a b s t r a c t "Inverse vaccination" refers to antigen-specific tolerogenic immunization treatments that are capable of inhibiting autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), initial trials using purified myelin antigens required repeated injections because of the rapid clearance of the antigens. This problem has been overcome by DNA-based vaccines encoding for myelin autoantigens alone or in combination with "adjuvant" molecules, such as interleukin (IL)-4 or IL-10, that support regulatory immune responses. Phase I and II clinical trials with myelin basic protein (MBP)-based DNA vaccines showed positive results in reducing magnetic resonance imaging (MRI)-measured lesions and inducing tolerance to myelin antigens in subsets of MS patients. However, DNA vaccination has potential risks that limit its use in humans. An alternative approach could be the use of protein-based inverse vaccines loaded in polymeric biodegradable lactic-glycolic acid (PLGA) nano/microparticles (NP) to obtain the sustained release of antigens and regulatory adjuvants. The aim of this work was to test the effectiveness of PLGA-NP loaded with the myelin oligodendrocyte glycoprotein (MOG) autoantigen and recombinant (r) IL-10 to inverse vaccinate mice with EAE. In vitro experiments showed that upon encapsulation in PLGA-NP, both MOG 35-55 and rIL-10 were released for several weeks into the supernatant. PLGA-NP did not display cytotoxic or proinflammatory activity and were partially endocytosed by phagocytes. In vivo experiments showed that subcutaneous prophylactic and therapeutic inverse vaccination with PLGA-NP loaded with MOG 35-55 and rIL-10 significantly ameliorated the course of EAE induced with MOG 35-55 in C57BL/6 mice. Moreover, they decreased the histopathologic lesions in the central nervous tissue and the secretion of IL-17 and interferon (IFN)-␥ induced by MOG in splenic T cells in vitro. These data suggest that subcutaneous PLGA-NP-based inverse vaccination may be an effective tool to treat autoimmune diseases.

Extracellular matrix remodeling after proatherosclerotic injury involves an increase in hyaluronan (HA) that is coupled with vascular smooth muscle cell (SMC) migration, proliferation, and with neointima formation. As such events are dependent on HA, in this study we assessed the effects on SMC behavior of 4-methylumbelliferone (4-MU). As previously described in other cell types, 4-MU reduced HA in cultures of primary human aortic SMCs (AoSMCs) as well as the cellular content of the HA precursor UDP-glucuronic acid. We found that SMCs increased UDP-glucuronyl transferase 1 enzymes, which can reduce the cellular content of UDP-glucuronic acid confirming that the availability of the UDP-sugar substrates can regulate HA synthesis. Interestingly, we reported that 4-MU reduced the transcripts coding for the three HA synthases as well as UDP glucose pyrophosphorylase and dehydrogenase. As HA synthase transcript reduction is common to other cell types, the 4-MU effect on gene expression may be considered a mechanism for HA synthesis inhibition. Moreover, we showed that 4-MU strongly inhibits AoSMCs migration, which was restored by the addition of exogenous HA indicating that the rescuing depends on the interaction of HA with its receptor CD44. Besides the decrease in HA synthesis and cell migration, 4-MU reduced AoSMCs proliferation, indicating that 4-MU may exert a vasoprotective effect.

UDP-glucose dehydrogenase (UGDH) supplies the cell with UDP-glucuronic acid (UDP-GlcUA), a precursor of glycosaminoglycan and proteoglycan synthesis. Here we reported the cloning and the characterization of the UGDH from the amphibian Xenopus laevis that is one of the model organisms for developmental biology. We found that X. laevis UGDH (xUGDH) maintained a very high degree of similarity with other known UGDH sequences both at the genomic and the protein levels. Also its kinetic parameters are similar to those of UGDH from other species. During X. laevis development, UDGH is always expressed but clearly increases its mRNA levels at the tail bud stage (i.e. 30 h post-fertilization). This result fits well with our previous observation that hyaluronan, a glycosaminoglycan that is synthesized using UDP-GlcUA and UDP-N-acetylglucosamine, is abundantly detected at this developmental stage. The expression of UGDH was found to be related to hyaluronan synthesis. In human smooth muscle cells the overexpression of xUGDH or endogenous abrogation of UGDH modulated hyaluronan synthesis specifically. Our findings were confirmed by in vivo experiments where the silencing of xUGDH in X. laevis embryos decreased glycosaminoglycan synthesis causing severe embryonic malformations because of a defective gastrulation process.

Ovarian cancer (OvCA) accounts for one of the leading causes of death from gynecologic malignancy. Despite progress in therapy improvements in OvCA, most patients develop a recurrence after first-line treatments, dependent on the tumor and non-tumor complexity/heterogeneity of the neoplasm and its surrounding tumor microenvironment (TME). The TME has gained greater attention in the design of specific therapies within the new era of immunotherapy. It is now clear that the immune contexture in OvCA, here referred as tumor immune microenvironment (TIME), acts as a crucial orchestrator of OvCA progression, thus representing a necessary target for combined therapies. Currently, several advancements of antitumor immune responses in OvCA are based on the characterization of tumor-infiltrating lymphocytes, which have been shown to correlate with a significantly improved clinical outcome. Here, we reviewed the literature on selected TIME components of OvCA, such as macrophages, neutrophils, γδ T lymphocytes, and natural killer (NK) cells; these cells can have a role in either supporting or limiting OvCA, depending on the TIME stimuli. We also reviewed and discussed the major (immune)-therapeutic approaches currently employed to target and/or potentiate macrophages, neutrophils, γδ T lymphocytes, and NK cells in the OvCA context.

