35 results on '"Hough, O."'
Search Results
2. CRISPR Genome Engineering in Donor Lung During Cyclic EVLP with 10°C Preservation: Usage of TorEx Lung Perfusion System
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Yoshiyasu, N., primary, Mariscal, A., additional, Salvador, A., additional, Hough, O., additional, Yamamoto, H., additional, Gokhale, H., additional, Shan, H., additional, Chen, M., additional, Guan, Z., additional, Suzuki, Y., additional, Yao, Y., additional, Mesaki, K., additional, Nagoya, A., additional, Juvet, S., additional, Waddell, T.K., additional, Liu, M., additional, Cypel, M., additional, and Keshavjee, S., additional
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- 2024
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3. (1276) Development of Mini-Circuit Ex-Vivo Lung Perfusion to Accelerate Human Lung Translational Research
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Yamamoto, H., primary, Mariscal, A., additional, Hough, O., additional, Mesaki, K., additional, Taniguchi, D., additional, Gokhale, H., additional, Chen, M., additional, Shan, H., additional, Suzuki, Y., additional, Yoshiyasu, N., additional, Yamanashi, K., additional, Aujla, T., additional, Bojic, D., additional, Sorbo, L. Del, additional, Yeung, J., additional, Liu, M., additional, Cypel, M., additional, and Keshavjee, S., additional
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- 2023
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4. Improved ex Vivo Lung Perfusion (EVLP) with Dialysis and Nutrition to Achieve Successful 36h EVLP and Lung Transplantation
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Hough, O., primary, Mariscal, A., additional, Yamamoto, H., additional, Mangat, H., additional, Taniguchi, D., additional, Gokhale, H., additional, Chen, M., additional, Shan, H., additional, Bojic, D., additional, Aulja, T., additional, Ali, A., additional, Main, K., additional, Yoshiyasu, N., additional, Chan, C., additional, Cypel, M., additional, Keshavjee, S., additional, and Liu, M., additional
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- 2023
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5. (714) Pulmonary Stress Index During Ex Vivo Lung Perfusion is Associated with Evlp and Lung Transplant Recipient Outcomes
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Hough, O., primary, Zhou, X., additional, Nardo, M. Di, additional, Ali, A., additional, Brambate, E., additional, Ribeiro, R., additional, Gomes, B., additional, Cypel, M., additional, Slutsky, A., additional, Sage, A., additional, Keshavjee, S., additional, and Sorbo, L. Del, additional
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- 2023
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6. (234) Human Pulmonary Endothelial Function is Improved by Nutrient Supplementation in Ex Vivo Lung Perfusion Perfusates
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Bojic, D., primary, Vellanki, R., additional, Hough, O., additional, Wouters, B., additional, Andreazza, A., additional, Keshavjee, S., additional, and Liu, M., additional
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- 2023
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7. Evaluation of 10°C as the Optimal Storage Temperature for Injured Donor Lungs in a Large Animal Transplant Model
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Abdelnour-Berchtold, E., primary, Ali, A., additional, Wang, A., additional, Hough, O., additional, Beroncal, E., additional, Kawashima, M., additional, Andreazza, A., additional, Keshavjee, S., additional, and Cypel, M., additional
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- 2022
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8. (555) - CRISPR Genome Engineering in Donor Lung During Cyclic EVLP with 10°C Preservation: Usage of TorEx Lung Perfusion System
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Mariscal, A., Salvador, A., Hough, O., Yamamoto, H., Gokhale, H., Shan, H., Chen, M., Guan, Z., Suzuki, Y., Yao, Y., Mesaki, K., Nagoya, A., Juvet, S., Waddell, T.K., Liu, M., Cypel, M., and Keshavjee, S.
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- 2024
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9. (318) - Towards the Digital Lung: A Deep Learning Approach to Simulating Physiological Lung Function During Ex Vivo Lung Perfusion
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Del Sorbo, L., Hough, O., Borrillo, T., Grubert Van Iderstine, M., Chao, B.T., Orsini, L., Valero, J., Cypel, M., Wang, B., Keshavjee, S., and Sage, A.T.
