Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

The progress in understanding and treatment of diabetic retinopathy

In most countries, diabetic retinopathy is the most frequently occurring complication of diabetes mellitus and remains a leading cause of vision loss globally. Its etiology and pathology have been extensively studied for half a century, yet there are disappointingly few therapeutic options. Although some new treatments have been introduced for diabetic macular edema (DME) (e.g. intravitreal vascular endothelial growth factor inhibitors ('anti-VEGFs') and new steroids), up to 50% of patients fail to respond. Furthermore, for people with proliferative diabetic retinopathy (PDR), laser photocoagulation remains a mainstay therapy, even though it is inherently a destructive procedure. This review summarizes the clinical features of diabetic retinopathy and its risk factors. It describes details of retinal pathology and the cell culture approaches and animal models that are used to mimic its key components, advance understanding of its pathogenesis, and enable identification of new therapeutic targets. We emphasise that although there have been significant advances, there is still a pressing need for a better understanding basic mechanisms to enable development of reliable and robust means to identify patients at highest risk, and to intervene effectively before vision loss occurs.

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke.

Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide

TMC 120 (Dapivirine) is a potent non-nucleoside reverse transcriptase inhibitor that is presently being developed as a vaginal HIV microbicide. To date, most vaginal microbicides under clinical investigation have been formulated as single-dose semi-solid gels, designed for application to the vagina before each act of intercourse. However, a clear rationale exists for providing long-term, controlled release of vaginal microbicides in order to afford continuous protection against heterosexually transmitted HIV infection and to improve user compliance. In this study we report on the incorporation of various pharmaceutical excipients into TMC 120 silicone, reservoir-type intravaginal rings (IVRs) in order to modify the controlled release characteristics of the microbicide. The results demonstrate that TMC 120 is released in zero-order fashion from the rings over a 28-day period and that release parameters could be modified by the inclusion of release-modifying excipients in the IVR. The hydrophobic liquid excipient isopropyl myristate had little effect on steady-state daily release rates, but did increase the magnitude and duration of burst release in proportion to excipient loading in the IVR. By comparison, the hydrophobic liquid poly(dimethylsiloxane) had little effect on TMC 120 release parameters. A hydrophilic excipient, lactose, had the surprising effect of decreasing TMC 120 burst release while increasing the apparent steady-state daily release in a concentration-dependent manner. Based on previous cell culture data and vaginal physiology, TMC120 is released from the various ring formulations in amounts potentially capable of maintaining a protective vaginal concentration. It is further predicted that the observed release rates may be maintained for at least a period of 1 year from a single ring device. TMC 120 release profiles and the mechanical properties of rings could be modified by the physicochemical nature of hydrophobic and hydrophilic excipients incorporated into the IVRs.

Prediction and Verification of miRNA Expression in Human and Rat Retinas.

PURPOSE: MicroRNAs (miRNAs) play a global role in regulating gene expression and have important tissue-specific functions. Little is known about their role in the retina. The purpose of this study was to establish the retinal expression of those miRNAs predicted to target genes involved in vision. METHODS: miRNAs potentially targeting important "retinal" genes, as defined by expression pattern and implication in disease, were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). The presence of candidate miRNAs in human and rat retinal RNA was assessed by RT-PCR. cDNA levels for each miRNA were determined by quantitative PCR. The ability to discriminate between miRNAs varying by a single nucleotide was assessed. The activity of miR-124 and miR-29 against predicted target sites in Rdh10 and Impdh1 was tested by cotransfection of miRNA mimics and luciferase reporter plasmids. RESULTS: Sixty-seven miRNAs were predicted to target one or more of the 320 retinal genes listed herein. All 11 candidate miRNAs tested were expressed in the retina, including miR-7, miR-124, miR135a, and miR135b. Relative levels of individual miRNAs were similar between rats and humans. The Rdh10 3'UTR, which contains a predicted miR-124 target site, mediated the inhibition of luciferase activity by miR-124 mimics in cell culture. CONCLUSIONS: Many miRNAs likely to regulate genes important for retinal function are present in the retina. Conservation of miRNA retinal expression patterns from rats to humans supports evidence from other tissues that disruption of miRNAs is a likely cause of a range of visual abnormalities.

