Sitagliptin Induces Tolerogenic Human Dendritic Cells.

Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-ß, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-ß) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-ß+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

Pomegranate Peel Extract Differently Modulates Gene Expression in Gingiva-Derived Mesenchymal Stromal Cells under Physiological and Inflammatory Conditions.

Pomegranate has shown a favorable effect on gingivitis/periodontitis, but the mechanisms involved are poorly understood. The aim of this study was to test the effect of pomegranate peel extract (PoPEx) on gingiva-derived mesenchymal stromal cells (GMSCs) under physiological and inflammatory conditions. GMSC lines from healthy (H) and periodontitis (P) gingiva (n = 3 of each) were established. The lines were treated with two non-toxic concentrations of PoPEX (low-10; high-40 µg/mL), with or without additional lipopolysaccharide (LPS) stimulation. Twenty-four genes in GMSCs involved in different functions were examined using real-time polymerase chain reaction (RT-PCR). PoPEx (mostly at higher concentrations) inhibited the basal expression of IL-6, MCP-1, GRO-α, RANTES, IP-10, HIF-1α, SDF-1, and HGF but increased the expression of IL-8, TLR3, TGF-ß, TGF-ß/LAP ratio, IDO-1, and IGFB4 genes in H-GMSCs. PoPEx increased IL-6, RANTES, MMP3, and BMP2 but inhibited TLR2 and GRO-α gene expression in P-GMSCs. LPS upregulated genes for proinflammatory cytokines and chemokines, tissue regeneration/repair (MMP3, IGFBP4, HGF), and immunomodulation (IP-10, RANTES, IDO-1, TLR3, COX-2), more strongly in P-GMSCs. PoPEx also potentiated most genes' expression in LPS-stimulated P-GMSCs, including upregulation of osteoblastic genes (RUNX2, BMP2, COL1A1, and OPG), simultaneously inhibiting cell proliferation. In conclusion, the modulatory effects of PoPEx on gene expression in GMSCs are complex and dependent on applied concentrations, GMSC type, and LPS stimulation. Generally, the effect is more pronounced in inflammation-simulating conditions.

Immunomodulatory activity of dipeptidyl peptidase-4 inhibitors in immune-related diseases.

Dipeptidyl peptidase-4 (DPP-4), also known as CD26, is a 110-kDa cell surface glycoprotein with enzymatic and signal transducing activity. DPP-4/CD26 is expressed by various cells, including CD4+ and CD8+ T cells, B cells, dendritic cells, macrophages, and NK cells. DPP-4 inhibitors (DPP-4i) were introduced to clinics in 2006 as new oral antihyperglycemic drugs approved for type 2 diabetes mellitus treatment. In addition to glucose-lowering effects, emerging data, from clinical studies and their animal models, suggest that DPP-4i could display anti-inflammatory and immunomodulatory effects as well, but the molecular and immunological mechanisms of these actions are insufficiently investigated. This review focuses on the modulatory activity of DPP-4i in the immune system and the possible application of DPP-4i in other immune-related diseases in patients with or without diabetes.

Dysfunctions of Neutrophils in the Peripheral Blood of Children with Cystic Fibrosis.

Dysfunction of neutrophils in patients with cystic fibrosis (CF) is best characterized in bronchoalveolar lavage (BAL), whereas peripheral blood neutrophils are less examined, and the results are contradictory, especially in younger populations. Therefore, this work aimed to study functional and phenotypic changes in circulating neutrophils in children with CF. The study included 19 CF children (5-17 years) and 14 corresponding age-matched healthy children. Isolated neutrophils were cultured either alone or with different stimuli. Several functions were studied: apoptosis, NET-osis, phagocytosis, and production of reactive oxygen species (ROS), neutrophil elastase (NE), and 11 cytokines. In addition, the expression of 20 molecules involved in different functions of neutrophils was evaluated by using flow cytometry. CF neutrophils showed reduced apoptosis and lower production of NE and IL-18 compared to the healthy controls, whereas IL-8 was augmented. All of these functions were further potentiated after neutrophil stimulation, which included higher ROS production and the up-regulation of CD11b and IL-10 expression. NET-osis was higher only when neutrophils from moderate-severe CF were treated with Pseudomonas aeruginosa, and the process correlated with forced expiratory volume in the first second (FEV1). Phagocytosis was not significantly changed. In conclusion, circulating neutrophils from children with CF showed fewer impaired changes in phenotype than in function. Functional abnormalities, which were already present at the baseline levels in neutrophils, depended on the type of stimuli that mimicked different activation states of these cells at the site of infection.

