Nanoparticle-mediated Delivery of IL-2 To T Follicular Helper Cells Protects BDF1 Mice from Lupus-like Disease.

We recently reported that poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with interleukin (IL)-2 and targeted to T cells inhibited the development of lupus-like disease in BDF1 mice by inducing functional T regulatory cells (Tregs). Here we show that the protection from disease and the extended survival of BDF1 mice provided by IL-2-loaded NPs targeted to T cells is not only due to an induction of Tregs but also contributed by an inhibition of T follicular helper (TFH) cells. These results identify a dual protective activity of IL-2 in the control of lupus autoimmunity, namely the inhibition of effector TFH cells, in addition to the previously known induction of Tregs. This newly recognized activity of IL-2 delivered by NPs can help better explain the beneficial effects of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE), and might be considered as a new strategy to slow disease progression and improve outcomes in lupus patients.

DNA Vaccination With Hsp70 Protects Against Systemic Lupus Erythematosus in (NZB × NZW)F1 Mice.

OBJECTIVE: To address whether a targeted modulation of the abnormal expression of Hsp70 and autoantibodies against this molecule in systemic lupus erythematosus can influence disease. METHODS: Lupus-prone (NZB × NZW)F1 mice that had been DNA-vaccinated with plasmids encoding Hsp70 and controls were monitored for lupus disease parameters including anti-double stranded DNA (anti-dsDNA) autoantibodies and cytokines using enzyme-linked immunosorbent assay, and for kidney function and pathology. The phenotypic and numerical changes in relevant immune cells were evaluated by flow cytometry, and cell function was assessed. RESULTS: Mice that had been DNA-vaccinated with Hsp70 displayed marked suppression of anti-dsDNA antibody production, reduced renal disease, and antiinflammatory responses that are associated with a significantly extended survival, compared to controls. These protective effects in Hsp70-vaccinated mice were associated with an induction of tolerogenic immune responses and an expansion of functional Treg cells. CONCLUSION: DNA vaccination with Hsp70 suppresses murine lupus by inducing tolerogenic immune responses and antiinflammatory immune responses associated with reduced disease manifestations and increased mouse survival.

Evidence Supporting a Paracrine Effect of IGF-1/VEGF on Human Mesenchymal Stromal Cell Commitment.

Healing of skeletal defects is strictly dependent on osteogenesis and efficient vascularization of engineered scaffolds. Insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) are both involved in these processes. The in vitro administration of IGF-1 in association with VEGF is able to modulate the osteoblastic or endothelial commitment of mesenchymal stromal cells (MSCs) of different origins (e.g. periosteum and skin). In the present study, in order to deepen a possible paracrine effect of IGF-1 and VEGF on periosteum-derived progenitor cells (PDPCs) and skin-derived MSCs (S-MSCs), a Transwell coculture approach was used. We explored the genes involved in endothelial and osteoblastic differentiation, those modulating mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K)-AKT signaling pathways as well as genes implicated in stemness (i.e. Sox2, Oct4, and Nanog). Periosteal cells, which are typically committed toward osteoblastogenesis, are driven in the direction of endothelial gene expression when influenced by S-MSCs. The latter, once influenced by PDPCs, lose their endothelial commitment and increase the expression of osteoblast-associated genes. PI3K/AKT and MAPK signaling pathways seem to be markedly involved in this behavior. Our results evidence that paracrine signals between MSCs may differently modulate their commitment in a bone microenvironment, opening stimulating viewpoints for skeletal tissue engineering strategies coupling angiogenesis and osteogenesis processes.

In vitro lifespan and senescent behaviour of human periosteal derived stem cells.

