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- ItemConjugated linoleic acid (CLA)-enriched milk fat inhibits growth and modulates CLA-responsive biomarkers in MCF-7 and SW480 human cancer cell lines(Cambridge University Press, 2003-07) Miller, Aine; Stanton, Catherine; Murphy, John; Devery, Rosaleen; Science Foundation IrelandMilk enriched in conjugated linoleic acid (CLA) was obtained from cows on pasture supplemented with full-fat rapeseeds (FFR; 2·26g cis 9, trans 11 (c9, t11)-CLA/100g fatty acid methyl esters) and full-fat soyabeans (1·83g c9, t11-CLA/100g fatty acid methyl esters). A control milk fat (1·69g c9, t11-CLA/100g fatty acid methyl esters) was obtained from cows fed on pasture only. The present study assessed the potency of the CLA-enriched milk fats to modulate biomarkers that had previously been observed to respond to c9, t11-CLA in the MCF-7 and SW480 cell lines. Cell numbers decreased (P<0·05) by up to 61 and 58% following the incubation of MCF-7 and SW480 cells, respectively, for 4d with milk fats (yielding CLA concentrations between 60·2 and 80·6μM). The FFR milk fat, containing the highest CLA content, increased (P<0·05) [14C]arachidonic acid (AA) uptake into the monoacylglycerol fraction of MCF-7 and SW480 cells while it decreased (P<0·05) uptake into the phospholipid fraction of the latter. This milk fat also decreased (P<0·05) [14C]AA conversion to prostaglandin (PG) E2 while increasing conversion to PGF2α in both cell lines. All milk-fat samples increased (P<0·05) lipid peroxidation as measured by 8-epi-PGF2α in both cell lines. In SW480 cells the milk-fat samples decreased (P<0·05) bcl-2 and cytosolic glutathione levels while increasing (P<0·05) membrane-associated annexin V levels. All milk-fat samples decreased (P<0·05) the expression of ras in SW480 cells. These data suggest that milk-fat CLA was effective at modulating synthetic CLA-responsive biomarkers.
- ItemMechanisms of adherence of a probiotic Lactobacillus strain during and after in vivo assessment in ulcerative colitis patients(Co-Action Publishing, 2004-07) Dunne, Colum P.; Kelly, Peter; O'Halloran, Sile; Soden, Declan; Bennett, Mary; von Wright, Atte; Vilpponen-Salmela, Terttu; Kiely, Barry; O'Mahony, Liam; Collins, J. Kevin; O'Sullivan, Gerald C.; Shanahan, Fergus; European Regional Development Fund; Department of Agriculture and Food, Ireland; Health Research Board; Science Foundation Ireland; European Commission; Higher Education AuthorityIn a pilot-scale, open-label study to determine the ability of well-characterized probiotic Lactobacillus salivarius UCC118 cells to adhere to human epithelial cells in situ , the bacterial strain was administered to ulcerative colitis patients at approximately 109 CFU/day for 12 days. Microbiological analysis of biopsy specimens demonstrated that the ingested bacteria effectively adhered to both inflamed and non-inflamed mucosa of the large bowel in significant numbers. In previous reports, we have described the ability of the lactobacilli to adhere to enterocytic epithelial cells in vitro. In this study, we found that the bacteria adhered at higher levels to differentiated rather than undifferentiated epithelial monolayers; and that stationary phase lactobacilli were found to adhere to eukaryotic HT-29 and Caco-2 epithelial cells at greater levels than log phase bacterial cells. Pretreatment of the Lactobacillus cells with proteolytic enzymes abolished attachment, indicating the potential involvement of surface/exposed protein(s) as bacterial adhesin(s). SDS-PAGE (denaturing) techniques determined that the proteolytic treatment resulted in degradation of a cell wall-associated protein of approximately 84 kDa. The proteinaceous factor was purified by both anion-exchange chromatography and by gel extraction after SDS-PAGE electrophoresis, and under in vitro assay conditions proved capable of adherence and significant inhibition of bacterial attachment to enterocytic epithelial cells. Key words: probiotic, Lactobacillus, adhesin, cell-borne, proteinaceous.