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- 2024
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10. Addition of Dialysis to Ex-Vivo Lung Perfusion Maintains Homeostasis and Stability of Donor Lungs: A Proof of Concept Study
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Hough, O., primary, Gao, X., additional, Yang, C., additional, Takahashi, M., additional, Mariscal, A., additional, Chen, M., additional, Gokhale, H., additional, Shan, H., additional, Nykanen, A., additional, Gomes, B., additional, Ali, A., additional, Cypel, M., additional, Chan, C., additional, Keshavjee, S., additional, and Liu, M., additional
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- 2021
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11. S02. TUMOUR RISKS AND GENOTYPE-PHENOTYPE ANALYSIS IN AN IRISH COHORT OF PATIENTS WITH GERMLINE MUTATIONS IN THE SUCCINATE DEHYDROGENASE SUBUNIT GENES SDHB, SDHC AND SDHD
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Hynds, P, Coghlan, D, Purcell, C, Green, A, Ward, A, Lynch, SA, Hough, O, Duff, M, Cody, N, Carroll, C, Bradley, L, Green, Andrew, Lynch, Sally-Ann, Crushell, E, Byrne, N, Gorman, K, King, M, Irvine, A, Monavari, A, Knerr, I, Cotter, M, McConnell, V, Browne, F, Lambert, D, Turner, J, Casey, J, Doyle, S, Nesbitt, IM, Fitzgibbon, M, Pastores, G, Kirk, R, Treacy, EP, Benson, KA, Kennedy, C, Yachnin, K, Cavalleri, G L, Conlon, P, McVeigh, Úna M, McVeigh, Terri P, Miller, Nicola, Morris, Derek W, Kerin, Michael J, Irwin, R., Caffrey, A., McLaughlin, M., McNulty, H., Cassidy, T., Pentieva, K., Walsh, C., Minguzzi, S, MacCooey, A, Brosnan, J, Brosnan, M, Henry, M, Meleady, P, Parle-McDermott, A, Gilbert, EH, O’Reilly, S, Merrigan, M, McGettigan, D, Molloy, AM, Brody, LC, Bodmer, W, Hutnik, K, Ennis, S, Lawson, DJ, Wilson, JF, Cavalleri, GL, Flynn, M, Whitton, L, Gill, M, Corvin, A, Donohoe, G, Morrison, C, Morris, D, Stapleton, CP., Birdwell, KA., Mark, PB., Sanders, ML., Phelan, PJ., Maxwell, AP., McKnight, AJ., Kennedy, C., Jardine, A., Traynor, J.P, Chapman, F., Keating, B., Conlon, PJ., Cavalleri, GL., Gunne, EA, Lambert, DM, Martin, R, Donnelly, DE, Callaghan, MB, Morrison, PJ, McConville, DO, Archbold, GP, Lewis, A, Morrison, P J, Das, S., Kelly, D., Moran, B., Harold, E., Han, K., Mulligan, N., Barrett, C., Buckley, P.G., Mc Mahon, P., McCaffrey, J, Van Essen, H. F., Connor, K., Ylstra, B., Lambrechts, D., Gallagher, W.M., O’Connor, D.P., Kelly, C.M., O’Neill, T, Power, C, de Franco, E, Ellard, S, Antao, B, O’Connell, SM, Dabir, T, Heggarty, S, Dockery, A, Carrigan, M, Wynne, N, Keegan, D, Stevenson, K, Silvestri, G, McCourt, J, Humphries, P, Kenna, PF., Farrar, GJ, Agbahovbe, R, Cohen, ASA, Gibson, WT, Cole, AM, Bohlender, R., Hu, H, Heinrich, E, Ramirez, C, Yu, Y, Powell, F, Gaio, E, Villafuerte, F., Taylor, C, Huff, C, Simonson, T., Cavalleri, G., Scullion, C, Irwin, R, Thakur, A, Walsh, C, Shortall, C, Palfi, A, Chadderton, N, Kenna, PF, Boomkamp, S, Shen, S, Hardcastle, AJ, Maloney, DM, Millington-Ward, S, Mackin, S-J, Irwin, R E., O’Neill, KM., Pollin, G, Apostolova, G, Dechant, G, Mackin, SJ, O’Neill, K, Walsh, CP, Sohedein, MNA, Morris, DW, Chaudhry, M, Segurado, R, Shields, D, Wilson, AG, Watkin, R.L., Piskareva, O., Madden, S., Stallings, R., Kerrigan, S.W., O’Neill, K M., Thursby, SJ, Bertens, C, Masala, L, Loughery, J, McArt, D, Amenyah, S. D., McMahon, A., Deane, J., Ward, M., Strain, J.J., Horigan, G., Purvis, J., Lees-Murdock, D., Lynch, SM., Ward, M, McNulty, H, Horigan, G, Purvis, J, Tackett, M, McKenna, DJ., Angel, Z, and Walsh, CP.
