Nausicaa Clemente

Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo

Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases.

Osteopontin Bridging Innate and Adaptive Immunity in Autoimmune Diseases

Osteopontin (OPN) regulates the immune response at multiple levels. Physiologically, it regulates the host response to infections by driving T helper (Th) polarization and acting on both innate and adaptive immunity; pathologically, it contributes to the development of immune-mediated and inflammatory diseases. In some cases, the mechanisms of these effects have been described, but many aspects of the OPN function remain elusive. This is in part ascribable to the fact that OPN is a complex molecule with several posttranslational modifications and it may act as either an immobilized protein of the extracellular matrix or a soluble cytokine or an intracytoplasmic molecule by binding to a wide variety of molecules including crystals of calcium phosphate, several cell surface receptors, and intracytoplasmic molecules. This review describes the OPN structure, isoforms, and functions and its role in regulating the crosstalk between innate and adaptive immunity in autoimmune diseases.

Enhanced cytotoxic effect of camptothecin nanosponges in anaplastic thyroid cancer cells in vitro and in vivo on orthotopic xenograft tumors

Abstract Anaplastic carcinoma of the thyroid (ATC) is a lethal human malignant cancer with median survival of 6 months. To date, no treatment has substantially changed its course, which makes urgent need for the development of novel drugs or novel formulations for drug delivery. Nanomedicine has enormous potential to improve the accuracy of cancer therapy by enhancing availability and stability, decreasing effective doses and reducing side effects of drugs. Camptothecin (CPT) is an inhibitor of DNA topoisomerase-I with several anticancer properties but has poor solubility and a high degradation rate. Previously, we reported that CPT encapsulated in β-cyclodextrin-nanosponges (CN-CPT) increased solubility, was protected from degradation and inhibited the growth of prostate tumor cells both in vitro and in vivo. The aim of this study was to extend that work by assessing the CN-CPT effectiveness on ATC both in vitro and in vivo. Results showed that CN-CPT significantly inhibited viability, clonogenic capacity and cell-cycle progression of ATC cell lines showing a faster and enhanced effect compared to free CPT. Moreover, CN-CPT inhibited tumor cell adhesion to vascular endothelial cells, migration, secretion of pro-angiogenic factors (IL-8 and VEGF-α), expression of β-PIX, belonging to the Rho family activators, and phosphorylation of the Erk1/2 MAPK. Finally, CN-CPT significantly inhibited the growth, the metastatization and the vascularization of orthotopic ATC xenografts in SCID/beige mice without apparent toxic effects in vivo. This work extends the previous insight showing that β-cyclodextrin-nanosponges are a promising tool for the treatment of ATC.

Triggering of B7h by the ICOS modulates maturation and migration of monocyte-derived dendritic cells

B7h, expressed by several cell types, binds ICOS expressed by activated T cells. We have previously shown that B7h triggering by ICOS-Fc inhibits human endothelial cell adhesiveness. This work investigated the effect of ICOS-Fc on human monocyte-derived dendritic cells (DCs). We found that DCs matured with LPS in the presence of ICOS-Fc (mDCsICOS) produced greater amounts of IL-23 and IL-10, and promoted a higher secretion of IL-17A and IL-17F in MLCs than did those DCs matured with LPS alone (mDCs). Moreover, mDCsICOS pulsed with the keyhole limpet hemocyanin Ag during the maturation phase were better stimulators of Ag-specific MHC class I–, but not class II–restricted T cells than mDCs. This was probably due to promotion of cross-presentation because it was not detected when the Flu-MA58–66 Ag was directly loaded on already matured DCs and mDCsICOS. Finally, ICOS-Fc inhibited the adhesion of both immature DCs and mDCs to vascular and lymphoid endothelial cells, their migratory activity, and the expression of the Rac-1 activator β-Pix involved in cell motility. These data suggest that B7h stimulation modulates DC function with effects on their maturation and recruitment into tissues. This opens a novel view on the use of interactors of the ICOS:B7h system as immunomodulatory drugs.