- ItemProcessing effects on the nutritional advancement of probiotics and prebiotics(Co-Action Publishing, 2004-07) Ananta, E.; Birkeland, S. E.; Corcoran, B.; Fitzgerald, Gerald F.; Hinz, S.; Klijn, A.; Matto, J.; Mercernier, A.; Nilsson, U.; Nyman, M.; O'Sullivan, Eilís; Parche, S.; Rautonen, N.; Ross, R. Paul; Saarela, M.; Stanton, Catherine; Stahl, U.; Suomalainen, T.; Vincken, J. P.; Virkajarvi, I.; Voragen, F.; Wesenfeld, J.; Wouters, R.; Knorr, D.; Science Foundation Ireland; European CommissionInvestigates the processing effects on the nutritional advancement of probiotics and prebiotics. Efforts of health researchers to overcome difficulties that impact on the performance of functional foods; Importance of characterizing the interactions between probiotics and prebiotics in starter cultures or in functional foods prior to human consumption; Role of prebiotics on the viability and stability of probiotic cultures within food matrices during processing and storage.
- ItemLactococcus lactis is capable of improving the riboflavin status in deficient rats(Cambridge University Press, 2005) LeBlanc, Jean Guy; Burgess, Catherine M.; Sesma, Fernando; de Giori, Graciela Savoy; van Sinderen, Douwe; Science Foundation Ireland; European Commission; Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina; Consejo de Investigaciones de la Universidad Nacional de Tucumán, ArgentinaLactococcus lactis is a commonly used starter strain that can be converted from a vitamin B2 consumer into a vitamin B2 'factory' by over-expressing its riboflavin biosynthesis genes. The present study was conducted to assess in a rat bioassay the response of riboflavin produced by GM or native lactic acid bacteria (LAB). The riboflavin-producing strains were able to eliminate most physiological manifestations of ariboflavinosis such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegalia that were observed using a riboflavin depletion–repletion model. Riboflavin status and growth rates were greatly improved when the depleted rats were fed with cultures of L. lactis that overproduced this vitamin whereas the native strain did not show the same effect. The present study is the first animal trial with food containing living bacteria that were engineered to overproduce riboflavin. These results pave the way for analysing the effect of similar riboflavin-overproducing LAB in human trials.
- ItemGastric electrical stimulation: a report of two cases(Irish Medical Organisation, 2005-12) Sibartie, Vikrant; Quigley, Eamonn M.; O'Donnell, Aonghus P.; Thompson, Christopher J.; Science Foundation IrelandGastroparesis refractory to prokinetic agents poses a major challenge to the physician and patient, alike. In the past 5 years, electrical methods to treat gastroparesis have emerged from animal and human experiments to a potentially valuable tool in clinical gastroenterology. One of these methods, known as gastric electrical stimulation (GES), is being increasingly used in specialized centres worldwide, but had never been tried in Ireland. We describe here our experience with the first two implantations of gastric neurostimulators performed in Ireland and the outcome with these 2 patients. Our results at 6 months show reduction in symptoms and improvement in quality of life, which is encouraging and should prompt further evaluation of GES for patients with gastroparesis refractory to medical therapy.