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Abstracts ,Poster Presentations ,Oral Presentations ,Article - Abstract
Neurofibromatosis (NF1) affects 1/2500 people throughout the world. Children with NF1 require a multidisciplinary service ideally, delivered on a single site. NF1 is a very variable condition with children requiring the expertise of genetics, paediatricians, ophthalmologists, dermatologists, neurologists and other specialities as required. Building such a service concentrates expertise, facilitates coordination of care and fosters ideal opportunities for research. Aims: 1) To develop a service ensuring children had access to a multidisciplinary clinic on an annual basis. 2) Hold monthly clinics offering ophthalmology, medical, developmental and dermatology follow up. 3) To create a registry of patients which captures the incidence and prevalence of NF1 in Ireland. To offer best possible care for the children attending the service by following international consensus guidelines. 4) To liaise with NF1 Association, families and research authorities. Methods: 1) Appointment of a CNS/CNM2 in Neurofibromatosis as funded by the NCH Foundation. 2) Visit to the complex NF1 Clinic in Manchester’s Children’s Hospital and learn from their service, MDT and guidelines. 3) Establish links with genetics, oncology, radiology and orthopaedic depts. in OLCHC. 4) Create a referral pathway for HCPs to ensure children with NF1 are referred to most appropriate service in a timely fashion. 5) To register the service on Orphanet and gain entry into an ERN as a multi-site service in conjunction with OLCHC. Results/Conclusion: To date, the service has been running for 9 months. The CNM2 provides telephone service and coordinates clinics. The Clinic has been registered in Orphanet and the process has begun to create a patient registry and enter the service in the ERN., Germline mutations in the succinate dehydrogenase subunit genes SDHB, SDHC and SDHD are the most frequent causes of inherited phaeochromocytomas and paragangliomas. Patients presenting with these tumours are usually offered genetic testing for these and other genes as part of standard clinical investigations. However, the information regarding penetrance and phenotype genotype correlations associated with SDHB/C/D mutations is variable, making it difficult to determine an optimum management strategy for this group. In order to address this issue we undertook a retrospective cohort study of patients who underwent genetic testing for SDHB, SDHC or SDHD. 195 patients were identified through the Irish Genetics laboratory electronic database as having had a genetic test for SDHB, SDHC or SDHD and referral source, referral reason and genetic test outcome were analysed. Analysis of penetrance and phenotype presentation was determined through a Clinical Genetics chart review of 147 patients from 40 separate families. Analysis of age-related tumour risks according to relevant gene and mutation type (for SDHB and SDHD) provided estimates of penetrance and genotype-phenotype correlations. Increased knowledge of the molecular basis of phenotypic variability commonly observed in individuals with germline SDHB/C/D mutations will facilitate the development of age-appropriate management protocols based on gene specific tumour risks., Irish Travellers are an endogamous, ethnically Irish population of ~40,000. Consanguinity is common. Knowledge of Traveller disorders exists but mainly in specialised Irish centres. Most Traveller disorders are published but ethnicity is not explicit, hampering diagnoses, particularly if the patient is overseas where knowledge about this population is poor. Aims: To catalogue inherited Irish Traveller disorders through identifying the disorders, detailing mutations, use of coding, (OMIM, Orphacodes & ICD10), publications, and help develop a database to facilitate diagnoses. Methods: A literature review was undertaken. Key national and international Clinician/scientists were contacted to identify relevant disorders and publications. Laboratory and clinical databases were searched to retrieve disorders & mutations. Annotations were updated. An Excel database was established listing each disorder, its appropriate code, associated mutation and relevant publication. Results: 86 distinct rare genetic disorders resulting in 75 phenotypes were identified; 78/86 were autosomal recessive; 4 of these were dominant disorders presenting only in the recessive state. Seven dominant disorders with no recessive phenotype were included as > one affected individual existed. One common 17q12 duplication was included, presenting in two unrelated families. Homozygous mutations were found in all recessive disorders bar one. The genetic basis of 78/86 was established. A further 2/76 have common haplotypes; the genetic basis of six disorders remains unclear. Linkage disequilibrium was observed in 4 families with co-existing McArdles disease and microcephaly & 11 individuals have co-existing Friedreich’s ataxia & galactosemia. Conclusion: Our work is the first step towards cataloguing inherited Irish Traveller disorders. Future challenges include development of an online mutation database., Primary Trimethylaminuria (TMA)(OMIM 136132), is an autosomal recessive rare disorder which results in diminished capacity to oxidise the dietary derived amine trimethylaminuria to its odourless metabolite Trimethylamine-n-oxide (TMA-n-Oxide). Severe primary TMA has been defined as the percentage of unmetabolised free TMA in urine being >40% and mild/moderate TMA range is 10-39%. More than 30 variants of the Flavin monooxygenase 3 (FMO3) have been reported to cause primary TMA. Diagnosis of primary TMA has implications for management of the patient in relation to treatment and genetic counselling. We sequenced the entire FMO3 gene coding region in 10 patients who had a biochemical diagnosis of TMA made in the past 5 years. Three of the patients had severe TMAU (% TMA range 39.4 to 45), (Group A) and 7 had mild to moderate TMAU (%TMA range 10-30), (Group B). We identified causative (loss of function) in 5/10 individuals. Homozygosity for loss of function mutations was detected for 2/3 cases with severe TMAuria (Group A). 