Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Plasma metabolomics reveals biomarkers of the atherosclerosis

Atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in industrialized societies. The lack of metabolite biomarkers has impeded the clinical diagnosis of atherosclerosis so far. In this study, stable atherosclerosis patients (n=16) and age- and sex-matched non-atherosclerosis healthy subjects (n=28) were recruited from the local community (Harbin, P. R. China). The plasma was collected from each study subject and was subjected to metabolomics analysis by GC/MS. Pattern recognition analyses (principal components analysis, orthogonal partial least-squares discriminate analysis, and hierarchical clustering analysis) commonly demonstrated plasma metabolome, which was significantly different from atherosclerotic and non-atherosclerotic subjects. The development of atherosclerosis-induced metabolic perturbations of fatty acids, such as palmitate, stearate, and 1-monolinoleoylglycerol, was confirmed consistent with previous publication, showing that palmitate significantly contributes to atherosclerosis development via targeting apoptosis and inflammation pathways. Altogether, this study demonstrated that the development of atherosclerosis directly perturbed fatty acid metabolism, especially that of palmitate, which was confirmed as a phenotypic biomarker for clinical diagnosis of atherosclerosis.

Characterisation of enterovirus 71 replication kinetics in human colorectal cell line, HT29

Hand, Foot and Mouth Disease (HFMD), a contagious viral disease that commonly affects infants and children with blisters and flu like symptoms, is caused by a group of enteroviruses such as Enterovirus 71 (EV71) and coxsackievirus A16 (CA16). However some HFMD caused by EV71 may further develop into severe neurological complications such as encephalitis and meningitis. The route of transmission was postulated that the virus transmit from one person to another through direct contact of vesicular fluid or droplet from the infected or via faecal-oral route. To this end, this study utilised a human colorectal adenocarcinoma cell line (HT29) with epithelioid morphology as an in vitro model for the investigation of EV71 replication kinetics. Using qPCR, viral RNA was first detected in HT29 cells as early as 12 h post infection (hpi) while viral protein was first detected at 48 hpi. A significant change in HT29 cells’ morphology was also observed after 48 hpi. Furthermore HT29 cell viability also significantly decreased at 72 hpi. Together, data from this study demonstrated that co-culture of HT29 with EV71 is a useful in vitro model to study the pathogenesis of EV71

Pharmacokinetics of isofraxidin in rat plasma after oral administration of the extract of acanthopanax senticosus using HPLC with solid phase extraction method

High-performance liquid chromatography coupled with solid phase extraction method was developed for determination of isofraxidin in rat plasma after oral administration of Acanthopanax senticosus extract (ASE), and pharmacokinetic parameters of isofraxidin either in ASE or pure compound were measured. The HPLC analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mm x 150 mm, 5 microm) with the isocratic elution of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v) (A : B = 22 : 78) and the detection wavelength was set at 343 nm. The calibration curve was linear over the range of 0.156-15.625 microg/ml. The limit of detection was 60 ng/ml. The intra-day precision was 5.8%, and the inter-day precision was 6.0%. The recovery was 87.30+/-1.73%. When the dosage of ASE is equal to pure compound caculated by the amount of isofraxidin, it has been found to have two maximum concentrations in plasma while the pure compound only showed one peak in the plasma concentration-time curve. The determined content of isofraxidin in plasma after oral administration of ASE is the total contents of free isofraxidin and its precursors in ASE in vitro. The pharmacokinetic characteristics of ASE showed the priority of the extract and the properities of traditional Chinese medicine.