Microbeam studies of the bystander response.

Microbeams have undergone a renaissance since their introduction and early use in the mid 60s. Recent advances in imaging, software and beam delivery have allowed rapid technological developments in microbeams for use in a range of experimental studies. The resurgence in the use of microbeams since the mid 90s has coincided with major changes in our understanding of how radiation interacts with cells. In particular, the evidence that bystander responses occur, where cells not directly irradiated can respond to irradiated neighbours, has brought about the evolution of new models of radiation response. Although these processes have been studied using a range of experimental approaches, microbeams offer a unique route by which bystander responses can be elucidated. Without exception, all of the microbeams currently active internationally have studied bystander responses in a range of cell and tissue models. Together these studies have considerably advanced our knowledge of bystander responses and the underpinning mechanisms. Much of this has come from charged particle microbeam studies, but increasingly, X-ray and electron microbeams are starting to contribute quantitative and mechanistic information on bystander effects. A recent development has been the move from studies with 2-D cell culture models to more complex 3-D systems where the possibilities of utilizing the unique characteristics of microbeams in terms of their spatial and temporal delivery will make a major impact.

Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] : Cholesterol (DOPS : Chol) liposomes for inhalation. Incubation of rSLPI with cathepsin L leads to complete loss of activity while encapsulation of rSLPI in DOPS : Chol liposomes retained 92.6 of its activity after challenge with cathepsin L. rSLPI-loaded liposomes were aerosolized efficiently using a standard nebulizer with a minimal loss of activity and stability. This formulation was biocompatible and encapsulation did not appear to diminish access to intracellular sites of action in in vitro cell culture studies. Liposome encapsulation of rSLPI therefore improves stability and potentially reduces the level and frequency of dosing required for therapeutic effect after inhalation.

Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma

To understand the molecular etiology of osteosarcoma, we isolated and characterized a human osteosarcoma cell line (OS1). OS1 cells have high osteogenic potential in differentiation induction media. Molecular analysis reveals OS1 cells express the pocket protein pRB and the runt-related transcription factor Runx2. Strikingly, Runx2 is expressed at higher levels in OS1 cells than in human fetal osteoblasts. Both pRB and Runx2 have growth suppressive potential in osteoblasts and are key factors controlling competency for osteoblast differentiation. The high levels of Runx2 clearly suggest osteosarcomas may form from committed osteoblasts that have bypassed growth restrictions normally imposed by Runx2. Interestingly, OS1 cells do not exhibit p53 expression and thus lack a functional p53/p21 DNA damage response pathway as has been observed for other osteosarcoma cell types. Absence of this pathway predicts genomic instability and/or vulnerability to secondary mutations that may counteract the anti-proliferative activity of Runx2 that is normally observed in osteoblasts. We conclude OS1 cells provide a valuable cell culture model to examine molecular events that are responsible for the pathologic conversion of phenotypically normal osteoblast precursors into osteosarcoma cells.

Relative quantification of the proteomeic changes associated with the mycotoxin zearalenone in the H295R steroidogenesis model

Zearalenone (ZEN) is a mycotoxin with endocrine disrupting effects having vast economic implications in e.g. pig farming. Structurally, ZEN resembles 17b-estradiol, and thus is able to bind to estrogen receptors (ER) in target cells. Because of this, it is also classified as a non-steroidal estrogen, a phytoestrogen, a mycoestrogen, and a growth promoter. Quantitative proteomic analysis was undertaken using stable-isotope labeling by amino acids in cell culture (SILAC) upon exposure of the steroidogenesis cell model H295R with ZEN to elucidate its effect on protein regulation. ZEN significantly regulated 21 proteins, including proteins with known endocrine disrupting effects and several oncogenes. In addition, network analysis using Ingenuity Pathway Analysis showed that ZEN affected the oxidative phosphorylation pathway and the mitochondrial dysfunction pathway, both previously reported to be involved in endocrine dysfunction.