3/7 of the patients with mild to moderate TMAuria biochemically had a genetic diagnosis. Two were homozygous for Glu158Lys/ Glu308Gly and the other was compound heterozygous for P153L and A232T. Primary TMAU is rare in Ireland and mutational analysis should not replace biochemical diagnosis.The rate of detection of pathogenic mutations was low using the recommended biochemical cut-offs. The E305X mutation the first FMO3 mutation described in OMIM (136132.0001) in an Irish Australian family may be an Irish Mutation. Two new apparent FMO3 mutations are described in this Irish population. A cut- off of free TMA levels higher than that suggested on the Gene Utility card may be more beneficial in directing genotyping., Background: As part of the Irish Kidney Gene project, 2000 people with renal disease were surveyed and >30% of participants reported a family history for their condition. This strongly suggests an underlying genetic component for the development of kidney disease. Blood and urine tests as well as kidney biopsies are frequently used to inform on aetiology of the disease. However, in around 10% of cases, aetiology is simply unknown, making it difficult for physicians to provide a clear diagnosis or prognosis to these patients. Aim: This project aims to utilise genomic sequencing to stratify patients with hereditary renal disease (HRD). In doing so we seek to aid clinical diagnosis, provide insight into pathogenesis and in some cases point to specific therapies. Methods: We developed a custom, targeted NGS panel for inherited kidney diseases which we have applied to 48 HRD patients. The panel includes 11 genes which are established causes of polycystic kidney disease, von Hippel Lindau syndrome, renal cysts and diabetes syndrome and Alport syndrome. The NimbleGen Heat-Seq kit was used for library preparation and samples were sequenced using an Illumina MiSeq platform at Beaumont Hospital. Data was analysed using a custom bioinformatics pipeline and variants were classified according to the ACMG guidelines. Results/Conclusions: To date, this panel has identified candidate pathogenic variation in a third of samples studied. Future work in this project will include the development of a larger targeted panel including >100 known renal disease genes., Breast cancer is the most common female malignancy worldwide. Up to 10% of cases are the result of an inherited monogenic mutation, while a further 25% appear in familial clusters. Only 30% of hereditary breast cancers are attributed to mutations in BRCA1 and BRCA2, identified as high-risk genes through linkage analysis. While BRCA mutational status is highly informative, and allows clinicians to modify surveillance, prevention and therapeutic strategies, the risk conferred by mutations in other genes is more difficult to define in light of variable penetrance. Next-generation sequencing has been rapidly evolving to advance testing sensitivity and throughput in a cost-effective manner. This progression has made multi-gene testing a practical option when looking to identify inherited mutation(s) in a clinical setting. However, current clinically available multi-gene panels generate many variants of unknown significance in genes that are presently not considered clinically useful. The aim of our study was to design a multi-gene panel to enable the detection of rare, probably pathogenic variants contributing to the susceptibility of breast cancer in an Irish population. An extensive literature review was conducted in order to generate a list of 282 genes with potential association to breast cancer. Targeted DNA enrichment and multiplexed next-generation sequencing was performed on a cohort of 167 samples from the west of Ireland. 90 breast cancer patients and 77 geographically-matched controls were included in this study. Bioinformatic analysis was performed following GATK best practices workflow. Variant data for our 282 selected genes will be presented and discussed., Increasingly accurate surveys of human health throughout the life course has led experts to propose that stresses on the developing child whilst in the mother’s womb can affect the individual’s health later in life. Such long-term effects on health are thought to be mediated by a semi-permanent trace on the genes called an epigenetic mark, mediated by processes such as DNA methylation. DNA methylation patterns may be altered by the mother’s diet, particularly folate – a key component in the DNA methylation cycle. Currently, mothers are recommended to supplement their diet with 400μg folic acid/day as a preventative measure against neural tube defects prior to/during the first trimester. However, there remains no clinical recommendation as to whether mothers should continue supplementation during the latter two trimesters and the potentially heritable effects. Thus, we analysed cord blood samples (n=93) from the Folic Acid Supplementation in the Second and Third Trimesters (FASSTT) randomised control trial for genome-wide DNA methylation. Offspring exposed to folic acid in later pregnancy had fewer highly methylated genomic regions and more intermediately methylated sites. Upon further interrogation, gene ontology analysis revealed these sites are enriched for genes associated with cognition and neurological system processes, and tissue analysis revealed enrichment of affected genes associated with the brain. Cognitive and psychosocial testing of the children at age 7 years, using standardised tests (WPPSI, TEIQue-CSF, RASP), showed that the children supplemented during pregnancy scored significantly higher for emotional intelligence, resilience and verbal IQ. Thus, this study offers a potential biological mechanism linking maternal folate levels with childhood cognition., Introduction: We previously identified the mitochondrial 10-formyltetrahydrofolate synthase enzyme, MTHFD1L, as a risk factor for human Neural Tube Defects (NTD). This association was further supported by a mouse model of mutant mthfd1l, that exhibited an NTD and was rescued with maternal formate supplementation. The abundance of MTHFD1L is also increased in a range of cancers. MTHFD1L performs the last step in mitochondrial one carbon metabolism to produce formate for transport into the cytoplasm. Aim: Given the pivotal role of MTHFD1L in human disease, we sought to decipher