- ItemCutaneous glucocorticoid receptor sensitivity and proinflammatory cytokine levels in antidepressant-resistant depression(Cambridge University Press, 2006-01) Fitzgerald, Peter; O'Brien, Sinead M.; Scully, Paul; Rijkers, Kim; Scott, Lucinda V.; Dinan, Timothy G.; Science Foundation Ireland; Health Research Board; Wellcome Trust, United KingdomABSTRACT Background. There is evidence to indicate that peripheral glucocorticoid receptor (GR) function is reduced in major depression, and a possible molecular explanation for this is the impact of raised pro-inflammatory cytokines. The topical steroid vasoconstriction assay provides a convenient probe of peripheral GR function. The present study sought to assess the sensitivity of peripheral GRs in antidepressant-resistant major depressives and investigate the association between GR sensitivity and circulating plasma cytokines. Method. Nineteen antidepressant-resistant depressives together with age- and sex-matched healthy controls underwent the steroid vasoconstriction assay using three commercial preparations of corticosteroids containing clobetasol propionate 0.05%, betamethasone valerate 0.1%, and clobetasone butyrate 0.05%, corresponding to very potent, potent, and moderately potent steroid creams respectively. The pro-inflammatory cytokines, tumour necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) were measured using enzyme-linked immunosorbent assays. The severity of the depressive episode was assessed using the Hamilton Depression Scale (HAMD). Results. Depressed subjects had a significantly reduced vasoconstriction response across all three strengths of steroid. They also had significantly higher concentrations of TNF-a and IL-6. There was a significant inverse correlation between TNF-a concentration and vasoconstriction response and also between the HAMD score and vasoconstriction response. Conclusions. These findings suggest that cutaneous GR function is abnormal in antidepressantresistant depression, that circulating TNF-a may play a significant role in this abnormality and that the efficacy of topical steroids in antidepressant-resistant depressives is reduced.
- ItemFas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data(Medknow Publications, 2006-02-02) Ryan, Aideen E.; Lane, Sinead; Shanahan, Fergus; O'Connell, Joe; Houston, Aileen M.; Science Foundation Ireland; Enterprise Ireland; Wellcome Trust; Health Research Board; Higher Education AuthorityBackground: During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L) expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of antitumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. Methods: RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM,HCT116, Jurkat) and three mouse (CMT93, CT26, B16F10) cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. Results: Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. Conclusion: These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.
- ItemA genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system(BioMed Central, 2006-04-28) O'Driscoll, Jonathan; Heiter, Daniel F.; Wilson, Geoffrey G.; Fitzgerald, Gerald F.; Roberts, Richard; van Sinderen, Douwe; Science Foundation IrelandBackground: Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from Lactococcus lactis recognizes an asymmetric,complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a prodigious barrier to bacteriophage infection. LlaJI is comprised of four similarly oriented genes, encoding two 5mC-MTases (M1.LlaJI and M2.LlaJI) and two subunits responsible for restriction activity R1.LlaJI and R2.LlaJI). Here we employ a detailed genetic analysis of the LlaJI restriction determinants in an attempt to characterize mechanistic features of this unusual hetero-oligomeric endonuclease. Results: Detailed bioinformatics analysis confirmed the presence of a conserved GTP binding and hydrolysis domain within the C-terminal half of the R1.LlaJI amino acid sequence whilst the N-terminal half appeared to be entirely unique. This domain architecture was homologous with that of the "B" subunit of the GTP-dependent, methyl-specific McrBC endonuclease from E.coli K-12. R1.LlaJI did not appear to contain a catalytic centre, whereas this conserved motif; PD....D/EXK, was clearly identified within the amino acid sequence for R2.LlaJI. Both R1.LlaJI and R2.LlaJI were found to be absolutely required for detectable LlaJI activity in vivo. The LlaJI restriction subunits were purified and examined in vitro, which allowed the assignment of R1.LlaJI as the sole specificity determining subunit, whilst R2.LlaJI is believed to mediate DNA cleavage. Conclusion: The hetero-subunit structure of LlaJI, wherein one subunit mediates DNA binding whilst the other subunit is predicted to catalyze strand hydrolysis distinguishes LlaJI from previously characterized restriction-modification systems. Furthermore, this distinction is accentuated by the fact that whilst LlaJI behaves as a conventional Type IIA system in vivo, in that it restricts un-methylated DNA, it resembles the Type IV McrBC endonuclease, an enzyme specific for methylated DNA. A number of similar restriction determinants were identified in the database and it is likely LlaJI together with these homologous systems, comprise a new subtype of the Type II class incorporating features of Type II and Type IV systems.