Phosphonate-Modified Cellulose Nanocrystals Potentiate the Th1 Polarising Capacity of Monocyte-Derived Dendritic Cells via GABA-B Receptor.

Purpose: Phosphonates, like 3-AminoPropylphosphonic Acid (ApA), possess a great potential for the therapy of bone tumours, and their delivery via cellulose nanocrystals (CNCs) seems a promising approach for their increased efficacy in target tissues. However, the immunological effects of CNC-phosphonates have not been investigated thoroughly. The main aim was to examine how the modification of CNCs with phosphonate affects their immunomodulatory properties in human cells. Methods: Wood-based native (n) CNCs were modified via oxidation (ox-CNCs) and subsequent conjugation with ApA (ApA-CNCs). CNCs were characterised by atomic force microscopy (AFM) and nanoindentation. Cytotoxicity and immunomodulatory potential of CNCs were investigated in cultures of human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs)/T cells co-cultures by monitoring phenotype, cytokines production, allostimulatory and Th/Treg polarisation capacity. Results: AFM showed an increase in CNCs' thickens, elasticity modulus and hardness during the modification with ApA. When applied at non-toxic doses, nCNCs showed a tolerogenic potential upon internalisation by MoDCs, as judged by their increased capacity to up-regulate tolerogenic markers and induce regulatory T cells (Treg), especially when present during the differentiation of MoDCs. In contrast, ox- and ApA-CNCs induced oxidative stress and autophagy in MoDCs, which correlated with their stimulatory effect on the maturation of MoDCs, but also inhibition of MoDCs differentiation. ApA-CNC-treated MoDCs displayed the highest allostimulatory and Th1/CTL polarising activity in co-cultures with T cells. These effects of ApA-CNCs were mediated via GABA-B receptor-induced lowering of cAMP levels in MoDCs, and they could be blocked by GABA-B receptor inhibitor. Moreover, the Th1 polarising and allostimulatory capacity of MoDCs differentiated with ApA-CNC were largely preserved upon the maturation of MoDCs, whereas nCNC- and ox-CNC-differentiated MoDCs displayed an increased tolerogenic potential. Conclusion: The delivery of ApA via CNCs induces potent DC-mediated Th1 polarisation, which could be beneficial in their potential application in tumour therapy.

Mesenchymal Stromal Cells from Healthy and Inflamed Human Gingiva Respond Differently to Porphyromonas gingivalis.

Gingiva-Derived Mesenchymal Stromal Cells (GMSCs) have been shown to play an important role in periodontitis. However, how P. gingivalis, one of the key etiological agents of the disease, affects healthy (H)- and periodontitis (P)-GMSCs is unknown. To address this problem, we established 10 H-GMSC and 12 P-GMSC lines. No significant differences in morphology, differentiation into chondroblasts and adipocytes, expression of characteristic MSCS markers, including pericyte antigens NG2 and PDGFR, were observed between H- and P-GMSC lines. However, proliferation, cell size and osteogenic potential were higher in P-GMSCs, in contrast to their lower ability to suppress mononuclear cell proliferation. P. gingivalis up-regulated the mRNA expression of IL-6, IL-8, MCP-1, GRO-α, RANTES, TLR-2, HIF-1α, OPG, MMP-3, SDF-1, HGF and IP-10 in P-GMSCs, whereas only IL-6, MCP-1 and GRO-α were up-regulated in H-GMSCs. The expression of MCP-1, RANTES, IP-10 and HGF was significantly higher in P-GMSCs compared to H-GMSCs, but IDO1 was lower. No significant changes in the expression of TLR-3, TLR-4, TGF-ß, LAP, IGFBP4 and TIMP-1 were observed in both types of GMSCs. In conclusion, our results suggest that P-GMSCs retain their pro-inflammatory properties in culture, exhibit lower immunosuppressive potential than their healthy counterparts, and impaired regeneration-associated gene induction in culture. All these functions are potentiated significantly by P. gingivalis treatment.