the cellular response to the expression level of MTHFD1L in HEK293 cells. Methods: Human MTHFD1L was overexpressed in a stably transfected line using a pcDNA3.2 vector and knocked down using two inducible shRNA constructs that were clonally selected. Cells were grown and sampled over a five-day period. Expression level was confirmed by RT-qPCR. Intracellular and media formate levels were measured using GC-MS. Proteomics analysis was performed on whole cell lysates using LC-MS/MS on an Ultimate 3000 nano LC system coupled to a LTQ Orbitrap XL. Results: Intracellular and media formate levels directly correlated with expression level of MTHFD1L compared to controls within an approximately 1.5 to 3 fold range. Our proteomics analysis showed that MTHFD1L expression level had an effect on proteins involved in DNA synthesis, replication and repair. Discussion: We have demonstrated that MTHFD1L expression level has a direct impact on both intra- and extra-cellular levels of formate and may act as a signal for uncontrolled cell proliferation., Ireland has remained relatively isolated from mainland Europe, notwithstanding historical migrations including the Norse-Vikings, Anglo-Normans, and the British Plantations. Although previous studies have shown the Irish to have elevated levels of homozygosity compared to mainland Europe, the extent of genetic structure within Ireland, and the genomic impact of historical migrations, is largely unknown. Here we illustrate fine-scale genetic structure across Ireland that follows sociological boundaries and present evidence of admixture events into Ireland. Utilising the ‘Irish DNA Atlas’, a DNA cohort (n = 194) of genealogically described Irish individuals with four generations of ancestry linked to specific regions in Ireland, we analysed in combination with 2,039 individuals of regional British ancestry (the PoBI dataset) and show that the Irish population subdivides into 10 distinct geographically-stratified genetic clusters; three of shared British/Irish ancestry, and seven of predominantly ‘Gaelic’ Irish ancestry. This structure is remarkably homogenous, and is associated with very little gene flow barriers within Ireland. Additionally, using a reference of 6,760 European individuals and two ancient Irish genomes, we quantified the ancestry of these Irish clusters within the context of Europe as well as ancient Ireland. We show high levels of north-west French-like and Norwegian-like ancestry within Ireland, and homogenous levels of ancient Irish ancestry in our ‘Gaelic’ Irish clusters. Finally we detect admixture events into Ireland, coinciding with the Plantations of Ulster, as well as Norse-Viking activity within Ireland. Our work informs both on Irish history, as well as the study of Mendelian and complex disease genetics involving populations of Irish ancestry., Schizophrenia affects 1% of adults and is a major global health problem. I am interested in the potential role of the centrosome in schizophrenia. The centrosome, an organelle within cells, plays a crucial role in brain development where it directs cell shape, polarity and motility. The centrosome also seeds the growth of antenna-like signalling structures called primary cilia. Rare mutations in centrosome genes cause disorders that present with severe cognitive deficits and variable neuropsychiatric phenotypes. GWAS data has implicated many genes in schizophrenia. We have shown that seven schizophrenia risk genes encode proteins with centrosomal functions. Of these, SDCCAG8 is also associated with educational attainment in GWAS and the genome-wide significant SNPs for the two phenotypes are in high linkage disequilibrium indicating a pleiotropic effect. We have found that a schizophrenia risk SNP in SDCCAG8 is significantly associated with poorer performance in a social cognition task, in a large Irish dataset of schizophrenia patients and controls (p=0.001). To analyse the molecular function of SDCCAG8 we have used genome editing to knock it out in neuronal and retinal cells. Preliminary data shows that loss of SDCCAG8 impairs cells’ ability to make primary cilia and that their capacity to repair genome damage is reduced. Current work is addressing whether SDCCAG8 affects activities that may contribute to schizophrenia, including cell migration and cell signalling. This could identify molecular mechanisms by which SDCCAG8 mutations contribute to schizophrenia risk and cognition, and help uncover the processes that implicate centrosome genes in neurodevelopmental phenotypes., Multiple genetic loci have been identified for non-melanoma skin cancer (NMSC) in the general population. Polygenic risk score (PRS) was defined as the sum of all alleles associated with a trait weighted by the effect size of that allele as determined by a previous genome-wide association study (GWAS). We tested whether PRS, calculated using a GWAS of NMSC in a non-transplant population, can be used to determine risk of developing and time to NMSC post-transplant. Post-kidney transplant NMSC cases (n=155) and controls (n=442) were collected from Tennessee, Ireland and Scotland. Genetic variants that reached pre-defined levels of significance were chosen from a squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) GWAS, both conducted in non-transplant populations. Using these GWAS results, BCC and SCC PRSs were calculated at each p-value threshold (pT) for each sample. PRSs were tested as a predictor of case:control status using logistic regression and time to NMSC post-transplant in a survival model. SCC PRS calculated at pT 1x10-6 was the most significant predictor of case: control status of NMSC post-transplant (OR per 1 stdev increase in PRS=2.3; corrected P (Pc)=0.04). When NMSC was subdivided into SCC and BCC, SCC PRS pT 1x10-6 significantly predicted case:control SCC (OR=2.5, Pc=0.02) and BCC status (OR=7.6, Pc=0.02). SCC PRS pT 1x10-5 also significantly predicted time to BCC (Pc=0.007, HR=1.8) and SCC (Pc=0.05, HR=1.4). PRS of non-transplant NMSC can be used to predict case:control status of post-transplant NMSC, SCC and BCC as well as time to developing BCC and SCC post-transplant., Introduction: Rare diseases are diseases, which affect a small number of people compared to the general population. In Europe, a disease is considered rare when it affects no more than 5 per 10,000 individuals. A disease can be rare in one region but common in another. The objective of this study was to derive a proxy estimate the number of childhood onset rare diseases through referrals to the country’s only Genetics center, as the Republic of Ireland does not have a centralized rare disease registry. Methods: A retrospective review of referrals to cytogenetics and clinical genetics for the years 2000-2016 for patients born in the year 2000 was undertaken. Anonymized data was catalogued into rare, common, normal, likely rare & unclassifiable by review of records, and assigned Orphacodes based on diagnosis. Census live birth data was used as the denominator. Results: 54,7891 live births were recorded by the census in 2000. 