Investigation into the molecular genetics of the inflammatory bowel diseases

The aims of this thesis were firstly to investigate gene expression profiles in human colonic and terminal ileal biopsies using microarray technology in a well phenotyped cohort of patients with Crohn's disease, ulcerative colitis and a control cohort. The role in disease pathogenesis of differentially expressed genes was investigated along with the expression of candidate genes identified by genome wide association study and cell lineage analysis. Parallel studies attempted to replicate the Nature Genetics publications of Peltekova and Stoll and colleagues who investigated the role the IBD5 locus and the DLG5 gene respectively in patients with inflammatory bowel disease.In the healthy adult colon cluster analysis showed differences in gene expression between the right and left colon. (x²=25.1, p<0.0001). Developmental genes HOXA13, (p=2.3x10⁻¹⁶, HOXB13 (p <1xl0⁻⁴⁵), GL11 (p=4.0xl0⁻²⁴), and GLI3 (p=2.1x10⁻²⁸) primarily drove this separation.Upregulated genes in the Crohn's disease biopsies compared to the controls included SAA1 (Fold change (FC) +7.5, p=1.47xl0⁻⁴¹) and REGL (FC +7.3, p=2.3xl0⁻¹⁶). Cellular detoxification genes including-SLC14A2 (FC -2.49, p= 0.00002) were downregulated. In the Crohn's disease terminal ileal biopsies diubiquitin (FC+11.3, p<1x10⁻⁴⁵), MMP3 (FC +7.4, p=1.3x10⁻¹¹) and IRTA1 (FC -11.4, p= 4.7xl0⁻¹²) were differentially expressed compared to controls. In the colon SAA1 (FC +6.3, p= 5.3xl0⁻⁸) was upregulated and TSLP (FC -2.3, p= 2.7xl0⁻⁶) was downregulated comparing non-inflamed Crohn's disease and control biopsies.Of the Crohn's disease susceptibility genes identified by genome wide association scan IL-23A, JAK2 and STAT3 were upregulated in Crohn's disease confirming dysregulation of Thl7 signalling. Modest differential expression was also observed in a number of the autophagy genes, notably ATG16L1. When clustering analysis was undertaken, terminal ileal Crohn's disease and terminal ileal control biopsies separated from colonic Crohn's disease and colonic control biopsies. Further clustering analysis of the terminal ileal biopsies showed separation between the terminal ileal Crohn's disease and control biopsies.When the ulcerative colitis biopsies and control biopsies were compared, differentially upregulated genes in ulcerative colitis included SAA1 (p<10⁻⁴⁵) the alpha defensins, DEFA5&6 (p=0.00003 and p=6.95xl0⁻⁷ respectively), MMP3 (p=5.6x10⁻¹⁰) and MMP7 (p=2.3x10⁻⁷). Increased DEFA5&6 expression was further characterized to Paneth cell metaplasia by immunohistochemistry and in-situ hybridization.Variants in all the examined IBD5 SNPs were associated with Crohn's disease (p<0.003). The IBD5 locus was also associated more severe Crohn's disease behaviour. In the absence of the IBD5 risk haplotype, no association of OCTN1/2 variants with Crohn's disease was detected. The analysis of the DLG5 variant 113A showed there were no associations with inflammatory bowel disease, Crohn's disease or ulcerative colitis.Overall these data emphasise the key role of a number of inflammatory molecules and pathways in pathogenesis of Crohn's disease and ulcerative colitis, and their potential for translation to therapeutic targets. The results also add considerably to the recent genome wide association studies in providing complimentary human colonic and ileal expression data along with detailed analysis of the IL-23 and autophagy pathways

