: In eight women (9.3%), the pH in the placental arteries from both twins was <7.15, indicating fetal acidosis. In the remaining 78 women the arterial pH was ≥7.15 in both twins. The median Tim-3 level was 361 pg/ml when arterial pH was <7.15 and 199 pg/ml when pH was ≥7.15 (p = .003). Similarly, gal-9 was 31.2 versus 12.4 ng/ml when pH was <7.15 or ≥7.15, respectively (p = .001). A Tim-3 concentration >260 pg/ml predicted arterial pH <7.15 with a sensitivity of 87.5%, specificity of 79.5% and negative predictive value of 98.4%. A gal-9 level >18.4 predicted arterial pH <7.15 with a sensitivity of 100%, specificity of 73.8% and a negative predictive value of 100%. Conclusion: We conclude that elevations in Tim-3 and gal-9 in PBMCs during gestation predict the subsequent occurrence of a pH <7.15 in the fetal arteries at delivery in twin gestations.