1872 referrals to Genetics (representing 1749 individuals born in 2000) were retrieved for review. 1007 had cytogenetic testing only, of which 51 had rare chromosomal anomalies. Review of 742 referrals to clinical genetics yielded 581 with a rare disease (78%), 7 with a likely rare disease, 56 with a common disorder, 83 who were normal (at risk relative) & 15 unclassified (hadn’t yet been seen). Of the 53/1749 who had died (3%), 51 had a rare disease with congenital malformations (24) the most common cause., Neurofibromatosis type 1 (NF1) is a relatively common autosomal dominant genetic condition, with an incidence of around 1 in 3000. All NF1 patients attend our regional NF1 clinic intermittently and our departmental database records clinical details. Currently, we have 468 living patients affected with NF1 in Northern Ireland. NF1 is caused by mutations, or occasionally deletions, of the neurofibromin tumour suppressor gene, which leads to over-activation of the RAS-MAPK pathway, and tumour formation. These vary from benign lesions, such as neurofibromas, through to malignant peripheral nerve sheath tumours (MPNSTs) and tumours in other sites, particularly the central nervous system, that can be associated with significant morbidity and mortality. MEK inhibitors have recently been shown to be an effective treatment modality in the tumours associated with NF1. We have studied our population to determine the number of patients with plexiform neurofibromas, who are at risk of MPNSTs, and the proportions of patients with tumours elsewhere. This will allow us to identify which patients could benefit from MEK inhibitors in the future., Tuberous Sclerosis complex (TSC) is an autosomal dominant genetic condition which results, in the majority of patients, from a mutation in the TSC1 or TSC2 genes. Many of the patients are affected by angiomyolipomas and sub-ependymal giant cell astrocytomas. There is evidence that mTOR inhibitors, particularly Everolimus, shrink such tumours. In addition, the recent EXIST-3 study showed that Everolimus led to a significant reduction in seizure frequency in TSC patients whose seizures had previously proved resistant to anti-epileptic drug treatment. Consequently, a European licence has been granted to prescribe Everolimus for this indication. In order to determine the potential number of patients who may be eligible for consideration of this treatment, we undertook a complete population survey of epilepsy in our TSC patients. Information was extracted from our database and descriptive statistics were carried out. We were particularly interested in obtaining numbers of those whose seizures were poorly-controlled, defined as requiring 3 or more anti-epileptic drugs to manage their seizures, or requiring neurosurgical intervention. Many of the TSC patients with a diagnosis of epilepsy were also diagnosed with learning difficulties. The possibility of an association between degree of seizure control and severity of learning difficulties was explored. Finally, the annual cost of prescribing Everolimus to Northern Ireland’s TSC patients with poorly-controlled seizures was estimated., Charcot neuroarthropathy is associated with neurological deficit and is often seen in patients with a history of diabetes. Zygodactyly is a common congenital malformation with cutaneous webbing of the second and third toes. To determine the frequency of Zygodactyly in midfoot (tarso-metatarsal) Charcot neuropathy due to diabetes, we analysed a prospective series of twenty-five patients with Charcot neuropathy referred to podiatry clinics from diabetes and vascular departments. Twenty-nine patients with diabetes (but no Charcot neuropathy) were used as controls. Nineteen of the twenty-five patients with type 2 diabetes, peripheral neuropathy, and midfoot Charcot neuroarthropathy, exhibited Zygodactyly as did one of the twenty-nine controls. There was a significant difference between the two groups (Chi squared test p< 0.001). None of the cases or controls had any dysmorphic features or other limb malformations. Zygodactyly occurred in association with midfoot Charcot neuroarthropathy (diabetic neuropathy) in 76% of cases. No association between Zygodactyly, diabetes and Charcot neuropathy has previously been recognised. Genes such as OPG and RANKL affect foot and bone development and MSX1 and PLA2G6 affect spinal and distal nerve development. The possibility of a genetic contribution in patients who develop type 2 diabetes, peripheral neuropathy and Charcot neuroarthropathy must be considered. Zygodactyly may act as a predictive marker for Charcot neuropathy and further identification of regulatory genes may be possible. Until then, recognition of Zygodactyly may allow early intervention and a reduction of complications in patients with Charcot neuropathy., Development of an unusual clinical phenotype across both common and rare cancer types presents a significant challenge from a diagnostic and therapeutic perspective. We describe two distinct cases involving an Ovarian adenocarcinoma and a Medullary Thyroid cancer (MTC) patient and wherein both patients presented with metastases at highly unusual locations, followed by development of an aggressive disease. In first case involving a patient diagnosed with ovarian adenocarcinoma presented with a rare solitary extracranial brain metastases with no other associated metastases after 2 years post-hysterectomy and chemotherapy. Despite surgical removal of the metastatic lesion and