Regulation of base excision repair during adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages. It has been shown that the high-efficiency DNA-repair capacity of hMSCs is decreased during their differentiation. However, the underlying its mechanism during adipogenesis and osteogenesis is unknown. Herein, we investigated how alkyl-damage repair is modulated during adipogenic and osteogenic differentiation, especially focusing on the base excision repair (BER) pathway. Response to an alkylation agent was assessed via quantification of the double-strand break (DSB) foci and activities of BER-related enzymes during differentiation in hMSCs. Adipocytes showed high resistance against methyl methanesulfonate (MMS)-induced alkyl damage, whereas osteoblasts were more sensitive than hMSCs. During the differentiation, activities, and protein levels of uracil-DNA glycosylase were found to be regulated. In addition, ligation-related proteins, such as X-ray repair cross-complementing protein 1 (XRCC1) and DNA polymerase ß, were upregulated in adipocytes, whereas their levels and recruitment declined during osteogenesis. These modulations of BER enzyme activity during differentiation influenced DNA repair efficiency and the accumulation of DSBs as repair intermediates in the nucleus. Taken together, we suggest that BER enzymatic activity is regulated in adipogenic and osteogenic differentiation and these alterations in the BER pathway led to different responses to alkyl damage from those in hMSCs.

TGF-ß secreted by human umbilical cord blood-derived mesenchymal stem cells ameliorates atopic dermatitis by inhibiting secretion of TNF-α and IgE.

Human mesenchymal stem cells (MSCs) are promising therapeutics for autoimmune diseases due to their immunomodulatory effects. In particular, human umbilical cord blood-derived MSCs (hUCB-MSCs) have a prominent therapeutic effect on atopic dermatitis (AD). However, the underlying mechanism is unclear. This study investigated the role of transforming growth factor-beta (TGF-ß) in the therapeutic effect of hUCB-MSCs on AD. Small interfering RNA (siRNA)-mediated depletion of TGF-ß disrupted the therapeutic effect of hUCB-MSCs in a mouse model of AD by attenuating the beneficial changes in histopathology, mast cell infiltration, tumor necrosis factor-alpha (TNF-α) expression, and the serum IgE level. To confirm that hUCB-MSCs regulate secretion of TNF-α, we investigated whether they inhibit TNF-α secretion by activated LAD2 cells. Coculture with hUCB-MSCs significantly inhibited secretion of TNF-α by LAD2 cells. However, this effect was abolished by siRNA-mediated depletion of TGF-ß in hUCB-MSCs. TNF-α expression in activated LAD2 cells was regulated by the extracellular signal-related kinase signaling pathway and was suppressed by TGF-ß secreted from hUCB-MSCs. In addition, TGF-ß secreted by hUCB-MSCs inhibited maturation of B cells. Taken together, our findings suggest that TGF-ß plays a key role in the therapeutic effect of hUCB-MSCs on AD by regulating TNF-α in mast cells and maturation of B cells.

Preconditioning with interleukin-1 beta and interferon-gamma enhances the efficacy of human umbilical cord blood-derived mesenchymal stem cells-based therapy via enhancing prostaglandin E2 secretion and indoleamine 2,3-dioxygenase activity in dextran sulfate sodium-induced colitis.

Preconditioning with inflammatory cytokines has improved mesenchymal stem cells characteristics, including differentiation and immunomodulating functions. In this study, we developed a preconditioning combination strategy using interleukin-1beta (IL-1ß) and interferon-gamma (IFN-γ) to enhance the immuneregulatory ability of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Our results showed that hUCB-MSCs preconditioned with IL-1ß and IFN-γ (primed hUCB-MSCs) created a statistically significant decrease in peripheral blood mononuclear cell proliferation, indicating that their immunosuppressive ability was increased. The secretion of PGE2, cyclooxygenase 2 mRNA expression, and indoleamine 2,3-dioxygenase (IDO) mRNA expression in primed hUCB-MSCs was significantly higher than those in the untreated hUCB-MSCs or the IL-1ß or IFN-γ only treated hUCB-MSCs. When inhibitors of IDO and PGE2 were treated, peripheral blood mononuclear cell proliferation, which is inhibited by primed hUCB-MSCs, was recovered. We found that Th1 T cell differentiation was also inhibited by PGE2 and IDO in the primed hUCB-MSCs, and Tregs differentiation was increased by PGE2 and IDO in the primed hUCB-MSCs. Furthermore, the primed hUCB-MSCs as well as supernatants increase CD4+ T cells migration. We demonstrated the therapeutic effects of primed hUCB-MSCs in dextran sulfate sodium-induced colitis model. In conclusion, we have demonstrated that primed hUCB-MSCs simultaneously enhance PGE2 and IDO and greatly improve the immunoregulatory capacity of MSCs, and we have developed an optimal condition for pretreatment of MSCs for the treatment of immune diseases. Our results raise the possibility that the combination of PGE2 and IDO could be therapeutic mediators for controlling immunosuppression of MSCs.