Periosteum derived progenitor cells (PDPCs) represent promising mesenchymal stem cells (MSCs) for skeletal regeneration and to test bone cell based tissue engineering strategies. Most of regenerative medicine approaches based on MSCs require a noteworthy amount of cells that must be expanded in vitro prior to their use. As culture expansion method may impact on cell behaviour, we assessed the replicative and metabolic capacity (nitric oxide production and glucose consumption), senescence hallmarks of PDPCs serially passaged as well as the expression of selected genes specifically related to early osteoblastic differentiation, bone remodelling and stemness during PDPC sequential passaging. We also scouted a Systems Biology approach to examine and elucidate the experimental results through mathematical modelling and in silico simulations. PDPC subculture led to a progressive proliferative decline but, despite this, PDPCs maintained almost constant their metabolic activity. In vitro, senescent PDPCs displayed the typical "replicative senescence" features, involving p16 and not p53 in the regulation of this phenomenon. Gene expression analysis evidenced the tendency of sub-cultured PDPCs to increase the expression of genes involved in bone resorption. The mathematical analysis of the experimental results showed a strict similarity between replicative senescence and age-related changes, enabling the definition of an in silico model mimicking PDPC behaviour in terms of nitric oxide (NO) production. The relationship between NO production and subculture passages could represent a cutting edge "replicative senescence index". Overall, our findings suggest the possibility to use early-passage PDPCs for bone regenerative approaches based on the local recruitment of stem cells, whilst the later cell passages could be a suitable in vitro tool to validate scaffolds intended for bone regeneration in elderly subjects.

Adaptive immune regulation in autoimmune diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the loss of insulin-producing pancreatic ß-cells. The pathogenesis of T1D is complex and multifactorial and involves a genetic susceptibility that predisposes to abnormal immune responses in the presence of ill-defined environmental insults to the pancreatic islets. This review discusses how adaptive immunoregulatory T cells contribute to the modulation of the development and evolution of T1D, together with potential approaches that target these cells for new therapies in the disease.

Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation.

Bone extracellular matrix (ECM) is a natural composite made of collagen and mineral hydroxyapatite (HA). Dynamic cell-ECM interactions play a critical role in regulating cell differentiation and function. Understanding the principal ECM cues promoting osteogenic differentiation would be pivotal for both bone tissue engineering and regenerative medicine. Altering the mineral content generally modifies the stiffness as well as other physicochemical cues provided by composite materials, complicating the "cause-effect" analysis of resultant cell behaviour. To isolate the contribution of mechanical cues from other HA-derived signals, we developed and characterised composite HA/gelatin scaffolds with different mineral contents along with a set of stiffness-matched HA-free gelatin scaffolds. Samples were seeded with human periosteal derived progenitor cells (PDPCs) and cultured over 7 days, analysing their resultant morphology and gene expression. Our results show that both stiffness and HA contribute to directing PDPC osteogenic differentiation, highlighting the role of stiffness in triggering the expression of osteogenic genes and of HA in accelerating the process, particularly at high concentrations.

Human Periosteal Derived Stem Cell Potential: The Impact of age.

There is a great deal of interest in the understanding of possible age-related changes in Mesenchymal Stem Cells in view of their use for regenerative medicine applications. Given to the outmost standing of periosteum in bone biology and to probe data for a cell-based therapy promoting graft osseointegration in the elderly, we tried to identify specific aging markers or pattern of expression in human periosteal precursor cells. Immunohistochemical detection of Ki67 and p53, Nitric Oxide production and qRT- PCR of a selected gene panel for osteoblastic differentiation, bone remodeling and stemness were evaluated. We confirmed that both Ki67 and p53 are noteworthy indicators of senescence in human periosteal precursor cells and their expression significantly correlate with cell NO production. Moreover, cell age affects genes involved in bone remodeling, with a significant increase in interleukin-6 mRNA expression and receptor activator of nuclear factor kappa-B ligand/osteoprotegerin ratio. The analysis of mRNAs of genes involved in pluripotency regulation and self-renewal of stem cells, evidenced changes at least in part related to bone remodeling. We believe that this is the first study taking on age-related changes in human periosteal precursor cells, and paving the way toward new regenerative medicine strategies in bone aging and/or bone metabolic diseases.

Periosteum derived stem cells for regenerative medicine proposals: Boosting current knowledge.

Periosteum is a thin fibrous layer that covers most bones. It resides in a dynamic mechanically loaded environment and provides a niche for pluripotent cells and a source for molecular factors that modulate cell behaviour. Elucidating periosteum regenerative potential has become a hot topic in orthopaedics. This review discusses the state of the art of osteochondral tissue engineering rested on periosteum derived progenitor cells (PDPCs) and suggests upcoming research directions. Periosteal cells isolation, characterization and migration in the site of injury, as well as their differentiation, are analysed. Moreover, the role of cell mechanosensing and its contribution to matrix organization, bone microarchitecture and bone stenght is examined. In this regard the role of periostin and its upregulation under mechanical stress in order to preserve PDPC survival and bone tissue integrity is contemplated. The review also summarized the role of the periosteum in the field of dentistry and maxillofacial reconstruction. The involvement of microRNAs in osteoblast differentiation and in endogenous tissue repair is explored as well. Finally the novel concept of a guided bone regeneration based on the use of periosteum itself as a smart material and the realization of constructs able to mimic the extracellular matrix features is talked out. Additionally, since periosteum can differentiate into insulin producing cells it could be a suitable source in allogenic transplantations. That innovative applications would take advantage from investigations aimed to assess PDPC immune privilege.