stereotactic radiotherapy, the patient showed a further relapse at the initial as well as two additional extracranial regions. Our current analysis of whole-genome sequencing of primary tumour and extracranial lesion, reveal a remarkable difference in the genomic aberration landscape between the primary tumour and the metastases. In addition, we also identify several structural variants including novel gene fusions as well as gross chromosomal abnormalities, which could be potentially utilized as targets for treating this patient further. In the second case, whole-exome sequencing of primary tumour and bone-marrow metastases in the MTC patient identified three germline single nucleotide polymorphisms (SNPs) within the RET proto-oncogene that remained undetected using routine hospital genetic testing procedures. More importantly, we report for the first time in thyroid cancer on the occurrence of a “chromothripsis-like pattern”, which involved shattering of chromosome 4 leading to complete abrogation of normal chromosomal function, along with dramatic widespread copy number aberrations across both primary tumour and bone marrow samples. These results provide a rationale for the application of comprehensive genomic analysis of cancers presenting with unusual and aggressive phenotypes to facilitate more appropriate therapeutic options and diagnoses., Transient Neonatal Diabetes (TNDM) is characterised by diabetes that develops in the first 6 weeks of life and resolves by 18 months. Approximately 70% of cases are classified as TNDM Type-1 (TNDM1), caused by methylation defects on chromosome 6q24. It is associated with some congenital anomalies, however associated hepatobiliary abnormalities are not described. Choledochal cysts are congenital dilations of part or all of the bile duct, occurring in 100,000-150,000 live births. The 5 major types are classified according to the extent of hepatobiliary involvement. Surgical excision of the cyst is indicated to prevent complications such as stone formation, malignancy, cyst rupture and pancreatitis. We describe a case of TNDM1 due to whole chromosome paternal uniparental disomy 6, with co-existence of a type 1a choledochal cyst in a female born following intrauterine growth retardation. Hyperglycaemia soon after birth led to insulin treatment and a diagnosis of TNDM1, with resolution of the diabetes by 4 months of life. Follow up of antenatal findings of a cystic anomaly demonstrated the presence of a type 1a choledochal cyst on ultrasound and magnetic resonance cholangiopancreatography. Sucessful surgical excision of the cyst and a roux-en-Y hepaticojejunostomy was undertaken at 6 months of age. To our knowledge the co-existence of these disorders has not previously been reported. Further genetic analysis by whole exome sequencing is now in progress to determine if a mutation in the PKHD1 gene, unmasked by the paternal UPD of the entire chromosome 6, explains the associated choledochal cyst in this case., Mosaic mutations can go unnoticed, underlie genetic disease or normal human variation, and may be transmitted as constitutional variants to future generations. Marfan syndrome (MFS) is a clinically variable systemic connective tissue disorder involving ocular, skeletal, and cardiovascular systems. The risk to siblings of an identified de novo variant in a proband remains above population risk but less than the 50% risk attributed probands (~75%) who have an affected parent. This is due to somatic and germline mutations reported in rare cases. We describe the phenotypic variability in three siblings with a confirmed heterozygous pathogenic exon 52 fibrillin1 (FBN1) gene variant with clinically unaffected parents Parental leucocyte DNA was tested and did not identify the FBN1 gene variant. Paternity has been unequivocally confirmed and subsequent testing of parental buccal samples failed to detect the variant. One brother had aortic valve replacement and aortic aneurysm repair at 35 while another brother had surgery of aortic dilatation at the sinuses of Valsalva at 32. The brothers had variable joint hypermobility, patellar dislocations and ophthalmic presentations involving subluxed lenses, myopia and ambylopia. Early onset of varicose veins as a teenager in one and thoracolumbar scolosis in another brother were present. Their 42 year old sister has apparently normal aortic and cardiac imaging and ophthalmology but has mild Marfanoid facial features. To our knowledge this is the first reported family in the literature of 3 siblings as a result of parental mosaicism for a FBN1 gene variant and highlights the impact for genetic counselling., The inherited retinal degeneration (IRD) patient cohort used in the study has been obtained via a collaborative network of opthamoloogists whereby if an IRD is suspected given consent, a DNA sample is taken and provided to a central laboratory for genetic analysis. The study seeks to detect previously identified, together with as yet undiscovered, pathological mutations in a panel of known retinal degeneration genes utilizing target capture next generation sequencing (NGS) for 264 IRD genes. The study to date includes over 700 IRD patients from more than 500 pedigrees. While clinical trials are in progress for patients with IRDs, many such trials require patients to have a known causative mutation to participate in these trials. The Target 5000 research project aims to genetically characterise the estimated 5,000 people in Ireland with IRDs. To date, as part of Target 5000, over 10% of the Irish IRD population has been sequenced providing real insights into the genetic architecture of IRDs in Ireland. Target 5000 offers not only a chance to discover new relevant and pathogenic mutations, but is vital to providing patients with information regarding the underlying genetic pathogenesis of their disease. Thus far, during the course of the study, genetic analysis of IRD patients has helped to resolve ambiguous phenotypes and to identify causative mutations in approximately 60% of IRD cases. The growing body of data from NGS studies of IRDs globally should facilitate better correlations between genotype and phenotype and refine methods for diagnoses and prognoses., Overgrowth syndromes are characterized by tall stature, macrocephaly