Therapeutic effect of long-interval repeated intravenous administration of human umbilical cord blood-derived mesenchymal stem cells in DBA/1 mice with collagen-induced arthritis.

Rheumatoid arthritis (RA) is a common inflammatory chronic disease. It has been reported that mesenchymal stem cells (MSCs) have the effect of immune suppression in collagen-induced arthritis (CIA) mice model. However, the in vivo therapeutic effect from the long-interval repeated intravenous administration of human umbilical cord blood-derived (hUCB)-MSCs had not been investigated in CIA mice model. This study was undertaken to investigate the effects of long-interval repeated intravenous administration of hUCB-MSCs at different doses in CIA mice model. Mice were intravenously injected with three different doses of hUCB-MSCs once every 2 weeks for three times. RA severity was assessed by clinical joint score and histologic analysis including hematoxylin and eosin staining, safranin-O staining, and toluidine blue staining. We used real-time polymerase chain reaction and flow cytometry to quantify differences in inflammatory cytokines and Tregs. Mice treated with hUCB-MSCs showed significant improvement in clinical joint score. Histologic analysis revealed that hUCB-MSCs definitely reduced joint inflammation, cartilage damage, and formation of pannus in multimedium and multihigh groups. These hUCB-MSCs also significantly decreased IL-1 beta protein levels in multimedium and multihigh groups and IL-6 protein levels in all hUCB-MSCs-treated groups. Furthermore, mRNA levels of IL-1 beta and IL-6 were decreased significantly in all hUCB-MSCs-treated groups, whereas the expression of anti-inflammatory cytokine IL-10 was increased in the multihigh group. Tregs known as suppressor T cells were also significantly increased in the multihigh group. Our findings suggest that long-interval repeated intravenous administration of hUCB-MSCs has therapeutic effects by improving symptoms of RA in CIA mice model in a dose-dependent manner.

Antibacterial Copper-Doped Calcium Phosphate Glasses for Bone Tissue Regeneration.

Calcium phosphate glasses are a promising new generation of biomaterials that can simultaneously induce tissue regeneration and controlled release of therapeutic molecules. In this work, novel calcium phosphate glasses containing 0, 2, 4, and 6 mol % Cu2+ were synthesized via room temperature precipitation reaction in aqueous solution. The effect of Cu2+ addition on the glass properties and structure was investigated using thermal analysis, 31P solid-state MAS NMR, Raman spectroscopy, and X-ray diffraction. All glasses crystallize at temperature >500 °C and are mainly formed by Q1 groups. The release of P, Ca, and Cu in solution over time was monitored via inductively coupled plasma-optical emission spectroscopy. It was found that with increasing Cu content, the amount of P and Ca released decreases whereas the amount of Cu released increases. The effect of Cu2+ release on the antibacterial activity against S. aureus, a bacterial strain commonly found in postsurgery infections, has been investigated. The addition of copper has been shown to infer the glasses antibacterial properties. As expected, the antibacterial activity of the glasses increases with increasing Cu2+ content. Cytocompatibility was assessed by seeding human osteoblast-like osteosarcoma cells Saos-2 (HTB85) on the glass particles. A significant increase in cell number was observed in all the glasses investigated. The copper-doped calcium phosphate glasses have proven to be multifunctional, as they combine bone regenerative properties with antibacterial activity. Therefore, they have great potential as antibacterial bioresorbable materials for hard tissue regeneration.

Phosphorus doped SnO2 thin films for transparent conducting oxide applications: synthesis, optoelectronic properties and computational models.