The glycosaminoglycan hyaluronan (HA) modulates cell proliferation and migration, and it is involved in several human vascular pathologies including atherosclerosis and vascular restenosis. During intima layer thickening, HA increases dramatically in the neointima extracellular matrix. Aging is one of the major risk factors for the insurgence of vascular diseases, in which smooth muscle cells (SMCs) play a role by determining neointima formation through their migration and proliferation. Therefore, we established an in vitro aging model consisting of sequential passages of human aortic smooth muscle cells (AoSMCs). Comparing young and aged cells, we found that, during the aging process in vitro, HA synthesis significantly increases, as do HA synthetic enzymes (i.e. HAS2 and HAS3), the precursor synthetic enzyme (UDP-glucose dehydrogenase), and the HA receptor CD44. In aged cells, we also observed increased CD44 signaling that consisted of higher levels of phosphorylated MAP kinase ERK1/2. Further, aged AoSMCs migrated faster than young cells, and such migration could be modulated by HA, which alters the ERK1/2 phosphorylation. HA oligosaccharides of 6.8 kDa and an anti-CD44 blocking antibody prevented ERK1/2 phosphorylation and inhibited AoSMCs migration. These results indicate that, during aging, HA can modulate cell migration involving CD44-mediated signaling through ERK1/2. These data suggest that age-related HA accumulation could promote SMC migration and intima thickening during vascular neointima formation. Hyaluronan (HA)2 is a linear, unsulfated glycosaminoglycan (GAG) that is composed of repeating units of D-glucuronic acid and N-acetylglucosamine linked together through alternating ␤1,4 and ␤1,3 glycosidic bonds. The amount and the molecular weight of HA are important factors that regulate the physiopathological effects that this molecule displays on cells (1). In mammals, three specific HA synthases (HAS1, -2, and -3) and three hyaluronidases (HYAL1, 2, and PH20) regulate HA synthesis and degradation with specific biochemical properties and distributions in adult as well as in embryonic tissues (2, 3). Therefore, these enzymes have a critical role in HA metabolism and are responsible for HA balance in the extracellular matrix (ECM).Hydrated HA makes the ECM an ideal environment in which cells can move and proliferate. Moreover, HA is an important space filling molecule as is evident in the vitreous humor, the dermis and the synovial fluid of joints. Besides its chemical and mechanical properties, HA interacts with several receptors at the cellular level that specifically trigger various signal transduction responses (4). The HA receptor CD44 is expressed on the surface of most cells, including immune system cells, and it mediates cell adhesion, proliferation and migration (5). Receptor for HA-mediated motility (RHAMM) mediates cellular motility (6). Lyve-1 is the specific HA receptor of the lymphatic system although very recent evidences indicate a more complex function of this protein unrelated to HA...

Organically modified mesoporous silica nanoparticles (MSNs) containing rose bengal (RB), a xanthene dye, were successfully synthesized. RB-modified MSNs have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen ((1)O2), which is particularly promising for photodynamic therapy (PDT) applications. In vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin cancer types (SK-MEL-28) after green-light irradiation.

Low-level laser therapy (LLLT) is widely used in regenerative medicine and in dental therapy by virtue of its beneficial effects in a plethora of pathological conditions. In this study, the effect of a 980 nm diode laser on pre-osteoblasts proliferation has been evaluated, along with reactive oxygen species (ROS) production. We hypothesized that ROS were a key factor in LLLT-induced pre-osteoblasts proliferation, as it is known that ROS can induce the activation of many biological pathways, leading to cell proliferation, differentiation or apoptosis. Murine pre-osteoblasts MC3T3 cells were irradiated with different energy outputs (1-50 J) in the absence or presence of the antioxidant N-Acetyl-L-cysteine (NAC). Laser treatment, in the absence of NAC, was able to induce a fluence-dependent statistically significant increase in ROS generation, while the presence of NAC strongly inhibited it. Cell proliferation, measured after laser stimulation, was significantly increased both at low and higher energy, with a peak at 10 J in the absence of the antioxidant. On the contrary, in the presence of NAC, laser irradiation was not able to induce any cell proliferation, suggesting a crucial role of ROS in this laser-induced cell effect. These results suggest that LLLT may be a useful tool for bone regeneration therapy and an effective range of fluences to be used is indicated.

As a direct correlation between aging and the risk of onset of vascular disease has been universally accepted, we prepared an in vitro aging model consisting in sequential passages of human aortic smooth muscle cells (AoSMC) in order to evaluate the cell behavior changes during aging. Because matrix metalloproteinases (MMP) are actively involved in matrix remodeling and disease outcome, in our model we found active MMP-2 only in the conditioned medium of young AoSMCs, whereas aged cells showed only the inactive zymogen form of MMP-2 (pro-MMP-2). We ascribed the pro-MMP-2 activation in young cells to an increase in membrane type 1 matrix metalloproteinase (MT1-MMP) content. Furthermore, we found that transcripts coding for tissue inhibitor of metalloproteinases (TIMPs) were up-regulated in aged cells, and this increase of TIMPs could also prevent pro-MMP-2 activation in aged cells. Moreover, we demonstrated that young AoSMCs possess higher migratory capabilities than aged cells. The young AoSMC migration can be inhibited by adding TIMP-1 and TIMP-2 to the cells reproducing aged AoSMC migratory behavior. Finally, the role of MMP-2 and TIMP-2 in AoSMC migration was confirmed silencing MMP-2 and TIMP-2 in young and aged AoSMCs, respectively; therefore, in this study we showed that these enzymes play a pivotal role in the regulation of the AoSMC migration during in vitro aging.