Vincenzo Mitolo

Membrane Microvesicles as Actors in the Establishment of a Favorable Prostatic Tumoral Niche: A Role for Activated Fibroblasts and CX3CL1-CX3CR1 Axis

Tumor microenvironment is enriched in plasma membrane microvesicles (MV) shed from all cell types that constitute the tumor mass, reflecting the antigenic profile of the cells they originate from. Fibroblasts and tumor cells mutually communicate within tumor microenvironment. Recent evidences suggest that tumor-derived MVs (TMV) exert a broad array of biological functions in cell-to-cell communication. To elucidate their role in cancer-to-fibroblast cell communication, TMV obtained from two prostate carcinoma cell lines with high and weak metastatic potential (PC3 and LnCaP, respectively) have been characterized. TMV exhibit matrix metalloproteinases (MMP) and extracellular MMP inducer at their surface, suggesting a role in extracellular matrix degradation. Moreover, TMV not only induce the activation of fibroblasts assessed through extracellular signal-regulated kinase 1/2 phosphorylation and MMP-9 up-regulation, increase motility and resistance to apoptosis but also promote MV shedding from activated fibroblasts able in turn to increase migration and invasion of highly metastatic PC3 cells but not LnCaP cells. PC3 cell chemotaxis seems, at least partially, dependent on membrane-bound CX3CL1/fractalkine ligand for chemokine receptor CX3CR1. The present results highlight a mechanism of mutual communication attributable not only to soluble factors but also to determinants harbored by MV, possibly contributing to the constitution of a favorable niche for cancer development.

Differential Contribution of Toll-Like Receptors 4 and 2 to the Cytokine Response to Salmonella enterica Serovar Typhimurium and Staphylococcus aureus in Mice

ABSTRACT The contribution of murine Toll-like receptors 2 and 4 (TLR2 and -4, respectively) to cytokine induction by heat-killed bacteria was analyzed in vitro and in vivo. Gram-negative bacteria induced cytokines primarily via TLR4; the contribution of TLR2 was only minor. Neither TLR4 nor, surprisingly, TLR2 was required in the MyD88-dependent response to Staphylococcus aureus.

Biological Role of the N-Formyl Peptide Receptors

Ligation of N-formyl-methionyl-leucyl-phenylalanine (fMLP) to its specific cell surface receptors triggers different cascades of biochemical events, eventually leading to cellular activation. The formyl peptide receptors (FPRs) are members of the seven-transmembrane, G-protein coupled receptors superfamily, expressed at high levels on polymorphonuclear and mononuclear phagocytes. The main responses elicited upon ligation of formylated peptides, referred to as cellular activation, are those of morphological polarization, locomotion, production of reactive-oxygen species and release of proteolytic enzymes. FPRs have in recent years been shown to be expressed also in several non myelocytic populations, suggesting other unidentified functions for this receptor family, independent of the inflammatory response. Finally, a number of ligands acting as exogenous or host-derived agonists for FPRs, as well as ligands acting as FPRs antagonists, have been described, indicating that these receptors may be differentially modulated by distinct molecules.

Macrophage chemotactic protein-1 and macrophage inflammatory protein-1α induce nitric oxide release and enhance parasite killing in leishmania infantum-infected human macrophages

Abstract. Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1α significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-γ. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L-NGmonomethylarginine (L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1α mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection.

MPTP-Induced Neuroinflammation Increases the Expression of Pro-Inflammatory Cytokines and Their Receptors in Mouse Brain

Parkinson’s disease (PD) is a common neurodegenerative disease characterised by a slow and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Despite intensive research, the cause of neuronal loss in PD is poorly understood. Inflammatory mechanisms have been implicated in the pathophysiology of PD. In this study, conducted on an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we investigated the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and their receptors (IL-1RI, TNF-αRI, IL-6Rα) at the SN and caudate-putamen (CP) levels. In MPTP-treated animals we observed a significant increase in IL-1β, TNF-α and IL-6 mRNA expression levels both in the SN and CP in comparison with untreated mice. In addition, both mRNA and protein levels of IL-1RI, TNF-αRI and IL-6Rα were significantly enhanced in the SN of MPTP-treated mice in comparison to controls, whereas no significant differences were observed in the CP between treated and untreated mice. Overall, these results indicate a role of both pro-inflammatory cytokines and their receptors in the pathogenesis of PD.

Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.

Expression of TLR4 and CD14 in the Central Nervous System (CNS) in a MPTP Mouse Model of Parkinson's-Like Disease

Systemic infections are often associated with neurodegenerative processes in many diseases of the central nervous system (CNS), including Parkinsons disease. Toll-like receptor (TLR)4 and CD14 act as receptors for lipopolysaccharide (LPS) released by gram-negative bacteria. In this contest, CD14 functions as the main LPS ligand and TLR4 transmits the LPS signal into the cell. In this paper, we investigated the expression of TLR4 and CD14, in different anatomical areas of the CNS, in an experimental model of Parkinsons-like disease, represented by MPTP-treated mouse. In particular, we analyzed the gene transcripts and proteins expression of CD14 and TLR4, in the substantia nigra and caudate-putamen nuclei of these animals. Results demonstrated an augmented expression of both CD14 and TLR4 in the substantia nigra of mice treated with MPTP in comparison to untreated animals, suggesting that the endotoxin receptors are over expressed in different manner in specific areas of the CNS during Parkinsons-like disease.

