Predictive Value of Plasma MicroRNA-216a/b in the Diagnosis of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a common human malignancy with poor survival, which was usually diagnosed at an advanced stage. MicroRNAs (miRNAs), a class of single stranded noncoding RNAs with only 17-25 ribonucleotides, were demonstrated to play an important role in lots of cancers. In the recent years, increasing evidence revealed that circulating miRNAs exhibited great potential in the diagnosis of various types of cancers. The present study was designed to evaluate the diagnostic value of plasma miRNA-216a/b for ESCC. Our results showed that the expression level of plasma miRNA-216a/b was significantly lower in ESCC patients compared with that of healthy controls. The receiver operating characteristic (ROC) curve analysis yielded an area under the ROC curve (AUC) value of 0.877 [95% CI (confidence interval): 0.818-0.922] for miRNA-216a and 0.756 (95% CI: 0.685-0.819) for miRNA-216b. Clinical data indicated that plasma miRNA-216a/b were inversely correlated with lymph node metastasis and TNM stage. Additionally, the plasma miRNA-216b expression level was significantly upregulated in postoperative samples compared to preoperative samples. Our study, for the first time, demonstrated that plasma miRNA-216a/b might serve as potential biomarkers for the diagnosis of ESCC and dysregulation of miRNA-216a/b might be involved in the progression of ESCC

Metabolic Disturbance and Th17/Treg Imbalance Are Associated With Progression of Gingivitis

ObjectiveThis study sought to explore the role of metabolic disturbance in immunoregulation of gingivitis targeting T helper 17 cells (Th17)/regulatory T cell (Treg).Materials and MethodsA total of 20 gingivitis patients and 19 healthy volunteers were recruited. Quantitative real time polymerase chain reaction (qRT-PCR) was used to evaluate expression patterns of Forkhead box protein P3 (Foxp3), transforming growth factor-β (TGF-β), retinoid-related orphan receptor-gammat (RORγt) and interleukin 17A (IL-17A) in the peripheral blood lymphocytes of subjects across the two groups. Moreover, the enzyme-linked immunosorbent assay (ELISA) technique was used to detect levels of TGF-β, IL-4, IL-6,TL-10 and L-17A secreted in the plasma as well as the SIgA secreted in saliva. Flow cytometry was used to detect the percentage of CD4+CD25+ Foxp3+Treg cells and the percentage of CD4+IL-17A+ Th17 cells in whole blood of subjects in both groups. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze the plasma metabolites in the gingivitis patient group. Statistical analysis was applied to determine whether the plasma metabolites and related metabolic pathways significantly differed between gingivitis patients and healthy controls. Ingenuity pathway analysis (IPA) was employed to identify the potential relation between the metabolites and the Th17 and Treg related pathway.ResultsThe percentages of CD4+IL17A+Th17 cells and IL-17 significantly increased in the peripheral blood in the gingivitis group. Moreover, the upregulation of IL-17A mRNA and RORγt mRNA were also found in the gingivitis group. However, the percentage of CD4+CD25+ Foxp3+Treg cells and Foxp3 mRNA in the whole blood did not significantly change. However, TGF-β mRNA as well as TGF-β, IL-4, IL-6, IL-10 in the periperial blood and SIgA in the saliva were higher in the gingivitis group. Notably, that the ratio of Th17/Treg cells was significantly increased during peripheral circulation. Furthermore, we identified 18 different metabolites which were differentially expressed in plasma between the gingivitis and healthy control groups. Notably, the levels of cholesterol, glycerol 1-octadecanoate, d-glucose, uric acid, cyclohexaneacetic acid, 3-pyridine, tryptophan, and undecane 2,4-dimethyl were significantly up-regulated. whereas the levels of lactic acid, glycine, linoleic acid, monopalmitic acid, glycerol, palmitic acid, pyruvate, 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene, 1 5-anhydro d-altrol, and boric acid were down-regulated in the gingivitis group, relative to healthy controls. IPA showed that these metabolites are connected to IL17 signaling, TGF-B signaling, and IL10 signaling, which are related closely to Th17 and Treg pathway.ConclusionOverall, these results showed that disturbance to glycolysis as well as amino and fatty acid metabolism are associated with Th17/Treg balance in gingivitis. Impaired immunometabolism may influence some periodontally involved systemic diseases, hence it is a promising strategy in targeted development of treatment therapies

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

<div><p>The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.</p></div

Porous chitosan scaffolds with embedded hyaluronic acid/chitosan/plasmid-DNA nanoparticles encoding TGF-β1 induce DNA controlled release, transfected chondrocytes, and promoted cell proliferation.