Ex vivo study of IL-6 expression and function in immune cell subsets from human periapical lesions.

AIM: Even though IL-6 is a key inflammatory cytokine in periapical lesions (PLs), its function in stable periapical disease is unknown. Therefore, the aim of this study was to investigate the following: first, the ex vivo production of IL-6 by clinically different PLs; next, subsets of immune cells expressing IL-6 in PLs according to their inflammatory status and finally, modulatory effect of IL-6 on T-cell cytokine production in cell cultures. METHODOLOGY: Inflammatory cells were isolated from a total of 95 human PLs. Detection of IL-6+ cells within the myeloid and lymphoid populations was performed by multicolour flow cytometry. ELISA and FlowCytomix Microbeads Assay were used to measure cytokine levels in culture supernatants. To study the role of IL-6 in PLs, mononuclear cells (MNC) from symptomatic (Sy) or asymptomatic (Asy) PLs were treated with IL-6 or Tocilizumab, an IL-6R blocking antibody. The differences between groups were tested by unpaired t-test, Mann-Whitney or Friedman tests. RESULTS: The levels of IL-6 in PL MNC culture supernatants were significantly higher compared with total PL cells and PL granulocytes (p < .001). MNC from Sy PLs produced significantly higher levels of IL-6 than MNC from Asy PLs (p < .001). Flow cytometry analysis showed that NKT cells, CD8+ T cells and M2 macrophages (MØ), were dominant IL-6+ cells, in contrast to CD4+ T cells. However, CD8+ and CD4+ T cells contributed the most to IL-6 production. IL-6hi producing MNC cultures had higher levels of Th1 (IFN-γ), Th17 (IL-17A), Tfh (IL-21) and RANKL, whereas Th2 (IL-4) and Tregs cytokines (IL-10, TGF-ß) were lower compared with IL-6low -producing cultures. Exogenous IL-6 stimulated 17A, IL-21 and RANKL, independently of PL activation status, but decreased IFN-γ, IL-4 and IL-33 levels in IL-6hi -producing cultures. Tocilizumab increased IL-10 and TGF-ß in IL-6low -producing cultures. All differences were p < .05. CONCLUSIONS: Most immune cells from Sy PLs expressed higher levels of IL-6 compared with Asy PLs, which correlated with IL-6 production in culture. Analysis of cytokines suggested a dominant pro-inflammatory T-cell response and osteodestructive function of IL-6 in PLs judging by Th17/Tfh cell activation, Tregs inhibition and increased RANKL/OPG ratio. Excessive IL-6 production decreased Th1/Th2 responses.

The Role of Nucleases Cleaving TLR3, TLR7/8 and TLR9 Ligands, Dicer RNase and miRNA/piRNA Proteins in Functional Adaptation to the Immune Escape and Xenophagy of Prostate Cancer Tissue.