Decreased function of Fas and variations of the perforin gene in adult patients with primary immune thrombocytopenia

A defective switching off of the immune response is involved in several autoimmune diseases. This switching off involves Fas‐mediated apoptosis, perforin‐mediated fratricide of activated lymphocytes, and the suppressive activity of regulatory T (Treg) cells. These mechanisms are altered in autoimmune lymphoproliferative syndrome that often displays autoimmune thrombocytopenia. The aim of this research was to evaluate these mechanisms in adult patients with primary immune thrombocytopenia (ITP), compared with healthy controls. The results show that a substantial subgroup of the ITP patients displayed a defective Fas function; most of them displayed decreased Fas expression in T cells activated in vitro. Moreover, ITP patients displayed an increased frequency of rare missense variations of the PRF1 gene and decreased levels of Treg. Immunological analysis showed that levels of Interleukin (IL)10 and IL17 were decreased and marginal zone B cells were increased. Moreover, myeloid and plasmacytoid dendritic cells were decreased in ITP patients. In conclusion, in adult ITP patients, several mechanisms involved in shutting off the immune response are defective and several immunological parameters are dysregulated; these alterations may play a role in the clinical heterogeneity of the disease.

ICOS-Ligand Triggering Impairs Osteoclast Differentiation and Function In Vitro and In Vivo

Osteoblasts, osteocytes, and osteoclasts (OCs) are involved in the bone production and resorption, which are crucial in bone homeostasis. OC hyperactivation plays a role in the exaggerated bone resorption of diseases such as osteoporosis, rheumatoid arthritis, and osteolytic tumor metastases. This work stems from the finding that OCs can express B7h (ICOS-Ligand), which is the ligand of the ICOS T cell costimulatory molecule. Because recent reports have shown that, in endothelial, dendritic, and tumor cells, B7h triggering modulates several activities of these cells, we analyzed the effect of B7h triggering by recombinant ICOS-Fc on OC differentiation and function. The results showed that ICOS-Fc inhibits RANKL-mediated differentiation of human monocyte-derived OC-like cells (MDOCs) by inhibiting the acquirement of the OC morphology, the CD14− cathepsin K+ phenotype, and the expression of tartrate-resistant acid phosphatase, OSCAR, NFATc1, and DC-STAMP. Moreover, ICOS-Fc induces a reversible decrease in the sizes of cells and nuclei and cathepsin K expression in mature MDOCs. Finally, ICOS-Fc inhibits the osteolytic activities of MDOCs in vitro and the development of bone loss in ovariectomized or soluble RANKL-treated mice. These findings open a novel field in the pharmacological use of agonists and antagonists of the ICOS–B7h system.

Role of Anti-Osteopontin Antibodies in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis

Osteopontin (OPN) is highly expressed in demyelinating lesions in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). OPN is cleaved by thrombin into N- (OPN-N) and C-terminal (OPN-C) fragments with different ligands and functions. In EAE, administering recombinant OPN induces relapses, whereas treatment with anti-OPN antibodies ameliorates the disease. Anti-OPN autoantibodies (autoAbs) are spontaneously produced during EAE but have never been detected in MS. The aim of the study was to evaluate anti-OPN autoAbs in the serum of MS patients, correlate them with disease course, and recapitulate the human findings in EAE. We performed ELISA in the serum of 122 patients collected cross-sectionally, and 50 patients with relapsing–remitting (RR) disease collected at diagnosis and followed longitudinally for 10 years. In the cross-sectional patients, the autoAb levels were higher in the RR patients than in the primary- and secondary-progressive MS and healthy control groups, and they were highest in the initial stages of the disease. In the longitudinal group, the levels at diagnosis directly correlated with the number of relapses during the following 10 years. Moreover, in patients with active disease, who underwent disease-modifying treatments, autoAbs were higher than in untreated patients and were associated with low MS severity score. The autoAb displayed neutralizing activity and mainly recognized OPN-C rather than OPN-N. To confirm the clinical effect of these autoAbs in vivo, EAE was induced using myelin oligodendrocyte glycoprotein MOG35–55 in C57BL/6 mice pre-vaccinated with ovalbumin (OVA)-linked OPN or OVA alone. We then evaluated the titer of antibodies to OPN, the clinical scores and in vitro cytokine secretion by spleen lymphocytes. Vaccination significantly induced antibodies against OPN during EAE, decreased disease severity, and the protective effect was correlated with decreased T cell secretion of interleukin 17 and interferon-γ ex vivo. The best effect was obtained with OPN-C, which induced significantly faster and more complete remission than other OPN vaccines. In conclusion, these data suggest that production of anti-OPN autoAbs may favor remission in both MS and EAE. Novel strategies boosting their levels, such as vaccination or passive immunization, may be proposed as a future strategy in personalized MS therapy.