Infection with Leishmania infantum Inhibits Actinomycin D‐Induced Apoptosis of Human Monocytic Cell Line U‐937

Abstract. Modulation of host cell apoptosis has been observed in many bacterial, protozoal, and viral infections. The aim of this work was to investigate the effect of viscerotropic Leishmania (L.) infantum infection on actinomycin D‐induced apoptosis of the human monocytic cell line U‐937. Cells were infected with L. infantum promastigotes or treated with the surface molecule lipophosphoglycan (LPG) or with parasite‐free supernatant of Leishmania culture medium and submitted to action of actinomycin D as the apoptosis‐inducing agent. Actinomycin D‐induced apoptosis in U‐937 cells was inhibited in the presence of both viable L. infantum promastigotes and soluble factors contained in Leishmania culture medium or purified LPG. Leishmania infantum affected the survival of U‐937 cells via a mechanism involving inhibition of caspase‐3 activation. Furthermore, protein kinase C δ (PKC δ) cleavage was increased in actinomycin D‐treated U‐937 cells and was inhibited by the addition of LPG. Thus, inhibition of the PKC‐mediated pathways by LPG can be implicated in the enhanced survival of the parasites.

Evidences for iNOS expression and nitric oxide production in the human macrophages.

Nitric oxide (NO) is a pleiotropic mediator of numerous biological processes, including smooth muscle relaxation, neurotransmission and defence against pathogens. In addition, NO is involved in the pathogenesis and control of inflammation, tumors, autoimmunity, and infectious and chronic degenerative diseases. NO, a highly reactive radical, is produced from L-arginine and oxygen by the enzyme NO synthase (NOS). Three NOS isoforms have been identified: two distinct NOS isoforms are constitutively expressed in cells, whereas a third isoform, inducible NOS (iNOS), is transcribed in response to specific stimuli. In particular, iNOS is responsible for the discontinuous synthesis of high amounts of NO and was originally characterized in murine macrophages after exposure to cytokines and/or microbial products. A wide range of microorganisms is sensibly inhibited in its development by NO, like fungi, bacteria, protozoa and viruses. Although NO production and its antimicrobial effect appear well established in rodent macrophages, the existence of L-arginine pathway in human mononuclear phagocytes has long been disputed. Recently, evidences showing the iNOS activity and NO production in other animal models, including humans, are now emerging, even if the NO induction has been more difficult to demonstrate. The present observations provide evidence for the occurrence of iNOS protein expression and NO production in human macrophages cultured in vitro.

Tumor Necrosis Factor Inhibitors Block Apoptosis of Human Epithelial Cells of the Salivary Glands

Inhibition of tumor necrosis factor‐α (TNF‐α) in organ‐specific autoimmune disease is proving efficacious for a large number of patients. A wide array of biological agents has been designed to inhibit TNF‐α, such as adalimumab (fully humanized) and etanercept (soluble TNF‐α receptor fusion constructs p75 subunit). Recently, we suggested that anti‐Ro and anti‐La autoantibodies (Abs) isolated from patients with Sjögrens syndrome, an autoimmune rheumatic disease, are able to trigger cell death through extrinsic apoptotic mechanisms in human salivary gland epithelial cells (SGEC). We analyzed if primary human SGEC cultures, established from biopsy of labial minor salivary glands, are able to produce TNF‐α, an inductor of the extrinsic apoptotic pathway, when treated with anti‐Ro autoantibodies. A comparative study was performed to test the efficacy of adalimumab and etanercept to block TNF‐α‐mediated apoptosis. ELISA assay and RT‐PCR were employed to visualize TNF‐α production, and apoptosis was evaluated by DNA ladder and flow cytometry. We found that cell treatment with anti‐Ro autoantibodies determines TNF‐α production that reaches a maximum at 16 h and is decreased (P < 0.05) at 24 and 48 h. Adalimumab seems to be more efficacious than etanercept in blocking TNF‐α‐mediated apoptosis. The YOPRO‐1 (+) and propidium iodide (−) method revealed 60% of apoptotic cells after 24 h of incubation with anti‐Ro compared with 15% of apoptotic cells treated with anti‐Ro plus adalimumab and 25% of apoptotic cells treated with anti‐Ro plus etanercept. The antiapoptotic effect of adalimumab and etanercept was supported by inhibition of DNA laddering induced by anti‐Ro Abs. These data validate the therapeutic efficacy of the anti‐TNF reagents in the treatment of autoimmune disorders.