Simultaneous determination of 6,7-dimethylesculetin and geniposide in rat plasma and its application to pharmacokinetic studies of Yin Chen Hao Tang preparation

A method for the rapid and simultaneous determination of 6,7-dimethylesculetin (CAS 120-08-1) and geniposide (CAS 24512-63-8) in rat plasma has been developed, using validated high performance liquid chromatography (HPLC) with solid phase extraction (SPE). The HPLC analysis was performed on a commercially available column (200 mm x 4.6 mm, 5 microm) with acetonitrile-methanol-0.1% aqueous formic acid as mobile phase and the UV detection at 343 nm and 238 nm for 6,7-dimethylesculetin and geniposide, respectively. The calibration curves for 6,7-dimethylesculetin and geniposide were linear over the range 0.4-25.6 microg/mL and 1.12-71.68 microg/mL, respectively. The lower limits of quantitation were 0.40 microg/ mL and 1.12 microg/mL, and the lower limits of detection were 0.06 microg/mL and 0.09 microg/ mL, respectively. The intra-day and inter-day precision for 6,7-dimethylesculetin and geniposide were < 5%, whereas the absolute recovery percentages were > 74%. A successful application of the developed HPLC analysis was demonstrated for the pharmacokinetic study of a Traditional Chinese Medicine formula of Yin Chen Hao Tang preparation.

Analysis of the constituents in the rat plasma after oral administration of Yin Chen Hao Tang by UPLC/Q-TOF-MS/MS

A UPLC/Q-TOF-MS/MS method for analyzing the constituents in rat plasma after oral administration of Yin Chen Hao Tang (YCHT), a traditional Chinese medical formula, has been established. The UPLC/MS fingerprints of the samples were established first in vitro and in vivo, with 45 compounds in YCHT and 21 compounds in rat plasma after oral administration of YCHT were detected. Of the 45 detected compounds in vitro, 30 were identified, and all of the 21 compounds detected in rat plasma were identified either by comparing the retention time and mass spectrometry data with that of reference compounds or by mass spectrometry analysis and retrieving the reference literatures. Of the identified 21 compounds in rat plasma, 19 were the original form of compounds absorbed from the 45 detected compounds in vitro, 2 were the metabolites of the compounds existed in YCHT. It is concluded that a rapid and validated method has been developed based on UPLC-MS/MS, which shows high sensitivity and resolution that is more suitable for identifying the bioactive constituents in plasma after oral administration of Chinese herbal medicines, and provides helpful chemical information for further pharmacology and active mechanism research on the Chinese medical formula.

Pharmacokinetics of cimifugin in rat plasma after oral administration of the extract of Saposhnikovia divaricatae root : Determination of cimifugin by high performance liquid chromatography coupled with solid phase extraction

High performance liquid chromatography (HPLC) coupled with the solid phase extraction method was developed for determining cimifugin (a coumarin derivative; one of Saposhnikovia divaricatae's constituents) in rat plasma after oral administration of Saposhnikovia divaricatae extract (SDE), and the pharmacokinetics of cimifugin either in SDE or as a single compound was investigated. The HPLC analysis was performed on a commercially available column (4.6 mm x 200 mm, 5 pm) with the isocratic elution of solvent A (Methanol) and solvent B (Water) (A:B=60:40) and the detection wavelength was set at 250 nm. The calibration curve was linear over the range of 0.100-10.040 microg/mL. The limit of detection was 30 ng/mL. At the rat plasma concentrations of 0.402, 4.016, 10.040 microg/mL, the intra-day precision was 6.21%, 3.98%, and 2.23%; the inter-day precision was 7.59%, 4.26%, and 2.09%, respectively. The absolute recovery was 76.58%, 76.61%, and 77.67%, respectively. When the dosage of SDE was equal to the pure compound calculated by the amount of cimifugin, it was found to have two maximum peaks while the pure compound only showed one peak in the plasma concentration-time curve. The pharmacokinetic characteristics of SDE showed the superiority of the extract and the properties of traditional Chinese medicine.

Polymer nanocarrier system for endosome escape and timed release of siRNA with complete gene silencing and cell death in cancer cells

An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U-2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA.