Phosphorus doped tin(iv) oxide (P:SnO2) films have been synthesised by an aerosol assisted chemical vapour deposition route. Triethyl phosphate was used as the phosphorus dopant source. The phosphorus concentration in solution was found to be key to electrical properties, with concentrations between 0.25-0.5 mol% phosphorus giving the lowest resistivities of the deposited films. The conductivity of the films synthesised improved on doping SnO2 with phosphorus, with resistivity values of 7.27 × 10-4 Ω cm and sheet resistance values of 18.2 Ω â–¡-1 achieved for the most conductive films. Phosphorus doping up to 1.0 mol% was shown to improve visible light transmission of the deposited films. The phosphorus doping also had a significant effect on film morphology, with varying microstructures achieved. The films were characterised by X-ray diffraction, scanning electron microscopy, UV/vis spectroscopy, Hall effect measurements and X-ray photoelectron spectroscopy. The data generated was used to build computational models of phosphorus as a dopant for SnO2, showing that the phosphorus acts as a shallow one-electron n-type donor allowing for good conductivities. Phosphorus does not suffer from self-compensation issues associated with other dopants, such as fluorine.

A reversible fluorescent probe for monitoring Ag(I) ions.

Silver-containing nanomaterials are of interest for their antibiotic properties, for a wide range of applications from medicine to consumer products. However, much remains to be learnt about the degradation of such materials and their effects on human health. While most analyses involve measurement of total silver levels, it is important also to be able to measure concentrations of active free Ag(I) ions. We report here the preparation of a coumarin-based probe, thiocoumarin silver sensor 1 (TcAg1), that responds reversibly to the addition of silver ions through the appearance of a new fluorescence emission peak at 565 nm. Importantly, this peak is not observed in the presence of Hg(II), a common interferent in Ag(I) sensing. To establish the utility of this sensor, we prepared silver-doped phosphate glasses with demonstrated bactericidal properties, and observed the Ag(I) release from these glasses in solutions of different ionic strength. TcAg1 is therefore a useful tool for the study of the environmental and medical effects of silver-containing materials.

Contrasting life histories contribute to divergent patterns of genetic diversity and population connectivity in freshwater sculpin fishes.

BACKGROUND: Life history characteristics are considered important factors influencing the evolutionary processes of natural populations, including the patterns of population genetic structure of a species. The sister species Cottus hangiongensis and C. koreanus are small bottom-dwelling freshwater sculpin fishes from South Korea that display marked life history divergence but are morphologically nearly indistinguishable. Cottus hangiongensis evolved an 'amphidromous' life history with a post-hatching pelagic larval phase. They spawn many small eggs in the low reaches of rivers, and hatched larvae migrate to the sea before returning to grow to maturity in the river mouth. In contrast, C. koreanus evolved a 'fluvial' landlocked type with benthic larvae. They release a smaller number of larger eggs, and the larvae undergo direct development, remaining benthic in the upstream rivers throughout their entire lives. We tested whether there were differences in patterns and levels of within-population genetic diversities and spatial population structure between the two closely related Korean sculpins using mitochondrial DNA control region sequences and seven nuclear microsatellite loci. RESULTS: The combined analyses of both marker sets revealed that C. hangiongensis harboured considerably higher levels of within-population genetic diversities (e.g. haplotype/allelic richness, heterozygosities) than C. koreanus. In contrast, the fluvial sculpin exhibited noticeably more spatial population structure than did the amphidromous sculpin, as suggested by pairwise FST statistics. The finding that C. hangiongensis individuals comprised a single random mating population across the east-flowing river basins in the Korean Peninsula, whereas C. koreanus individuals comprised genetically discrete individual populations, was further supported by an individual-based Bayesian population assignment and also factorial correspondence analyses. CONCLUSIONS: The higher genetic diversity, but lower population structure, of the amphidromous sculpin relative to the fluvial sculpin may have resulted from its greater larval dispersal and also possibly, higher fecundity accompanied by an amphidromous life history. Hence, we conclude that contrasting early life histories - including the presence or absence of the pelagic larval phase - may have led to divergent patterns of within-population genetic diversities and spatial population structure between the sister Cottus species following speciation from a common ancestor of marine sculpin.

MMPP Attenuates Non-Small Cell Lung Cancer Growth by Inhibiting the STAT3 DNA-Binding Activity via Direct Binding to the STAT3 DNA-Binding Domain.

