Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA).</p> <p>Methods</p> <p>Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium.</p> <p>Results</p> <p>The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44^high, CD24^low, and AC133⁺. These cells also demonstrated high BMP-2, BMP4, TGF-β, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α₂β₁, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α₂β₁, and CD146 surface marker expression than the epithelial type cells.</p> <p>Conclusion</p> <p>The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian cancer treatments.</p

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

<p>Abstract</p> <p>Background</p> <p><it>Arctium lappa </it>(<it>Niubang</it>), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from <it>A. lappa</it>, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes.</p> <p>Methods</p> <p>Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction.</p> <p>Results</p> <p>AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression.</p> <p>Conclusion</p> <p>AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.</p

Detection of Cartilage Oligomeric Matrix Protein Using a Quartz Crystal Microbalance

Current methods for diagnosing early stage osteoarthritis (OA) based on the magnetic resonance imaging and enzyme-linked immunosorbent assay methods are specific, but require specialized laboratory facilities and highly trained personal to obtain a definitive result. In this work, a user friendly and non-invasive quartz crystal microbalance (QCM) immunosensor method has been developed to detect Cartilage Oligomeric Matrix Protein (COMP) for early stage OA diagnosis. This QCM immunosensor was fabricated to immobilize COMP antibodies utilizing the self-assembled monolayer technique. The surface properties of the immunosensor were characterized by its FTIR and electrochemical impedance spectra (EIS). The feasibility study was based on urine samples obtained from 41 volunteers. Experiments were carried out in a flow system and the reproducibility of the electrodes was evaluated by the impedance measured by EIS. Its potential dynamically monitored the immunoreaction processes and could increase the efficiency and sensitivity of COMP detection in laboratory-cultured preparations and clinical samples. The frequency responses of the QCM immunosensor changed from 6 kHz when testing 50 ng/mL COMP concentration. The linear regression equation of frequency shift and COMP concentration was determined as: y = 0.0872 x + 1.2138 (R2 = 0.9957). The COMP in urine was also determined by both QCM and EIS for comparison. A highly sensitive, user friendly and cost effective analytical method for the early stage OA diagnosis has thus been successfully developed

Nonionic Polymeric Micelles for Oral Gene Delivery in Vivo

The main aim of this study was to investigate the feasibility of using nonionic polymeric micelles of poly( ethylene oxide) -poly(propylene oxide ) - poly(ethylene oxide ) (PEO-PPO-PEO) as a carrier for oral DNA delivery in vivo. The size and appearance of DNA/PEO-PPO-PEO polymeric micelles were examined, respectively, by dynamic light scattering and atomic force microscopy, and their zeta potential was measured. Expression of the delivered lacZ gene in various tissues of nude mice was assessed qualitatively by 5-bromo-4-chloro-3-indolyl-beta-D- galactopyranoside staining of sections and quantitatively by measuring enzyme activity in tissue extracts, using the substrate of beta-galactosidase, chlorophenol red-beta-D- galactopyranoside. In addition, the types of cells expressing the lacZ gene in the duodenum were identified by histological analysis. DNA/PEO-PPO-PEO polymeric micelles are a single population of rounded micelles with a mean diameter of 170 nm and a zeta potential of -4.3 mV. Duodenal penetration of DNA/PEO-PPO- PEO polymeric micelles was evaluated in vitro by calculating the apparent permeability coefficient. The results showed a dose-independent penetration rate of (5.75 +/- 0.37) x 10(-5) cm /sec at low DNA concentrations (0.026 - 0.26 mug/mul), but a decrease to ( 2.89 +/- 0.37) x 10(-5) cm/sec at a concentration of 1.3 mug/mul. Furthermore, when 10 mM RGD peptide or 10 mM EDTA was administered before and concurrent with the administration of DNA/PEO-PPO-PEO polymeric micelles, transport was inhibited ([0.95 +/- 0.57] x 10(-5) cm/sec) by blocking endocytosis or enhanced ([29.8 +/- 5.7] x 10(-5) cm/sec) by opening tight junctions, respectively. After oral administration of six doses at 8-hr intervals, the highest expression of transferred gene lacZ was seen 48 hr after administration of the first dose, with gene expression detected in the villi, crypts, and goblet cells of the duodenum and in the crypt cells of the stomach. Reporter gene activity was seen in the duodenum, stomach, and liver. Activity was also seen in the brain and testis when mice were administered 10 mM EDTA before and concurrent with DNA/ PEO-PPO- PEO polymeric micelle administration. lacZ mRNA was detected in these five organs and in the blood by reverse transcription-polymerase chain reaction. Taken together, these results show efficient, stable gene transfer can be achieved in mice by oral delivery of PEO-PPO-PEO polymeric micelles