The prototypic sensors for the induction of innate and adaptive immune responses are the Toll-like receptors (TLRs). Unusually high expression of TLRs in prostate carcinoma (PC), associated with less differentiated, more aggressive and more propagating forms of PC, changed the previous paradigm about the role of TLRs strictly in immune defense system. Our data reveal an entirely novel role of nucleic acids-sensing Toll-like receptors (NA-TLRs) in functional adaptation of malignant cells for supply and digestion of surrounding metabolic substrates from dead cells as specific mechanism of cancer cells survival, by corresponding ligands accelerated degradation and purine/pyrimidine salvage pathway. The spectrophotometric measurement protocols used for the determination of the activity of RNases and DNase II have been optimized in our laboratory as well as the enzyme-linked immunosorbent method for the determination of NF-κB p65 in prostate tissue samples. The protocols used to determine Dicer RNase, AGO2, TARBP2 and PIWIL4 were based on enzyme-linked immunosorbent assay. The amount of pre-existing acid-soluble oligonucleotides was measured and expressed as coefficient of absorbance. The activities of acid DNase II and RNase T2, and the activities of nucleases cleaving TLR3, TLR7/8 and TLR9 ligands (Poly I:C, poly U and unmethylated CpG), increased several times in PC, compared to the corresponding tumor adjacent and control tissue, exerting very high sensitivity and specificity of above 90%. Consequently higher levels of hypoxanthine and NF-κB p65 were reported in PC, whereas the opposite results were observed for miRNA biogenesis enzyme (Dicer RNase), miRNA processing protein (TARB2), miRNA-induced silencing complex protein (Argonaute-AGO) and PIWI-interacting RNAs silence transposon. Considering the crucial role of purine and pyrimidine nucleotides as energy carriers, subunits of nucleic acids and nucleotide cofactors, future explorations will be aimed to design novel anti-cancer immune strategies based on a specific acid endolysosomal nuclease inhibition.

Fecal microbiota composition associates with the capacity of human peripheral blood monocytes to differentiate into immunogenic dendritic cells in vitro.

Although promising for active immunization in cancer patients, dendritic cells (DCs) vaccines generated in vitro display high inter-individual variability in their immunogenicity, which mostly limits their therapeutic efficacy. Gut microbiota composition is a key emerging factor affecting individuals' immune responses, but it is unknown how it affects the variability of donors' precursor cells to differentiate into immunogenic DCs in vitro. By analyzing gut microbiota composition in 14 healthy donors, along with the phenotype and cytokines production by monocyte-derived DCs, we found significant correlations between immunogenic properties of DC and microbiota composition. Namely, donors who had higher α-diversity of gut microbiota and higher abundance of short-chain fatty acid (SCFAs) and SCFA-producing bacteria in feces, displayed lower expression of CD1a on immature (im)DC and higher expression of ILT-3, costimulatory molecules (CD86, CD40) proinflammatory cytokines (TNF-α, IL-6, IL-8) and IL-12p70/IL-10 ratio, all of which correlated with their lower maturation potential and immunogenicity upon stimulation with LPS/IFNγ, a well-known Th1 polarizing cocktail. In contrast, imDCs generated from donors with lower α-diversity and higher abundance of Bifidobacterium and Collinsella in feces displayed higher CD1a expression and higher potential to up-regulate CD86 and CD40, increase TNF-α, IL-6, IL-8 production, and IL-12p70/IL-10 ratio upon stimulation. These results emphasize the important role of gut microbiota on the capacity of donor precursor cells to differentiate into immunogenic DCs suitable for cancer therapy, which could be harnessed for improving the actual and future DC-based cancer therapies.

Plasma-Activated Medium Potentiates the Immunogenicity of Tumor Cell Lysates for Dendritic Cell-Based Cancer Vaccines.