Characterization and cytocompatibility of an antibiotic/chitosan/cyclodextrins nanocoating on titanium implants.

A novel ciprofloxacin loaded chitosan nanoparticle-based coating onto titanium substrates has been developed and characterized to obtain an orthopaedic implant surface able to in situ release the antibiotic for the prevention of post-operative infections. Ciprofloxacin loaded chitosan nanoparticles were obtained using the combination of sulfobutyl ether-beta-cyclodextrin and gamma-cyclodextrin. The resulting nanoparticulate system was characterized by TEM, HPLC and XPS. Particle size was in the range 426-552 nm and zeta potential values were around +30 mV. This antibacterial coating was able to in vitro inhibit two nosocomial Staphylococcus aureus strains growth, with a reduction of about 20 times compared to controls. No impairment in MG63 osteoblast-like cells viability, adhesion and gene expression were detected at 48 h, 7 and 14 days of culture. Overall, the investigated coating represents a promising candidate for the development of a new antibiotic carrier for titanium implants.

Role of IGF1 and IGF1/VEGF on human mesenchymal stromal cells in bone healing: two sources and two fates.

In the repair of skeletal defects one of the major obstacles still remains an efficient vascularization of engineered scaffolds. We have examined the ability of insulin growth factor-1, alone or in association with vascular endothelial growth factor, to modulate the osteoblastic or endothelial commitment of periosteum-derived progenitor cells (PDPCs) and skin-derived multipotent stromal cells (S-MSCs). A selected gene panel for endothelial and osteoblastic differentiation as well as genes that can affect MAPK and PI3K/AKT signaling pathways were investigated. Moreover, gene expression profile of Sox2, Oct4, and Nanog transcription factors was assessed. Our results showed that under growth factor stimulation PDPCs are induced toward an osteoblastic differentiation, while S-MSCs seem to move along an endothelial phenotype. This different commitment seems to be linked to a diverse MAPK or PI3K/AKT signaling pathway activation. The analysis of genes for stemness evidenced that at least in PDPCs multipotency and differentiation could coexist. These results open interesting perspective for the development of innovative bone tissue engineering approaches based on a good network of angiogenesis and osteogenesis processes.

mRNAs and miRNAs profiling of mesenchymal stem cells derived from amniotic fluid and skin: the double face of the coin.

Mesenchymal stem cells (MSCs) can be isolated from different adult sources and, even if the minimal criteria for defining MSCs have been reported, the scientific question about the potential distinctions among MSCs derived from different sources is still open. In particular, it is debated whether MSCs of different origin have the same grade of stemness or whether the source affects their undifferentiated status. Here, we report not only the isolation and the traditional characterization of MSCs derived from amniotic fluid (AF-MSCs) and skin (S-MSCs) but also a molecular characterization based on mRNAs and miRNAs profiling. Our results show that, even if both AF- and S-MSCs are mostly regulated by the same pathways (such as Wnt, MAPK and TGF-ß), there are some important differences at the molecular level that directly affect important cellular features, such as the ability to differentiate into adipocytes. In conclusion, even if further studies are necessary to improve the knowledge about the role of each dysregulated miRNAs gene, these differences may actually strengthen the question about the importance of tissue origin.

Bioactive glass/polymer composite scaffolds mimicking bone tissue.