Cartilage defects resulting from traumatic injury or degenerative diseases have very limited spontaneous healing ability. Recent progress in tissue engineering and local therapeutic gene delivery systems has led to promising new strategies for successful regeneration of hyaline cartilage. In the present study, tissue engineering and local therapeutic gene delivery systems are combined with the design of a novel gene-activated matrix (GAM) embedded with hybrid hyaluronic acid(HA)/chitosan(CS)/plasmid-DNA nanoparticles encoding transforming growth factor (TGF)-β1. A chitosan scaffold functioned as the three-dimensional carrier for the nanoparticles. Results demonstrated that scaffold-entrapped plasmid DNA was released in a sustained and steady manner over 120 days, and was effectively protected in the HA/CS/pDNA nanoparticles. Culture results demonstrated that chondrocytes grown in the novel GAM were highly proliferative and capable of filling scaffold micropores with cells and extracellular matrix. Confocal laser scanning microscopy indicated that chondrocytes seeded in the GAM expressed exogenous transgenes labeled with green fluorescent protein. ELISA results demonstrated detectable TGF-β1 expression in the supernatant of GAM cultures, which peaked at the sixth day of culture and afterwards showed a moderate decline. Histological results and biochemical assays confirmed promotion of chondrocyte proliferation. Cell culture indicated no affects on phenotypic expression of ECM molecules, such as GAG. The results of this study indicate the suitability of this novel GAM for enhanced in vitro cartilage tissue engineering

<i>In vitro</i> transfection efficiency of CP/DNA nanoparticles.

<p>(a) <i>In vitro</i> transfection efficiency of CP/DNA nanoparticles in both chondrocytes and synoviocytes compared to that of naked pDNA, CS/DNA nanoparticles, PEI (25 kDa)/DNA nanoparticles, and Lipofectamine™ 2000 (n = 3; 48 h post-transfection; error bars represent standard deviation). *<i>P</i><0.01 when CP/DNA nanoparticles compared to CS/DNA nanoparticles transfected towards chondrocytes (n = 3); **<i>P</i><0.01 when CP/DNA nanoparticles compared to PEI (25 kDa)/DNA nanoparticles transfected towards synoviocytes (n = 3); # or ## <i>P</i>>0.05 when CP/DNA nanoparticles compared to Lipofectamine™ 2000 transfected towards chondrocytes or synoviocytes (n = 3). (b) Percentage of chondrocytes or synoviocytes transfected <i>in vitro</i> using CP/DNA nanoparticles as measured by flow cytometry 48 h post-transfection. The influence of CP:DNA weight ratios on the transfection efficiency was assessed 48 h post-transfection (n = 3; error bars represent standard deviation).</p

Gel retarding analysis of CP/DNA nanoparticles.

<p>Lane 1: DNA marker. Lane 2: naked DNA control. Lane 3–8: CP/DNA nanoparticles prepared at CP:DNA weight ratios of 1∶2, 1∶1, 2∶1, 3∶1, 4∶1, and 5∶1 (a); electrophoresis photo of CP/DNA nanoparticles prepared with CP:DNA weight ratio = 3 at different pH levels (b); electrophoresis of CS/DNA nanoparticles prepared with the CS:DNA weight ratio = 3 at different pH levels (c).</p

Cell viabilities of CP/DNA nanoparticles, CS/DNA nanoparticles, PEI/DNA nanoparticles, and Lipofectamine™ 2000 in primary chondrocytes (a) and synoviocytes (b).

<p>*<i>P</i><0.01 compared to PEI/DNA nanoparticles; **<i>P</i><0.01 compared to Lipofectamine™ 2000.</p

Representative ¹H NMR spectra of chitosan (CS) and CS-<i>g</i>-PEI (CP) in a mixture solution (D₂O/CD₃COOD (V_D2O: V_CD3COOD = 1∶1) at 40°C.

<p>Representative ¹H NMR spectra of chitosan (CS) and CS-<i>g</i>-PEI (CP) in a mixture solution (D₂O/CD₃COOD (V_D2O: V_CD3COOD = 1∶1) at 40°C.</p

Characteristic of prepared CS-g-PEI (CP).

<p>^a calculated from GPC.</p><p>^b calculated from ¹H NMR.</p