Autologous dendritic cells (DCs)-based vaccines are considered quite promising for cancer immunotherapy due to their exquisite potential to induce tumor antigen-specific cytotoxic T cells. However, a lack of efficient protocols for inducing immunogenic tumor antigens limits the efficacy of DC-based cancer vaccines. Here, we found that a plasma-activated medium (PAM) induces immunogenic cell death (ICD) in tumor cells but not in an immortalized L929 cell line or human peripheral blood mononuclear cells. PAM induced an accumulation of reactive oxygen species (ROS), autophagy, apoptosis, and necrosis in a concentration-dependent manner. The tumor lysates prepared after PAM treatment displayed increased immunogenicity in a model of human monocyte-derived DCs, compared to the lysates prepared by a standard freezing/thawing method. Mature DCs loaded with PAM lysates showed an increased maturation potential, as estimated by their increased expression of CD83, CD86, CD40, IL-12/IL-10 production, and attenuated PDL1 and ILT-4 expression, compared to the DCs treated with control tumor lysates. Moreover, in co-culture with allogeneic T cells, DCs loaded with PAM-lysates increased the proportion of cytotoxic IFN-γ+ granzyme A+ CD8+ T cells and IL-17A-producing T cells and preserved the Th1 response. In contrast, control tumor lysates-treated DCs increased the frequency of Th2 (CD4+IL-4+), CD4, and CD8 regulatory T cell subtypes, none of which was observed with DCs loaded with PAM-lysates. Cumulatively, these results suggest that the novel method for preparing immunogenic tumor lysates with PAM could be suitable for improved DC-based immunotherapy of cancer patients.

Fermentation characteristics of novel Coriolus versicolor and Lentinus edodes kombucha beverages and immunomodulatory potential of their polysaccharide extracts.

Medicinal mushrooms, Coriolus versicolor and Lentinus edodes are extremely attractive as nutraceuticals. Here we used fruiting bodies to prepare novel kombucha beverage. Microbiological, physicochemical and chemical properties were monitored for eleven days, while the immunological properties of kombucha polysaccharide extracts were determined in peripheral blood mononuclear cell (PBMC) cultures. FTIR analysis of polysaccharide extracts showed dominant presence of polysaccharides, in addition to phenols, lipids and proteins. C. versicolor kombucha extract displayed more complex polysaccharides, and a higher content of total polysaccharides, phenols and flavonoids compared to L. edodes kombucha extract. The extracts were not cytotoxic for PBMC in vitro up to 500 µg/ml, while immunomodulatory effects depended on their chemical compositions. The most prominent effect was on the reduction of Th2 cytokines and IL-10 in PBMC cultures. Based on these results, novel kombucha products could be recommended as functional beverages or nutraceuticals with potentially beneficial immunomodulatory effects in allergies.

Biocompatible Iron-Boron Nanoparticles Designed for Neutron Capture Therapy Guided by Magnetic Resonance Imaging.

The combination of multiple functions in a single nanoparticle (NP) represents a key advantage of nanomedicine compared to traditional medical approaches. This is well represented by radiotherapy in which the dose of ionizing radiation should be calibrated on sensitizers biodistribution. Ideally, this is possible when the drug acts both as radiation enhancer and imaging contrast agent. Here, an easy, one-step, laser-assisted synthetic procedure is used to generate iron-boron (Fe-B) NPs featuring the set of functions required to assist neutron capture therapy (NCT) with magnetic resonance imaging. The Fe-B NPs exceed by three orders of magnitude the payload of boron isotopes contained in clinical sensitizers. The Fe-B NPs have magnetic properties of interest also for magnetophoretic accumulation in tissues and magnetic hyperthermia to assist drug permeation in tissues. Besides, Fe-B NPs are biocompatible and undergo slow degradation in the lysosomal environment that facilitates in vivo clearance through the liver-spleen-kidneys pathway. Overall, the Fe-B NPs represent a new promising tool for future exploitation in magnetic resonance imaging-guided boron NCT at higher levels of efficacy and tolerability.

Immunological aspects of nanocellulose.