Rationale: Signal transducer and activator of transcription-3 (STAT3) plays a pivotal role in cancer biology. Many small-molecule inhibitors that target STAT3 have been developed as potential anticancer drugs. While designing small-molecule inhibitors that target the SH2 domain of STAT3 remains the leading focus for drug discovery, there has been a growing interest in targeting the DNA-binding domain (DBD) of the protein. Methods: We demonstrated the potential antitumor activity of a novel, small-molecule (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) that directly binds to the DBD of STAT3, in patient-derived non-small cell lung cancer (NSCLC) xenograft model as well as in NCI-H460 cell xenograft model in nude mice. Results: MMPP effectively inhibited the phosphorylation of STAT3 and its DNA binding activity in vitro and in vivo. It induced G1-phase cell cycle arrest and apoptosis through the regulation of cell cycle- and apoptosis-regulating genes by directly binding to the hydroxyl residue of threonine 456 in the DBD of STAT3. Furthermore, MMPP showed a similar or better antitumor activity than that of docetaxel or cisplatin. Conclusion: MMPP is suggested to be a potential candidate for further development as an anticancer drug that targets the DBD of STAT3.

Comparison of tumor vascularity and hemodynamics in three rat hepatoma models.

PURPOSE: To compare tumor vascularity and hemodynamics in three rat hepatoma models: N1-S1 cells in Sprague-Dawley rats, McA-RH7777 cells in Sprague-Dawley rats, and 13762 MAT B III cells in F344 rats. METHODS: The three rat hepatoma models were induced in five rats per group. After confirming that the tumors grew up to 10 mm on magnetic resonance imaging, the rats underwent dynamic contrast-enhanced ultrasonography (DCE-US). Afterward, the rats were euthanized for histologic analyses. The Kruskal-Wallis test was used to compare the rat hepatoma models. Correlation coefficients were calculated between the microvessel density (MVD) and DCE-US parameters. RESULTS: On DCE-US imaging, arterial enhancement and washout were demonstrated in all N1-S1 tumors, while persistent peripheral enhancement on arterial to portal phases was shown in all 13762 MAT B III tumors. The McA-RH7777 tumors presented diverse enhancement patterns on arterial and portal phases. There were no significant differences in DCE-US parameters among the three hepatoma groups, while MVD was correlated with peak intensity (r = 0.565, p = 0.044), mean transit time (r = -0.559, p = 0.047), and time to peak (r = - 0.617, p = 0.025) of individual rats. The necrosis ratio was significantly different between the models (p = 0.031); 13762 MAT B III showed a significantly higher necrosis ratio than N1-S1 (p < 0.050 by post hoc test). CONCLUSION: The N1-S1 tumor may be suitable as a model to investigate hypervascular hepatic tumors of the liver in DCE-US such as hepatocellular carcinoma among the three tumors.

Transcription factor IRF8 controls Th1-like regulatory T-cell function.

Recent studies have suggested that regulatory T (Treg) cells comprise a heterogeneous population that regulates various aspects of the immune response, and that Treg cells use the factors that are expressed in their target cells to regulate them. We searched for factors that regulate Th1 response in Treg cells using a meta-analysis. In the process, we discovered that transcription factor interferon regulatory factor 8 (IRF8) was selectively expressed in Treg and Th1 cells. IRF8-deficient Treg cells showed defective expression of CXCR3 and aberrant expression of the Il4 and Il17 genes. Upon treatment with alpha galactosyl-C18-ceramide (αGal-C18-Cer), IRF8-deficient mice showed defective Treg cell recruitment in the liver. Eliciting Th1 immune response by anti-CD40 antibody injection in mice induced IRF8 expression in Treg cells. The expression of IRF8 was induced by Foxp3 in Treg cells. IRF8 had no effect on T-bet expression in Treg and vice versa. Thus, our results strongly suggest that IRF8 controls Th1 immune response in Treg cells independent of T-bet.

Doxorubicin-loaded poly(lactic-co-glycolic acid) microspheres prepared using the solid-in-oil-in-water method for the transarterial chemoembolization of a liver tumor.