A meta-analysis of pigmentary characteristics, sun sensitivity, freckling and melanocytic nevi and risk of basal cell carcinoma of the skin

Objective: To calculate pooled risk estimates of the association between pigmentary characteristics and basal cell carcinoma (BCC) of the skin. Methods: We searched three electronic databases and reviewed the reference lists of the retrieved articles until July 2012 to identify eligible epidemiologic studies. Eligible studies were those published in between 1965 and July 2012 that permitted quantitative assessment of the association between histologically-confirmed BCC and any of the following characteristics: hair colour, eye colour, skin colour, skin phototype, tanning and burning ability, and presence of freckling or melanocytic nevi. We included 29 studies from 2236 initially identified. We calculated summary odds ratios (ORs) using weighted averages of the log OR, using random effects models. Results: We found strongest associations with red hair (OR 2.02; 95% CI: 1.68, 2.44), fair skin colour (OR 2.11; 95% CI: 1.56, 2.86), and having skin that burns and never tans (OR 2.03; 95% CI: 1.73, 2.38). All other factors had weaker but positive associations with BCC, with the exception of freckling of the face in adulthood which showed no association. Conclusions: Although most studies report risk estimates that are in the same direction, there is significant heterogeneity in the size of the estimates. The associations were quite modest and remarkably similar, with ORs between about 1.5 and 2.5 for the highest risk level for each factor. Given the public health impact of BCC, this meta-analysis will make a valuable contribution to our understanding of BCC.

Circulating microRNAs involved in multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated, demyelinating and neurodegenerative disease of the central nervous system. After traumatic brain injury, it is the leading cause of neurology disability in young adults. Considerable advances have been made in identifying genes involved in MS but the genetic and phenotypic complexity associated with this disease significantly hinders any progress. A novel class of small RNA molecules, microRNAs (miRNAs) has acquired much attention because they regulate the expression of up to 30% of protein-coding genes and may play a pivotal role in the development of many, if not all, complex diseases. Seven published studies investigated miRNAs from peripheral blood mononuclear cells, CD4+, CD8+ T cell, B lymphocytes, peripheral blood leukocytes, whole blood and brain astrocytes with MS risk. The absence of MS studies investigating plasma miRNA prompted the current investigation of identifying a circulating miRNA signature in MS. We conducted a microarray analysis of over 900 known miRNA transcripts from plasma samples collected from four MS individuals and four sex-aged and ethnicity matched healthy controls. We identified six plasma miRNA (miR-614, miR-572, miR-648, miR-1826, miR-422a and miR-22) that were significantly up-regulated and one plasma miRNA (miR-1979) that was significantly down-regulated in MS individuals. Both miR-422a and miR-22 have previously been implicated in MS. The present study is the first to show a circulating miRNA signature involved in MS that could serve as a potential prognostic and diagnostic biomarker for MS.

Heparan sulfate proteoglycans, tumour progression and the cancer stem cell niche

The cancer stem cell hypothesis states that tumours arise from cells with the ability to self-renew and differentiate into multiple cell types, and that these cells persist in tumors as a distinct population that can cause disease relapse and hence metastasis. The crux of this hypothesis is that these cells are the only cells capable of, by themselves, giving rise to new tumours. What proportion of a tumour consists of these stem cells, where are they localised, how are they regulated, and how can we identify them? The stromal cells embedded within the extracellular matrix (ECM) not only provide a scaffold but also produce ECM constituents for use by stem cells. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to this cell niche and interact with a large number of ligands including growth factors, their receptors, and ECM structural components. It is still unclear whether ECM degradation and subsequent metastasis is a result of proteases produced by the tumour cells themselves or by cells within the stromal compartment. The identification of the cellular origin of cancer stem cells along with microenvironmental changes involved in the initiation, progression and the malignant conversion of all cancers is critical to the development of targeted therapeutics. As ubiquitous members of the ECM microenvironment and hence the cancer cell niche, HSPGs are candidates for a central role in these processes.

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

Background Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. Methods We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. Results In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. Conclusion We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

An assessment of MMP and TIMP gene expression in cell lines and stroma : tumour differences in microdissected breast cancer biopsies

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans’ ρ correlation analysis, a significant association (p < 0.05, ρ = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.