Cellulose is the most abundant natural polymer in the world. Nanoscale forms of cellulose, including cellulose nanofibers (CNF), cellulose nanocrystals (CNC) and bacterial nanocellulose (BC), are very attractive in industry, medicine and pharmacy. Biomedical applications of nanocellulose in tissue engineering, regenerative medicine, and controlled drug delivery are the most promising. Nanocellulose is considered a biocompatible nanomaterial and relatively safe for biomedical applications. However, more studies are needed to prove this hypothesis, especially those related to chronic exposure to nanocellulose. Besides toxicity, the response of the immune system is of particular importance in this sense. This paper provides a comprehensive and critical review of the current-state knowledge of the impact of nanocellulose on the immune system, especially on macrophages and dendritic cells (DC), as the central immunoregulatory cells, which has not been addressed in the literature sufficiently. Nanocellulose, especially CNC, can induce the inflammatory response upon the internalization by macrophages, but this reaction may be significantly modulated by introducing different functional groups on their surface. Our original results showed that nanocellulose has a potent immunotolerogenic potential. Native CNF potentiated the capacity of DC to induce conventional Tregs. When carboxyl groups were introduced on the CNF surface, the tolerogenic potential of DC was shifted towards the induction of regulatory CD8+ T cells, whereas the introduction of phosphonates on CNF surface potentiated DCs' capacity to induce both regulatory CD8+ T cells and Type 1 regulatory (Tr-1) cells. These results are extremely important when considering the application of nanocellulose in vivo, especially for tissue regeneration and wound healing.

The Effect of Stabilisation Agents on the Immunomodulatory Properties of Gold Nanoparticles Obtained by Ultrasonic Spray Pyrolysis.

Gold nanoparticles (GNPs) have been investigated extensively as drug carriers in tumour immunotherapy in combination with photothermal therapy. For this purpose, GNPs should be stabilised in biological fluids. The goal of this study was to examine how stabilisation agents influence cytotoxicity and immune response in vitro. Spherical GNPs, 20 nm in size, were prepared by ultrasonic spray pyrolysis (USP). Three types of stabilising agents were used: sodium citrate (SC), polyvinyl-pyrrolidone (PVP), and poly-ethylene glycol (PEG). Pristine, non-stabilised GNPs were used as a control. The culture models were mouse L929 cells, B16F10 melanoma cells and human peripheral blood mononuclear cells (PBMNCs), obtained from healthy donors. Control SC- and PEG-GNPs were non-cytotoxic at concentrations (range 1-100 µg/mL), in contrast to PVP-GNPs, which were cytotoxic at higher concentrations. Control GNPs inhibited the production of IFN-ϒ slightly, and augmented the production of IL-10 by PHA-stimulated PBMNC cultures. PEG-GNPs inhibited the production of pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α) and Th1-related cytokines (IFN-ϒ and IL-12p70), and increased the production of Th2 cytokines (IL-4 and IL-5). SC-PEG inhibited the production of IL-8 and IL-17A. In contrast, PVP-GNPs stimulated the production of pro-inflammatory cytokines, Th1 cytokines, and IL-17A, but also IL-10. When uptake of GNPs by monocytes/macrophages in PBMNC cultures was analysed, the ingestion of PEG- GNPs was significantly lower compared to SC- and PVP-GNPs. In conclusion, stabilisation agents modulate biocompatibility and immune response significantly, so their adequate choice for preparation of GNPs is an important factor when considering the use of GNPs for application in vivo.

Prostaglanin-E2 Potentiates the Suppressive Functions of Human Mononuclear Myeloid-Derived Suppressor Cells and Increases Their Capacity to Expand IL-10-Producing Regulatory T Cell Subsets.