Doxorubicin (DOX)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MSs) were fabricated using the solid-in-oil-in-water (S/O/W) emulsification method for transarterial chemoembolization (TACE) of a liver tumor. DOX-loaded PLGA MSs with a mean diameter of 26 µm and a spherical shape were prepared. The biodegradation of PLGA MSs was observed in serum using a scanning electron microscope (SEM). Drug release from the PLGA MSs was accelerated at an acidic pH (pH 5.5) compared to a normal physiological pH (pH 7.4). According to the results of a pharmacokinetic study in rats, the area under the curve (AUC) value of a drug, which indicates the systemic exposure extent of the drug, of the PLGA MSs group was 29.9% of that of a hepatic arterial injection (HAI) group. The DOX concentration ratio for liver tumors compared to normal livers was significantly higher in the PLGA MSs group than that of the HAI group (p<0.05). After the TACE procedure was performed with DOX-PLGA MSs in a rat hepatoma model, the mean size increment of tumor in DOX-PLGA MSs group was found to be lower than that of the HAI group, and the viable portion of the DOX-PLGA MSs group was less than the other groups (p<0.05). All these findings suggested that the developed DOX-loaded PLGA MSs fabricated with the S/O/W method can be used as a promising drug delivery system in TACE for liver tumors.

A metastatic hepatoma model of rats using the 13762-MAT-B-III cell line: basic characteristics and potential as a tool for interventional oncology experiments.

AIM: To evaluate the characteristics of a 13762-MAT-B-III model in the rat liver and to assess the adequacy of the model for transarterial embolization (TAE) study. MATERIALS AND METHODS: One hundred thousand 13762-MAT-B-III rat breast cancer cells were inoculated into the livers of 11 Fisher 344 rats. Natural course via magnetic resonance imaging (MRI) follow-up, histological characteristics and tumor response after TAE were evaluated. RESULTS: The tumor induction rate of the 13762-MAT-B-III hepatoma model was 100%. Except for one tumor that started to regress after 14 days, the 13762-MAT-B-III hepatomas showed rapid growth, up to 13.1±1.4 mm, at 21 days. Peritoneal seeding was observed in all rats. TAE was conducted successfully in three rats on day 11. The TAE-treated hepatomas were significantly smaller on day 21 (p=0.040) and had a significantly greater apoptosis ratio (p=0.046). CONCLUSION: The 13762-MAT-B-III hepatoma model can be useful in many interventional oncology studies by providing consistent and rapidly growing tumors.

Comparison of drug release and pharmacokinetics after transarterial chemoembolization using diverse lipiodol emulsions and drug-eluting beads.

In many studies for chemoembolization of hepatocellular carcinoma, the Lipiodol emulsion preparation protocols, especially the mixing steps, were unclear or even unrevealed at all. However, doxorubicin (DOX) release may depend on the composition and volume ratio (Lipiodol to DOX solution) of a Lipiodol emulsion. Therefore, we conducted a preclinical study to compare in-vitro drug release and in-vivo pharmacokinetics of DOX from diverse Lipiodol emulsions and drug-eluting beads (DEBs) and to compare the tumor response in a rabbit VX2 carcinoma model. DOX release profiles of four types of Lipiodol emulsions with different media (normal saline or Pamiray as an iodinated contrast medium), volume ratio (Lipiodol to DOX solution), and DEBs were investigated in-vitro. For the in-vivo study, 15 rabbits bearing VX2 carcinoma in the liver were treated with 4∶1 volume ratio Lipiodol emulsion (group A), 1∶1 volume ratio Lipiodol emulsion (group B), and DEBs (group C) chemoembolization. Blood and tissue sampling was conducted to evaluate DOX concentration in plasma and tissues, histological changes, and liver toxicity. The most stable emulsion was formed with Pamiray (including DOX) at a 4∶1 volume ratio. The AUC value of group A was significantly lower than that of group B (p = 0.003) but comparable to that of group C (p = 0.071). The Cmax value of group A was significantly different compared with those of group B (p = 0.004) and C (p = 0.015). The tissue drug concentration in group A was comparable to that in group C (p = 0.251). No viable tumor was detected in rabbits of group A and B. In group C, viable tumor less than 10% was seen in two of the five rabbits. There were no significant differences in liver enzyme levels after the procedure. In conclusion, DOX release and pharmacokinetics of presented emulsion systems depend substantially on their composition. Therefore, Lipiodol emulsion type should be considered when interpreting data and designing new studies dealing with chemoembolization.