Fatigue and biocompatibility properties of a poly(methyl methacrylate) bone cement with multi-walled carbon nanotubes

Composites of multi-walled carbon nanotubes (MWCNT) of varied functionality (unfunctionalised and carboxyl and amine functionalised) with polymethyl methacrylate (PMMA) were prepared for use as a bone cement. The MWCNT loadings ranged from 0.1 to 1.0 wt.%. The fatigue properties of these MWCNT–PMMA bone cements were characterised at MWCNT loading levels of 0.1 and 0.25 wt.% with the type and wt.% loading of MWCNT used having a strong influence on the number of cycles to failure. The morphology and degree of dispersion of the MWCNT in the PMMA matrix at different length scales were examined using field emission scanning electron microscopy. Improvements in the fatigue properties were attributed to the MWCNT arresting/retarding crack propagation through the cement through a bridging effect and hindering crack propagation. MWCNT agglomerates were evident within the cement microstructure and the degree of agglomeration was dependent on the level of loading and functionality of the MWCNT. The biocompatibility of the MWCNT–PMMA cements at MWCNT loading levels upto 1.0 wt.% was determined by means of established biological cell culture assays using MG-63 cells. Cell attachment after 4 h was determined using the crystal violet staining assay. Cell viability was determined over 7 days in vitro using the standard colorimetric MTT assay. Confocal scanning laser microscopy and SEM analysis was also used to assess cell morphology on the various substrates.

Apo J/clusterin expression and secretion: Evidence for 15-deoxy-Δ12,14-PGJ2-dependent mechanism

Cyclooxygenase-2 (Cox-2) and Apo J/clusterin are involved in inflammatory resolution and have each been reported to inhibit NF-?B signalling. Using a well-validated rat pheochromocytoma (PC12) cell culture model of Cox-2 over-expression the current study investigated inter-dependence between Cox-2 and clusterin with respect to induction of expression and impact on NF-?B signalling. Both gene expression and immunoblot analysis confirmed that intracellular and secreted levels of clusterin were elevated in Cox-2 over-expressing cells (PCXII). Clusterin expression was increased in control (PCMT) cells in a time- and dose-dependent manner by 15-deoxy-? 12,14-prostaglandin J 2 (15d-PGJ 2), but not PGE 2, and inhibited in PCXII cells by pharmacological Cox inhibition. In PCXII cells, inhibition of two transcription factors known to be activated by 15d-PGJ 2, heat shock factor 1 (HSF-1) and peroxisome proliferator activated receptor (PPAR)?, by transcription factor oligonucleotide decoy and antagonist (GW9662) treatment, respectively, reduced clusterin expression. While PCXII cells exhibited reduced TNF-a-induced cell surface ICAM-1 expression, IkB phosphorylation and degradation were similar to control cells. With respect to the impact of Cox-2-dependent clusterin upregulation on NF-?B signalling, basal levels of I?B were similar in control and PCXII cells, and no evidence for a physical association between clusterin and phospho-I?B was obtained. Moreover, while PCXII cells exhibited reduced NF-?B transcriptional activity, this was not restored by clusterin knock-down. These results indicate that Cox-2 induces clusterin in a 15d-PGJ 2-dependent manner, and via activation of HSF-1 and PPAR?. However, the results do not support a model whereby Cox-2/15d-PGJ 2-dependent inhibition of NF-?B signalling involves clusterin.

Cytosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, α- and β-zearalenol.

a- and b-zearalenol (a-ZOL and b-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of a-ZOL and b-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by a-ZOL and b-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities.

Production, characterisation and applications of monoclonal antibodies to two novel porcine bocaviruses from swine in Northern Ireland

The production, preliminary characterisation and applications of monoclonal antibodies (mAbs) against two novel swine bocaviruses isolated in cell culture from swine in Northern Ireland are described. Of the 17 stable final clones produced, four were characterised. All were of the IgG2a isotype and showed no cross-reactivity with either bocavirus strain. Partial neutralisation was observed with PBoV4 mAbs and homologous virus. The two mAbs selected for use in antigen-detecting ELISAs were successful in highlighting those fractions containing infectious virus within sucrose gradients. This is the first report of the production of specific reagents that will prove useful in the study of the biology of these viruses and swine bocavirus-associated diseases.