Myeloid-derived suppressor cells (MDSC) emerged as major factors driving the tumor progression due to numerous immunosuppressive mechanisms they possess. Prostaglandin (PG)E2 is shown critical for the induction of MDSC and their suppressive functions in vivo, but it is poorly understood how it affects the capacity of MDSC to induce different subsets of regulatory T cells (Treg). By using a novel protocol for the generation of mononuclear (M)-MDSC, we showed that PGE2 potentiates the GM-CSF/IL-6-dependent induction of CD33+CD11b+HLA-DR-CD14+ M-MDSC in vitro. PGE2 diminished the capacity of GM-CSF/IL-6 M-MDSC to produce proinflammatory cytokines upon activation and augmented their capacity to produce IL-27, IL-33, and TGF-ß. These results correlated with an increased potential of GM-CSF/IL-6/PGE2 M-MDSC to suppress T cell proliferation, expand alloreactive Th2 cells, and reduce the development of alloreactive Th17 and cytotoxic T cells. Interestingly, GM-CSF/IL-6/PGE2 M-MDSC displayed a lower capacity to induce TGF-ß-producing FoxP3+ regulatory Treg compared to GM-CSF/IL-6 M-MDSC, as a consequence of reduced IDO-1 expression. In contrast, GM-CSF/IL-6/PGE2 M-MDSC potentiated IL-10 production by CD8+T, Th2, and particularly CD4+FoxP3- type 1 Treg, the latter of which depended on ILT3 and ILT4 expression. Cumulatively, PGE2 potentiated the suppressive phenotype and functions of GM-CSF/IL-6-induced M-MDSC and changed the mechanisms involved in Treg induction, which could be important for investigating new therapeutic strategies focused on MDSC-related effects in tumors and autoimmune diseases.

Production of Soluble Receptor Activator of Nuclear Factor Kappa-Β Ligand and Osteoprotegerin by Apical Periodontitis Cells in Culture and Their Modulation by Cytokines.

RANKL, a bone-destructive cytokine, and OPG, its osteoprotective counterpart, are expressed in periapical lesions (PLs), which represent hystopatological manifestations of apical periodontitis. However, their regulation in PLs has not been elucidated yet. Therefore, our aim was to study the production of RANKL and OPG and their modulation by pro- and anti-inflammatory cytokines in PL cell cultures. Isolated PL cells were cultured alone or with addition of TNF-α, IFN-ϒ, IL-17, IL-4, IL-10, and IL- 33, respectively. The levels of RANKL and OPG in supernatants were measured by ELISA. The proportion of CD3+ (T cells) and CD19+/CD138+ (B cells/plasma cells) within isolated PLs was determined by immunocytochemistry. The levels of RANKL were higher in cultures of symptomatic PLs compared to asymptomatic PLs and PLs with the dominance of T cells (T-type lesions) over B cells/plasma cells (B-type lesions). A higher proportion of osteodestructive processes (RANKL/OPG ratio > 1.0) were detected in symptomatic PLs. The production of RANKL was upregulated by IFN-ϒ and IL-17 and higher concentrations of IL-33. IL-10 and lower concentrations of IL-33 augmented the production of OPG. The addition of either RANKL or anti-RANKL antibody to the cultures did not modify significantly the production of OPG. In conclusion, this original PL cell culture model suggests that increased bone destruction through upregulated production of RANKL could be associated with exacerbation of inflammation in PLs with the predominance of Th1 and Th17 responses and increased secretion of IL-33. In contrast, IL-10 and lower levels of IL-33, through upregulation of OPG, may suppress osteolytic processes.

Poly (ε-caprolactone) microspheres for prolonged release of selenium nanoparticles.