SB203580 increases G-CSF production via a stem-loop destabilizing element in the 3 ' untranslated region in macrophages independently of its effect on p38 MAPK activity

Background: Granulocyte-colony stimulating factor (G-CSF) is a major regulator of the production and survival of neutrophils. Regulation of G-CSF expression is complex and occurs at both transcription and post-transcription levels. Two distinct types of cis-acting elements in the 3' untranslated region (3' UTR) of G-CSF mRNA have been identified as destabilizing elements; these consist of adenylate uridylate-rich elements (AUREs) and a stem-loop destabilizing element (SLDE). Regulation of the stability of mRNA by p38 mitogen-activated protein kinase (MAPK) has been indicated to be linked to AUREs in the 3' UTR. However, whether p38 MAPK is involved in the regulation of the stability of G-CSF mRNA has not been elucidated. This study investigated the effect of SB203580, an inhibitor of p38 MAPK, on the lipopolysaccharide-induced G-CSF expression in macrophages at the post-transcription level. Results: Our study showed surprising results that SB203580 augmented the lipopolysaccharide-induced increase in the G-CSF mRNA levels in RAW264.7 mouse macrophages, mouse bone marrow-derived macrophages and in THP-1 human macrophages. This effect was also seen in p38a MAPK knockdown RAW264.7 cells, showing that it was not due to inhibition of p38 MAPK activity. In the presence of actinomycin D, the decay of G-CSF mRNA was slower in SB203580-treated cells than in control cells, showing that SB203580 increased the stability of G-CSF mRNA. Reporter genes containing luciferase with or without the 3' UTR of G-CSF were constructed and transfected into RAW264.7 cells and the results showed that the presence of the 3' UTR reduced the luciferase mRNA levels and luciferase activity. Furthermore, SB203580 increased the luciferase mRNA levels and activity in RAW264.7 cells transfected with the luciferase reporter containing the 3' UTR, but not in cells transfected with the luciferase reporter without the 3' UTR. Mutations of the highly conserved SLDE in the 3' UTR abolished these effects, showing that the SLDE was essential for the SB203580-induced increase in the stability of mRNA. Conclusions: SB203580 increases G-CSF expression in macrophages by increasing the stability of G-CSF mRNA via its 3' UTR, and the effect was not due to its inhibition of p38 MAPK activity. The results of this study also highlight a potential target for boosting endogenous production of G-CSF during neutropenia

LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1

Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-alpha. Reporter assays showed that ERK2 and C/EBP beta synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-kappa B (p50/p65) and C/EBP beta to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBP beta-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression

Anti-Apoptotic Gene Delivery with cyclo-(d-Trp-Tyr) Peptide Nanotube via Eye Drop Following Corneal Epithelial Debridement

Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carriers on corneal injury. First, the PNTs were found within 2 μm in length and 300 nm in width by an atomic force microscope and confocal laser microscope system. Plasmid DNAs were observed to be associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were associated with tyrosine of PNTs with a binding constant of 2.7 × 108 M−1. The stability of plasmid DNA with PNTs against the DNase was found at 60 min. Using thioflavin T pre-stained PNTs on the corneal eye drop delivery, the distribution of PNTs was in the epithelial and stroma regions. After corneal debridement, the rhodamine-labeled plasmid DNA and thioflavin T pre-stained PNTs were also delivered and could be observed in the stroma of cornea. PNTs complexed with anti-apoptotic plasmid caspase 3 silencing shRNA eye drop delivery decreased 41% of caspase 3 activity after the first dose by caspase 3 activity and Western blot analysis

In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO–PPO–PEO polymeric micelles on cornea

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO–PPO–PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO–PPO–PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB–DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB–DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB–DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO–PPO–PEO polymers as a vehicle for drug delivery. Keywords: Cornea, Eye drops, Polymeric micelles, Stabilit