Solid Lipid Nanoparticles Carrying Temozolomide for Melanoma Treatment. Preliminary In Vitro and In Vivo Studies

Aim: To develop an innovative delivery system for temozolomide (TMZ) in solid lipid nanoparticles (SLN), which has been preliminarily investigated for the treatment of melanoma. Materials and Methods: SLN-TMZ was obtained through fatty acid coacervation. Its pharmacological effects were assessed and compared with free TMZ in in vitro and in vivo models of melanoma and glioblastoma. Results: Compared to the standard free TMZ, SLN-TMZ exerted larger effects, when cell proliferation of melanoma cells, and neoangiogeneis were evaluated. SLN-TMZ also inhibited growth and vascularization of B16-F10 melanoma in C57/BL6 mice, without apparent toxic effects. Conclusion: SLN could be a promising strategy for the delivery of TMZ, allowing an increased stability of the drug and thereby its employment in the treatment of aggressive malignacies.

A double blind randomized experimental study on the use of IgM-enriched polyclonal immunoglobulins in an animal model of pneumonia developing shock

BACKGROUND Patients with severe pneumonia often develop septic shock. IgM-enriched immunoglobulins have been proposed as a potential adjuvant therapy for septic shock. While in vitro data are available on the possible mechanisms of action of IgM-enriched immunoglobulins, the results of the in vivo experimental studies are non-univocal and, overall, unconvincing. We designed this double blinded randomized controlled study to test whether IgM-enriched immunoglobulins administered as rescue treatment in a pneumonia model developing shock, could either limit lung damage and/or contain systemic inflammatory response. METHODS Thirty-eight Sprague Dawley rats were ventilated with injurious ventilation for 30min to prime the lung. The rats were subsequently randomized to received intratracheal instillation of either lipopolysaccharide (LPS) (12mg/kg) or placebo followed by 3.5h of protective mechanical ventilation. IgM-enriched immunoglobulins at 25mg/h (0.5mL/h) or saline were intravenously administered in the last hour of mechanical ventilation. During the experiment, gas exchange and hemodynamic measurements were recorded. Thereafter, the animals were sacrificed, and blood and organs were stored for cytokines measurements. RESULTS Despite similar lung and hemodynamic findings, the administration of IgM-enriched immunoglobulins compared to placebo significantly modulates the inflammatory response by increasing IL-10 levels in the bloodstream and by decreasing TNF-α in bronchoalveolar lavage (BAL) fluid. Furthermore, in vitro data suggest that IgM-enriched immunoglobulins induce monocytes production of IL-10 after LPS stimulation. CONCLUSIONS In an in vivo model of pneumonia developing shock, IgM-enriched immunoglobulins administered as rescue treatment enhance the anti-inflammatory response by increasing blood levels of IL-10 and reducing TNF-α in BAL fluid.

Longitudinal Assessment of Transorbital Sonography, Visual Acuity, and Biomarkers for Inflammation and Axonal Injury in Optic Neuritis

Background and Objective To investigate the relationship between optic nerve sheath diameter, optic nerve diameter, visual acuity and osteopontin, and neurofilament heavy chain in patients with acute optic neuritis. Patients and Methods Sonographic and visual acuity assessment and biomarker measurements were executed in 23 patients with unilateral optic neuritis and in 19 sex- and age-matched healthy controls. Results ONSD was thicker on the affected side at symptom onset (median 6.3 mm; interquartile range 6.0–6.5) than after 12 months (5.3 mm; 4.9–5.6; p < 0.001) or than in controls (5.2 mm; 4.8–5.5; p < 0.001). OND was significantly increased in the affected side (3.4 mm; 2.9–3.8) compared to healthy controls (2.7 mm; 2.5–2.9; p < 0.001) and was thicker at baseline than after 12 months (2.8 mm; 2.7–3.0; p < 0.01). Visual acuity improved significantly after 12 months (1.00; 0.90–1.00) compared to onset of symptoms (0.80; 0.40–1.00; p < 0.001). OPN levels were significantly higher in patients at presentation (median 6.44 ng/ml; 2.05–10.06) compared to healthy controls (3.21 ng/ml, 1.34–4.34; p < 0.03). Concentrations of NfH were significantly higher in patients than in controls. Conclusion ONSD and OND are increased in the affected eye. OPN and NfH are elevated in patients, confirming the presence of any underlying inflammation and axonal injury.