The aim of this work was the preparation and characterization of scaffolds with mechanical and functional properties able to regenerate bone. Porous scaffolds made of chitosan/gelatin (POL) blends containing different amounts of a bioactive glass (CEL2), as inorganic material stimulating biomineralization, were fabricated by freeze-drying. Foams with different compositions (CEL2/POL 0/100; 40/60; 70/30 wt %/wt) were prepared. Samples were crosslinked using genipin (GP) to improve mechanical strength and thermal stability. The scaffolds were characterized in terms of their stability in water, chemical structure, morphology, bioactivity, and mechanical behavior. Moreover, MG63 osteoblast-like cells and periosteal-derived stem cells were used to assess their biocompatibility. CEL2/POL samples showed interconnected pores having an average diameter ranging from 179 ± 5 µm for CEL2/POL 0/100 to 136 ± 5 µm for CEL2/POL 70/30. GP-crosslinking and the increase of CEL2 amount stabilized the composites to water solution (shown by swelling tests). In addition, the SBF soaking experiment showed a good bioactivity of the scaffold with 30 and 70 wt % CEL2. The compressive modulus increased by increasing CEL2 amount up to 2.1 ± 0.1 MPa for CEL2/POL 70/30. Dynamical mechanical analysis has evidenced that composite scaffolds at low frequencies showed an increase of storage and loss modulus with increasing frequency; furthermore, a drop of E' and E″ at 1 Hz was observed, and for higher frequencies both moduli increased again. Cells displayed a good ability to interact with the different tested scaffolds which did not modify cell metabolic activity at the analyzed points. MTT test proved only a slight difference between the two cytotypes analyzed.

Cell behaviour on bioactive polymeric coatings.

Researchers are testing different treatments to induce modifications in both chemical composition and topography of implant surface, with the aim to ameliorate bone-to-implant contacts and hence improve osseointegration processes and biomechanical properties in the short and long-term. Aim of the present research was the evaluation of MG63 osteoblast-like cells behaviour on polymeric coatings, electrosynthesized on titanium substrates, differently modified in order to improve implant performances both in terms of osseointegration and infection prevention. Cell viability data and scanning electron microscopy morphological observations were consistent with a good compatibility of modified electrosynthesized coatings and suggest the use of this procedure to produce new bioactive titanium coatings for implant surgery.

BRCA1 expression in triple negative sporadic breast cancers.

OBJECTIVE: To study how to identify patients with "triple negative" sporadic breast cancers (BCs) having BRCA1 silenced or down-regulated due to epigenetic BRCA1 inactivation. STUDY DESIGN: We selected, from our database, patients diagnosed with BC between 1995 and 2001 with tumors exhibiting the "triple negative" phenotype. "Triple positive" tumors were used as controls. BRCA1 protein expression was determined by immunohistochemistry. Methylation specific polymerase chain reaction (PCR) and bisulfite sequencing on genomic DNA were used to assess BRCA1 promoter methylation. BRCA1 m-RNA expression analysis was conducted by real-time PCR. RESULTS: Forty-four triple negative and 68 controls (triple positive) were eligible for our analysis. BRCA1 promoter methylation was present in 31.8% of triple negative and in 20.6% of triple positive cases. BRCA1 was inactivated (absent BRCA1 m-RNA expression and lack of BRCA1 protein) in 21.4% of tumors with BRCA1 promoter methylation, as compared with 6% of non-methylated ones (p = 0.0453). CONCLUSION: BRCA1 inactivation due to promoter methylation could play an important role in some sporadic BC cases. Patients with this signature could represent the basis for prospective studies aiming to compare clinical response to different drugs.

CAT25 is a mononucleotide marker to identify HNPCC patients.

Mismatch repair mutations are the cause of generalized genomic instability and are particularly evident at microsatellite loci, which is known as microsatellite instability (MSI). MSI is present in 85% to 90% of colorectal cancers and occurs in hereditary non-polyposis colorectal cancer (HNPCC). The National Cancer Institute recommends the "Bethesda panel" for MSI screening. Recently, a novel T(25) mononucleotide marker was described, termed CAT25. This microsatellite marker displays a quasi-monomorphic pattern in normal tissues. The aim of our study was to evaluate the performance of CAT25 in HNPCC patients and to compare its reliability with the results of the Bethesda panel. We tested 55 tumor tissues from HNPCC patients using both the Bethesda panel and the CAT25 mononucleotide marker. One hundred healthy blood donors were used as controls. The CAT25 microsatellite was found to be altered in all 13 colorectal cancers classified as MSI-H using the standard Bethesda panel. Colorectal tumors that showed a stable Bethesda pattern did not show altered CAT25 repeats. Additionally, CAT25 showed a monomorphic allele pattern in all tissue samples. In our series, the concordance between the Bethesda panel and CAT25 in identifying colorectal cancers with high MSI reached 100%. Our results suggest that the CAT25 microsatellite represents a sensitive and specific marker for MSI and could be, at least, included in the panel of markers for the identification of HNPCC patients.