- ItemConcomitant deficits in working memory and fear extinction are functionally dissociated from reduced anxiety in metabotropic glutamate receptor 7-deficient mice(Society for Neuroscience, 2006-06) Callaerts-Vegh, Zsuzsanna; Beckers, Tom; Ball, Simon M.; Baeyens, Frank; Callaerts, Patrick F.; Cryan, John F.; Molnar, Elek; D’Hooge, Rudi; Science Foundation Ireland; Fonds voor Wetenschappelijk Onderzoek, Belgium; Katholieke Universiteit Leuven, BelgiumMetabotropic glutamate receptor 7 (mGluR7), a receptor with a distinct brain distribution and a putative role in anxiety, emotional responding, and spatial working memory, could be an interesting therapeutic target for fear and anxiety disorders. mGluR7-deficient (mGluR7 / ) mice showed essentially normal performance in tests for neuromotor and exploratory activity and passive avoidance learning but prominent anxiolytic behavior in two anxiety tests. They showed a delayed learning curve during the acquisition of the hidden-platform water maze, and three interspersed probe trials indicated that mGluR7 / mice were slower to acquire spatial information. Working memory in the water maze task and the radial arm maze was impaired in mGluR7 / mice compared with mGluR7 / . mGluR7 / mice also displayed a higher resistance to extinction of fear-elicited response suppression in a conditioned emotional response protocol. In a non-fear-based water maze protocol, mGluR7 / mice displayed similar delayed extinction. These observed behavioral changes are probably not attributable to changes inAMPAorNMDAreceptor function because expression levels of AMPAand NMDA receptors were unaltered. Extinction of conditioned fear is an active and context-dependent form of inhibitory learning and an experimental model for therapeutic fear reduction. It appears to depend on glutamatergic and higher-level brain functions similar to those involved in spatial working memory but functionally dissociated from those that mediate constitutional responses in anxiety tests.
- ItemA general method for selection of riboflavin-overproducing food grade micro-organisms(BioMed Central, 2006-07) Burgess, Catherine M.; Smid, Eddy J.; Rutten, Ger; van Sinderen, Douwe; Science Foundation IrelandBackground: This study describes a strategy to select and isolate spontaneous riboflavin overproducing strains of Lactobacillus (Lb.) plantarum, Leuconostoc (Lc.) mesenteroides and Propionibacterium (P.) freudenreichii. Results: The toxic riboflavin analogue roseoflavin was used to isolate natural riboflavinoverproducing variants of the food grade micro-organisms Lb. plantarum, Lc. mesenteroides and P. freudenreichii strains. The method was successfully employed for strains of all three species. The mutation(s) responsible for the observed overproduction of riboflavin were identified for isolates of two species. Conclusion: Selection for spontaneous roseoflavin-resistant mutants was found to be a reliable method to obtain natural riboflavin-overproducing strains of a number of species commonly used in the food industry. This study presents a convenient method for deriving riboflavin-overproducing strains of bacterial starter cultures, which are currently used in the food industry, by a nonrecombinant methodology. Use of such starter strains can be exploited to increase the vitamin content in certain food products.
- ItemBounds on the distribution of the number of gaps when circles and lines are covered by fragments: theory and practical application to genomic and metagenomic projects(BioMed Central, 2007-03-02) Moriarty, John; Marchesi, Julian R.; Metcalfe, Anthony; Science Foundation Ireland; Irish GovernmentBackground: The question of how a circle or line segment becomes covered when random arcs are marked off has arisen repeatedly in bioinformatics. The number of uncovered gaps is of particular interest. Approximate distributions for the number of gaps have been given in the literature, one motivation being ease of computation. Error bounds for these approximate distributions have not been given. Results: We give bounds on the probability distribution of the number of gaps when a circle is covered by fragments of fixed size. The absolute error in the approximation is typically on the order of 0.1% at 10× coverage depth. The method can be applied to coverage problems on the interval, including edge effects, and applications are given to metagenomic libraries and shotgun sequencing.
- ItemRelatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity(BioMed Central, 2007-04-02) O'Connor, Eileen B.; Cotter, Paul D.; O'Connor, Paula M.; O'Sullivan, Orla; Tagg, John R.; Ross, R. Paul; Hill, Colin; Science Foundation Ireland; Irish Government; TeagascBackground: Two component lantibiotics, such as the plasmid-encoded lacticin 3147 produced by Lactococcus lactis DPC3147 and staphylococcin C55 produced by Staphylococcus aureus C55, represent an emerging subgroup of bacteriocins. These two bacteriocins are particularly closely related, exhibiting 86% (LtnA1 and C55α) and 55% (LtnA2 and C55β) identity in their component peptides. The aim of this study was to investigate, for the first time for any two component bacteriocins, the significance of the relatedness between these two systems. Results: So close is this relatedness that the hybrid peptide pairs LtnA1:C55β and C55α:LtnA2 were found to have activities in the single nanomolar range, comparing well with the native pairings. To determine whether this flexibility extended to the associated post-translational modification/processing machinery, the staphylococcin C55 structural genes were directly substituted for their lacticin 3147 counterparts in the ltn operon on the large conjugative lactococcal plasmid pMRC01. It was established that the lacticin LtnA1 post-translational and processing machinery could produce functionally active C55α, but not C55β. In order to investigate in closer detail the significance of the differences between LtnA1 and C55α, three residues in LtnA1 were replaced with the equivalent residues in C55α. Surprisingly, one such mutant LtnA1-Leu21Ala was not produced. This may be significant given the positioning of this residue in a putative lipid II binding loop. Conclusion: It is apparent, despite sharing striking similarities in terms of structure and activity, that these two complex bacteriocins display some highly dedicated features particular to either system.
- ItemHuman methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis(BioMed Central, 2008) Scanlan, Pauline D.; Shanahan, Fergus; Marchesi, Julian R.; Health Research BoardBackground: The incidence and diversity of human methanogens are insufficiently characterised in the gastrointestinal tract of both health and disease. A PCR and clone library methodology targeting the mcrA gene was adopted to facilitate the two-fold aim of surveying the relative incidence of methanogens in health and disease groups and also to provide an overview of methanogen diversity in the human gastrointestinal tract. Results: DNA faecal extracts (207 in total) from a group of healthy controls and five gastrointestinal disease groups were investigated. Colorectal cancer, polypectomised, irritable bowel syndrome and the control group had largely equivalent numbers of individuals positive for methanogens (range 45-50%). Methanogen incidence in the inflammatory bowel disease groups was reduced, 24% for ulcerative colitis and 30% for Crohn's disease. Four unique mcrA gene restriction fragment length polymorphism profiles were identified and bioinformatic analyses revealed that the majority of all sequences (94%) retrieved from libraries were 100% identical to Methanobrevibacter smithii mcrA gene. In addition, mcrA gene sequences most closely related to Methanobrevibacter oralis and members of the order Methanosarcinales were also recovered. Conclusion: The mcrA gene serves as a useful biomarker for methanogen detection in the human gut and the varying trends of methanogen incidence in the human gut could serve as important indicators of intestinal function. Although Methanobrevibacter smithii is the dominant methanogen in both the distal colon of individuals in health and disease, the diversity of methanogens is greater than previously reported. In conclusion, the low incidence of methanogens in Inflammatory Bowel Disease, the functionality of the methanogens and impact of methane production in addition to competitive interactions between methanogens and other microbial groups in the human gastrointestinal tract warrants further investigation.