Dysregulation of Human β-Defensin-2 Protein in Inflammatory Bowel Disease

BACKGROUND:Human beta-defensin-2 (HBD2) is an antimicrobial peptide implicated in the pathogenesis of inflammatory bowel disease (IBD). Low copy number and concomitant low mRNA expression of the HBD2 gene have been implicated in susceptibility to colonic Crohn's Disease (CD). We investigated the colonic distribution of HBD2 mRNA expression, and the contributions of genetic and environmental factors on HBD2 protein production. METHODOLOGY/PRINCIPAL FINDINGS:We examined HBD2 mRNA expression at three colonic locations by microarray analysis of biopsies from 151 patients (53 CD, 67 ulcerative colitis [UC], 31 controls). We investigated environmental and genetic influences on HBD2 protein production using ex vivo cultured sigmoid colon biopsies from 69 patients (22 CD, 26 UC, 21 controls) stimulated with lipopolysaccharide (LPS) and/or nicotine for 24 hours. HBD2 and cytokines were measured in culture supernatants. Using DNA samples from these patients, regions in the HBD2 gene promoter were sequenced for NF-kappaB binding-sites and HBD2 gene copy number was determined. HBD2 mRNA expression was highest in inflamed (vs. uninflamed p = 0.0122) ascending colon in CD and in inflamed (vs. uninflamed p<0.0001) sigmoid colon in UC. HBD2 protein production was increased in inflamed UC biopsies (p = 0.0078). There was no difference in HBD2 protein production from unstimulated biopsies of CD, UC and controls. LPS-induced HBD2 production was significantly increased in CD (p = 0.0375) but not UC (p = 0.2017); this LPS-induced response was augmented by nicotine in UC (p = 0.0308) but not CD (p = 0.6872). Nicotine alone did not affect HBD2 production. HBD2 production correlated with IL8 production in UC (p<0.001) and with IL10 in CD (p<0.05). Variations in the HBD2 promoter and HBD2 gene copy number did not affect HBD2 production. SIGNIFICANCE/CONCLUSIONS:Colonic HBD2 was dysregulated at mRNA and protein level in IBD. Inflammatory status and stimulus but not germline variations influenced these changes

Report and Recommendations on Strategies for Engaging Young Adults in the Historic Environment

This report is submitted by HEACS, the Historic Environment Advisory Council for Scotland, which was established in 2003 to provide Scottish Ministers with strategic advice on issues affecting the historic environment. In its second term HEACS was asked by Scottish Ministers: 'What strategies could be developed for attracting young adults to get engaged in protecting, conserving, understanding and enjoying the historic environment?' Young adults have been defined for the purposes of this report as those aged between 16 and 24 inclusive. Research was commissioned from Applejuice Consultants in 2008 to identify case studies, from the UK, Europe and North America, of good practice and successful outcomes in engaging young adults in the historic environment, with a view to assessing which approaches might be most applicable in Scotland. The case studies report should be read in conjunction with this report and has also been submitted to the Minister. The wording of the task set for HEACS implies that young adults are insufficiently engaged in the historic environment. HEACS sought to test this assumption by gathering baseline data on young adults' involvement. This proved surprisingly difficult. The lack of data is a problem in itself, and there is also a lack of consistency in the data that has been collected. There is a need to improve baseline data in order to monitor trends and evaluate the impact of policies, strategies and initiatives. A number of recommendations are made to address this issue. In Scotland we have an annual cohort of 50-55,000 young Scots making a total of about 450,000 within the 16-24 age range (inclusive) at any one time. Young adults therefore form a significant proportion of the population of Scotland

The contribution of OCTN1/2 variants within the IBD5 locus to disease susceptibility and severity in Crohn's disease

Background and Aims: Recent data suggest that polymorphisms in the organic cation transporter (OCTN) genes OCTN1 (SLC22A4) and OCTN2 (SLC22A5) represent disease-causing mutations within the IBD5 locus (chromosome 5q31). We investigated associations with disease susceptibility, phenotype, and evidence for epistasis with CARD15 in 679 patients with Crohn’s disease (CD) or ulcerative colitis (UC). Methods: A total of 374 patients with CD, 305 patients with UC, and 294 healthy controls (HCs) were studied. Genotyping for single nucleotide polymorphisms IGR2096, IGR2198, and IGR2230, OCTN1 variant (SLC22A4 1672C→T), and OCTN2 variant (SLC22A5 −207G→C) was performed using the TaqMan system. Results: The IBD5 OCTN1 and OCTN2 polymorphisms were in strong linkage disequilibrium (D′, >0.959). IGR2198 variant allele frequency (49.1% vs 40.8%; P = .0046) and homozygosity (21% vs 14.8%; P = .044) were associated with CD versus HCs. Variant allelic frequency of OCTN1 (53.6% vs 43%; P = .0008) and OCTN2 (56.1% vs 48.4%; P = .0092) polymorphisms and homozygosity for the OCTN1/2-TC haplotype (28.4% vs 16%; P = .0042) were associated with CD versus HCs. IGR2198 homozygosity and TC homozygosity were associated with stricturing/penetrating disease at follow-up (P = .011 and P = .011, respectively) and disease progression (P = .038 and P = .049, respectively) on univariate analysis and with need for surgery on multivariate analysis (P = .016 and P = .004, respectively). In the absence of the IBD5 risk haplotype, no association of OCTN1/2 variants with CD was detected. No associations were seen with UC. Conclusions: The IBD5 locus influences susceptibility, progression, and need for surgery in CD. However, the contribution of OCTN1/2 variants is not independent of the IBD5 haplotype; a causative role for these genes remains plausible but is not yet proven. Further genetic, functional, and expression data are now required. </p