Poly (ε-caprolactone) (PCL) microspheres as a carrier for sustained release of antibacterial agent, selenium nanoparticles (SeNPs), were developed. The obtained PCL/SeNPs microspheres were in the range 1-4 µm with the encapsulation efficiency of about 90%. The degradation process and release behavior of SeNPs from PCL microspheres were investigated in five different degradation media: phosphate buffer solution (PBS), a solution of lipase isolated from the porcine pancreas in PBS, 0.1 M hydrochloric acid (HCl), Pseudomonas aeruginosa PAO1 cell-free extract in PBS and implant fluid (exudate) from the subcutaneously implanted sterile polyvinyl sponges which induce a foreign-body inflammatory reaction. The samples were thoroughly characterized by SEM, TEM, FTIR, XRD, PSA, DSC, confocal microscopy, and ICP-OES techniques. Under physiological conditions at neutral pH, a very slow release of SeNPs occurred (3 and 8% in the case of PBS or PBS + lipase, respectively and after 660 days), while in the acidic environment their presence was not detected. On the other hand, the release in the medium with bacterial extract was much more pronounced, even after 24 h (13%). After 7 days, the concentration of SeNPs reached a maximum of around 30%. Also, 37% of SeNPs have been released after 11 days of incubation of PCL/SeNPs in the implant exudate. These results suggest that the release of SeNPs from PCL was triggered by Pseudomonas aeruginosa PAO1 bacterium as well as by foreign body inflammatory reaction to implant. Furthermore, PCL/SeNPs microspheres were investigated in terms of their biocompatibility. For this purpose, cytotoxicity, the formation of reactive oxygen species (ROS), and genotoxicity were evaluated on HepG2 cell line. The interaction of PCL/SeNPs with phagocytic cell line (Raw 264.7 macrophages) was monitored as well. It was found that the microspheres in investigated concentration range had no acute cytotoxic effects. Finally, SeNPs, as well as PCL/SeNPs, showed a considerable antibacterial activity against Gram-positive bacteria: Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 1228). These results suggest that PCL/SeNPs-based system could be an attractive platform for a prolonged prevention of infections accompanying implants.

Nanoaggregates of iron poly-oxo-clusters obtained by laser ablation in aqueous solution of phosphonates.

Laser ablation in liquid (LAL) emerged as a versatile technique for the synthesis of nanoparticles with various structures and compositions, although the control over products remains challenging in most cases. For instance, it is still difficult to drive the size of metal oxide crystalline domains down to the level of few atom clusters with LAL. Here we demonstrate that laser ablation of a bulk iron target in aqueous solution of phosphonates gives phosphonate-grafted iron oxo-clusters polymerized into nanoaggregates with Fe:ligand ratio of 2:1, instead of the usual nanocrystalline iron oxides. We attribute this result to the strong ability of phosphonate groups to bind iron oxide clusters and prevent their further growth into crystalline iron oxide. These laser generated poly-oxo-clusters are biocompatible and trackable by magnetic resonance imaging, providing interesting features for use in biological environments, such as nano-vehicles for iron administration. Besides, this method is promising for the generation of atom-scale metal-oxide clusters, which are ubiquitary in chemistry and of interest in biochemistry, catalysis, molecular magnetism and materials science.

Aesthetic Improvement of Undeveloped Calves After Treatment of Congenital Clubfoot Deformity.

BACKGROUND: Even when clubfoot deformity is treated in a timely manner, the consequences observed in adulthood include hypoplasia of the calf muscles, gait impairment, decreases in foot size, and it can also affect the tibial length. These consequences may have negative impacts on the patient's subjective appraisal of long-term outcomes, and can influence the patient's self-esteem in both male and female patients. OBJECTIVES: We present our experience in the treatment of undeveloped calves after surgical treatment of congenital clubfoot. METHODS: In total, 72 patients underwent corrective surgery in order to improve undeveloped calves resulting from a congenital clubfoot deformity. We used calf silicone implants in combination with fat grafting in multistaged procedures, in order to decrease complication rates and improve aesthetic outcome. RESULTS: Amongst our patients there were 54 (75%) females and 18 (25%) males. All of the patients, except one, had unilateral calf hypoplasia. The procedures were divided into several groups: (1) medial calf augmentation with silicone implants; (2) medial calf augmentation with silicone implants and fat grafting; and (3) medial and lateral calf augmentation with silicone implants and fat grafting. We had one case of a hyperpigmented scar and one case of partial scar dehiscence. There were no cases of compartment syndrome. The average follow-up period was 9.8 months. CONCLUSIONS: Calf enhancement surgery in patients with congenital clubfoot deformity is very gratifying. When combining calf implants with fat grafting in multistaged procedures, we can achieve excellent results with low complication rates.