- ItemIntramammary infusion of a live culture of Lactococcus lactis for treatment of bovine mastitis: comparison with antibiotic treatment in field trials(Cambridge University Press, 2008-04) Klostermann, Katja; Crispie, Fiona; Flynn, James; Ross, R. Paul; Hill, Colin; Meaney, William; Science Foundation Ireland; European CommissionA treatment containing a live food-grade organism, Lactococcus lactis DPC3147, was compared with conventional antibiotic therapy for its potential to treat bovine chronic subclinical or clinical mastitis in two separate field trials. Effects on disease symptoms and bacteriology were monitored in response to infusion with the culture in each trial. In the first trial, the live culture treatment was compared with an intramammary antibiotic (n=11 quarters for each treatment). Results from this small trial demonstrated that the live culture had potential to be as effective at eliminating chronic subclinical infections as an antibiotic treatment. By day 12, 7 of the 11 quarters treated with the live culture were pathogen-free compared with 5 of the 11 antibiotic-treated infected quarters. Somatic cell counts (SCC) remained relatively unchanged regardless of treatment: average log SCC pre- and post-treatment in the lactococci-treated group were 6·33±0·41 (day 0) and 6·27±0·43 cells/ml (day 12) and average log SCC pre- and post-treatment in the antibiotic-treated group were 6·34±0·37 and 6·22±0·46 cells/ml on day 0 and on day 12, respectively. In the second trial, the live culture was compared with an intramammary antibiotic for the treatment of naturally occurring clinical mastitis cases (n=25 quarters for each treatment). Following a 14-d experimental period, similar bacteriological responses were observed in 7 out of 25 live culture treated quarters and 9 out of 25 antibiotic-treated quarters. Additionally, 15 of 25 cases treated with the culture and 18 of 25 cases treated with the antibiotic did not exhibit clinical signs of the disease following treatment. The results of these trials suggest that live culture treatment with Lc. lactis DPC3147 may be as efficacious as common antibiotic treatments in some instances.
- ItemEffect of dietary enrichment with either n-3 or n-6 fatty acids on systemic metabolite and hormone concentration and ovarian function in heifers(Cambridge University Press, 2008-05) Lynch, C. O.; Hennessy, A. A.; Stanton, Catherine; Wathes, D. C.; Sreenan, J. M.; Diskin, M. G.; Kenny, D. A.; Childs, Stuart; Science Foundation IrelandThe objective of this experiment was to examine the effects of dietary n-3 or n-6 fatty acid (FA) supplementation on blood FA, metabolite and hormone concentrations, follicle size and dynamics and corpus luteum (CL) size. Reproductively normal heifers (n = 24) were individually fed diets of chopped straw and concentrate containing either (i) no added lipid (CON; n = 8); (ii) 2% added fat as whole raw soya beans (WSB, n-6; n = 8); or (iii) 2% added fat as fish oil (FO, n-3; n = 8). Following oestrous cycle synchronisation, blood samples were collected at appropriate times and intervals for the measurement of hormones, FAs and metabolites. On days 15 and 16 of the cycle, animals were subjected to an intravenous oxytocin challenge and prostaglandin F2α (PGF2α) response, measured as venous concentrations of 13,14-dihydro-15-keto PGF2α (PGFM). Dry matter intake and average daily gain were similar among treatments (P > 0.05). Plasma concentration of linoleic acid was highest on WSB (P < 0.05), while eicosapentaenoic (EPA, n-3; P < 0.0001) and docosahexaenoic acid (DHA, n-3; P < 0.0001) were greatest in the FO group. Plasma concentrations of arachidonic acid were higher on FO (P < 0.05) compared with CON and WSB. Plasma triglyceride concentrations increased, while β-hydroxybutyrate (BHBA) decreased with time on all diets (P < 0.05). There was a diet × time interaction (P < 0.01) for non-esterified fatty acid (NEFA) concentrations. Plasma cholesterol was higher on WSB and FO (P < 0.01) compared with CON. Progesterone (P4) and oestradiol (E2) concentrations, as well as follicle growth rate and CL diameter were similar across diets (P > 0.05). There was a diet × day interaction for PGFM (P < 0.01). When corrected for systemic E2 : P4 ratio, day 15 concentrations of PGFM were higher in the WSB group at 15 and 30 min (P < 0.01) post oxytocin administration compared with CON and FO, which were similar (P > 0.05). Concentrations of PGFM on day 16 were similar for WSB and FO and were greater than CON at 15 (P < 0.01) and 45 min (P < 0.05) post oxytocin administration, and at 30 min for FO (P < 0.05). With the exception of PGFM, dietary lipid source did not affect the reproductive variables measured.