Numerical and Experimental Investigation of Joule Heating in a Carbon Fibre Powder Epoxy Towpregging Line

Powder epoxy based towpregs offer favourable processing and storage properties, thanks to the low viscosity and thermal stability of the powder epoxy. Low-cost, high-quality towpregs, which are suitable for automated fibre placement or filament winding applications, can be produced at a high production rate with an automated towpregging line. This study focuses on improving the towpregging process by analysing the heating charac-teristics of a towpregging line that employs Joule heating to impregnate carbon fibre tows with powder epoxy. A finite element analysis heat transfer model was developed to identify the relationship between processing pa-rameters and heating of the carbon fibre tows. Model predictions matched well with experimental results. Using the temperature distribution predicted by the model, powder epoxy melting and sintering behaviour was investigated using semi-empirical equations. Results revealed that Joule heating provides efficient heating with very low power consumption. It was found that while it is possible to produce towpregs at high production speeds (15 m/min), slower speeds might yield more consistent quality. Using parametric studies in the model, it was shown that it is possible to increase towpregging line production rate without compromising the towpreg quality, by altering some of the key process parameters (supplied current, electrode distance etc.)

Early Contrast Enhancement: a novel Magnetic Resonance Imaging biomarker of pleural malignancy

Introduction: Pleural Malignancy (PM) is often occult on subjective radiological assessment. We sought to define a novel, semi-objective Magnetic Resonance Imaging (MRI) biomarker of PM, targeted to increased tumour microvessel density (MVD) and applicable to minimal pleural thickening. Materials and methods: 60 consecutive patients with suspected PM underwent contrast-enhanced 3-T MRI then pleural biopsy. In 58/60, parietal pleura signal intensity (SI) was measured in multiple regions of interest (ROI) at multiple time-points, generating ROI SI/time curves and Mean SI gradient (MSIG: SI increment/time). The diagnostic performance of Early Contrast Enhancement (ECE; which was defined as a SI peak in at least one ROI at or before 4.5 min) was compared with subjective MRI and Computed Tomography (CT) morphology results. MSIG was correlated against tumour MVD (based on Factor VIII immunostain) in 31 patients with Mesothelioma. Results: 71% (41/58) patients had PM. Pleural thickening was &#60;10 mm in 49/58 (84%). ECE sensitivity was 83% (95% CI 61–94%), specificity 83% (95% CI 68–91%), positive predictive value 68% (95% CI 47–84%), negative predictive value 92% (78–97%). ECE performance was similar or superior to subjective CT and MRI. MSIG correlated with MVD (r = 0.4258, p = .02). Discussion: ECE is a semi-objective, perfusion-based biomarker of PM, measurable in minimal pleural thickening. Further studies are warranted

Contact Resistance Heating of Unidirectional Carbon Fibre Tows in a Powder-Epoxy Towpregging Line

Contact resistances play a major role in Joule heating systems. Identifying contact resistances provides a better understanding of the heating process and increases the accuracy of the process models. In this study, contact resistances of the Joule heating section in a powder-epoxy-based towpregging line were explored. For different process parameters, such as production speed, tension, and temperature, contact resistances occurring between metal roller electrodes and dry carbon fibre tows (i.e. without powder-epoxy) were measured to gain an insight into the heating process. Moreover, the effect of contact resistance heating on the temperature profile of the carbon fibre tows was determined with an infrared thermal camera. A simplified finite element model was developed to calculate the temperature distribution onthe carbon fibre tow, which showed a good agreement with the experimental data. Results suggest that contact resistances in the towpregging line were significant and contact resistance heating changed the heating profile