Examining acute and chronic effects of short- and long-chain fatty acids on peptide YY (PYY) gene expression, cellular storage and secretion in STC-1 cells

PURPOSE: Peptide YY (PYY) is a gastrointestinal hormone with physiological actions regulating appetite and energy homoeostasis. The cellular mechanisms by which nutrients stimulate PYY secretion from intestinal enteroendocrine cells are still being elucidated.

METHODS: This study comprehensively evaluated the suitability of intestinal STC-1 cells as an in vitro model of PYY secretion. PYY concentrations (both intracellular and in culture media) with other intestinal peptides (CCK, GLP-1 and GIP) demonstrated that PYY is a prominent product of STC-1 cells. Furthermore, acute and chronic PYY responses to 15 short (SCFAs)- and long-chain (LCFAs) dietary fatty acids were measured alongside parameters for DNA synthesis, cell viability and cytotoxicity.

RESULTS: We found STC-1 cells to be reliable secretors of PYY constitutively releasing PYY into cell culture media (but not into non-stimulatory buffer). We demonstrate for the first time that STC-1 cells produce PYY mRNA transcripts; that STC-1 cells produce specific time- and concentration-dependent PYY secretory responses to valeric acid; that linoleic acid and conjugated linoleic acid 9,11 (CLA 9,11) are potent PYY secretagogues; and that chronic exposure of SCFAs and LCFAs can be detrimental to STC-1 cells.

CONCLUSIONS: Our studies demonstrate the potential usefulness of STC-1 cells as an in vitro model for investigating nutrient-stimulated PYY secretion in an acute setting. Furthermore, our discovery that CLA directly stimulates L-cells to secrete PYY indicates another possible mechanism contributing to the observed effects of dietary CLA on weight loss.

Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?

Aims/hypothesis: Blood–retina barrier leakage in diabetes results in extravasation of plasma lipoproteins. Intra-retinal modified LDLs have been implicated in diabetic retinopathy (DR), but their effects on retinal pigment epithelial (RPE) cells and the added effects of extravasated modified HDLs are unknown.

Methods: In human retinas from individuals with and without diabetes and DR, immunohistochemistry was used to detect ApoB, ApoA1 and endoplasmic reticulum (ER) stress markers. In cell culture, human RPE cells were treated with native LDL (N-LDL) or heavily-oxidised glycated LDL (HOG-LDL) with or without pretreatment with native HDL (N-HDL) or heavilyoxidised glycated HDL (HOG-HDL). Cell viability, oxidative stress, ER stress, apoptosis and autophagy were assessed by Cell Counting Kit-8 assay, dichlorofluorescein assay, western blotting, immunofluorescence and TUNEL assay. In separate
experiments, RPE cells were treated with lipid oxidation products, 7-ketocholesterol (7-KC, 5–40 µmol/l) or 4-hydroxynonenal (4-HNE, 5–80 µmol/l), with or without pretreatment with N-HDL or HOG-HDL.

Results: ApoB, ApoA1 staining and RPE ER stress were increased in the presence of DR. HOG-LDL but not N-LDL significantly decreased RPE cell viability and increased reactive oxygen species generation, ER stress, apoptosis and autophagy. Similarly, 4-HNE and 7-KC decreased viability and induced ER stress. Pretreatment with N-HDL mitigated these effects, whereas HOG-HDL was less effective by most, but not all, measures.

Conclusions/interpretation: In DR, extravascular modified LDL may promote RPE injury through oxidative stress, ER stress, autophagy and apoptosis. N-HDL has protective effects, but HOG-HDL is less effective. Extravasation and modification of HDL may modulate the injurious effects of extravasated modified LDL on the retinal pigment epithelium.