- ItemDevelopment of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector(BioMed Central, 2008-06) Monk, Ian R.; Casey, Pat G.; Cronin, Michael S.; Gahan, Cormac G.; Hill, Colin; Irish Research Council for Science, Engineering and TechnologyThe foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.
- ItemNisin inducible production of listeriolysin O in Lactococcus lactis NZ9000(BioMed Central, 2008-07) Bahey-El-Din, Mohammed; Griffin, Brendan T.; Gahan, Cormac G.; Science Foundation IrelandBackground: Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter. Results: The constructed vector (pNZPnisA:CYTO-LLO) was expressed in L. lactis NZ9000 and was best induced at mid-log phase with 0.2% v/v nisin for 4 h statically at 30°C. Purification of the His-tagged LLO was accomplished by Ni-NTA affinity chromatography and functionality was confirmed through haemolytic assays. Total LLO yield (measured as total protein content) was 4.43–5.9 mg per litre culture and the haemolytic activity was still detectable after 8 months of storage at 4°C. Conclusion: The LLO production method described in this work provides an approach to efficient LLO production in the Gram-positive Lactococcus bacterium to yield a significant source of the protein for research and diagnostic applications. Expression of LLO in L. lactis has a number of benefits over E. coli which may facilitate both in vivo and in vitro applications of this system.
- ItemCommensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-κB activation(PLOS, 2008-08-01) O'Mahony, Caitlin; Scully, Paul; O'Mahony, David; Murphy, Sharon; O'Brien, Frances; Lyons, Anne; Sherlock, Graham; MacSharry, John; Kiely, Barry; Shanahan, Fergus; O'Mahony, Liam; Ausubel, Frederick M.; Science Foundation Ireland; Health Research Board; Higher Education Authority; Alimentary HealthHost defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-kB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-kB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naı¨ve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-kB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-kB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.
- ItemProstaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells(Springer Nature, 2008-08-05) O'Callagan, Grace; Kelly, Jacquie; Shanahan, Fergus; Houston, Aileen M.; Health Research Board; Science Foundation IrelandFas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E2 (PGE2), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE2 increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E2-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE2 positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE2.
- ItemListeriolysin S, a novel peptide haemolysin associated with a subset of lineage I Listeria monocytogenes(PLOS, 2008-09-12) Cotter, Paul D.; Draper, Lorraine A.; Lawton, Elaine M.; Daly, Karen M.; Groeger, David S.; Casey, Pat G.; Ross, R. Paul; Hill, Colin; Cossart, Pascale; Science Foundation IrelandStreptolysin S (SLS) is a bacteriocin-like haemolytic and cytotoxic virulence factor that plays a key role in the virulence of Group A Streptococcus (GAS), the causative agent of pharyngitis, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. Although it has long been thought that SLS and related peptides are produced by GAS and related streptococci only, there is evidence to suggest that a number of the most notorious Gram-positive pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus, produce related peptides. The distribution of the L. monocytogenes cluster is particularly noteworthy in that it is found exclusively among a subset of lineage I strains; i.e., those responsible for the majority of outbreaks of listeriosis. Expression of these genes results in the production of a haemolytic and cytotoxic factor, designated Listeriolysin S, which contributes to virulence of the pathogen as assessed by murine- and human polymorphonuclear neutrophil–based studies. Thus, in the process of establishing the existence of an extended family of SLS-like modified virulence peptides (MVPs), the genetic basis for the enhanced virulence of a proportion of lineage I L. monocytogenes may have been revealed.