and other congenital features. These disorders typically arise sporadically through de novo dominant mutations in a growing list of genes. Although whole-exome sequencing (WES) allows us to examine all genes at once in a cost effective manner, we are left with a very large number of possible disease-causing variants to sift through. In addition, we must identify at least two patients with mutations in the same novel gene for the finding to be significant. To address this, we utilized detailed phenotyping of patients with undiagnosed overgrowth to group patients with significant phenotypic overlap and to help us interpret and prioritize the variants identified via WES. We performed WES for 12 undiagnosed patients from our overgrowth cohort. For most patients, there were no obvious causative variants in genes that were previously associated with human overgrowth. Therefore we analysed the participants’ clinical records to look for phenotypic traits that may lead us to new candidate genes. After further mining of the WES data, we prioritized possible disease causing variants based on a number of factors including biological function of the gene, predicted effect on protein function and a minor allele frequency, Living the ‘high life’ presents challenging conditions of extreme cold, hypobaric hypoxia and a restrictive diet that forces populations to adapt to survive. The Quechua are an indigenous high altitude population of Peru and Bolivia. They have resided at altitudes greater than 2500 meters above sea level (m.a.s.l) for the past 10,000 years, following their arrival in South America. Previous studies have characterised their adaptive physiology and identified genes under natural selection (ref). However our understanding of their genetic adaptation to hypoxia is incomplete, as previous studies focused on common genetic variation and applied a limited number of selection tests. To shed further light on genetic adaptation in the Quechua, we established a cohort of 43 Quechua individuals from Cerro de Pasco, Peru (4330 m.a.s.l). We performed whole genome sequencing to a mean depth of 34X. We detailed the demographic history of Quechua using principal components analysis, Admixture and Treemix. We performed five tests of selection, (iHS, XP-EHH, ΔiHH, FST and ΔDAF) on real, and simulated Quechua data incorporating details of the demographic history of the population. We performed a composite of multiple signals (CMS), which aggregates information from the five tests of selection, and identified robust signals of positive selection in high altitude Quechua individuals. The Quechua appear as a relatively homogenous population, with 10% European ancestry. We report the top 1% of genes under selection identified by CMS. We identify putative hypoxia associated genes under selection as well as the previously reported well-characterised hypoxia gene EGLN1., DNA methylation is an important epigenetic mechanism of regulating gene expression that is affected in certain human diseases including imprinting disorders and cancer. In mouse, UHRF1 is an essential cofactor of DNMT1, the enzyme responsible for maintaining methylation patterns. To investigate the effects of loss of UHRF1 on methylation patterns in human cells, UHRF1 levels were decreased in immortalized hTERT fibroblast cell lines using short hairpin RNA. Genome-wide effects on methylation were investigated by the Illumina Infinium HumanMethylation450 BeadChip array. Online bioinformatics software tools were used to identify FDR-significant hypomethylated gene classes, which were then verified by pyrosequencing. Transcriptional effects on these gene classes were investigated by the genome-wide Illumina HumanHT-12 v4 Expression BeadChip array, and verified by RT-qPCR. While UHRF1 depletion caused widespread demethylation, the replication-dependent histone gene cluster and the cancer testis antigen genes were identified as most significantly hypomethylated in UHRF1 knockdown cells. Pyrosequencing confirmed hypomethylation in promoter regions of cancer testis antigen genes TSPY2, MAGEC1, MAGEC2 and MAGEA12, and histone gene HIST2H2AA4 in knockdown cell lines. Hypomethylation in these gene classes correlated with an increase in expression in the knockdown cell line. In addition, cells were rescued using UHRF1 cDNA and showed a return to wild type transcription levels in the rescue cell line. We have shown that these genes are regulated by promoter DNA methylation, confirming the sensitivity of cancer-testis genes to demethylation, supporting possible use of methyltransferase inhibitors to boost antigen presentation in cancers, and the crucial role of UHRF1 in cell cycle regulation., X-linked Retinitis Pigmentosa (XLRP) is a severe, early-onset form of inherited retinal degeneration (IRD). It is estimated that approximately 15% of XLRP cases are due to mutations in RP2 (Retinitis Pigmentosa 2). The ubiquitously expressed RP2 protein is involved in ciliary trafficking of lipid-modified proteins – a process vital for photoreceptor function and survival. Most pathogenic RP2 mutations are suggested to result in truncation or complete loss of the protein. The most common stop mutation, R120X, appears to trigger nonsense-mediated decay of the transcript. RP2 is therefore an excellent candidate for gene augmentation therapy. In recent years, personalised cell models have emerged as invaluable tools for the elucidation of disease pathogeneses and have greatly enhanced pre-clinical proof of concept studies. Through the Target 5000 programme, a project focused on genetic characterisation of the 5,000 IRD patients in Ireland, a male patient harbouring the R120X RP2 mutation was identified. A patient-derived dermal fibroblast cell model of the disease was thus generated and characterised. The transduction efficiencies of AAV vectors of various serotypes in fibroblasts were tested and compared, after which it was decided to proceed with an AAV2/2.CAG.RP2 vector to explore RP2 delivery in this patient-derived cell model. In addition, the effects of RP2 overexpression in vivo in murine photoreceptors and retinal pigment epithelium cells were analysed., Mitochondrial dysfunction leads to a lack of energy production and ultimately the death of the cell. Recently a number of disorders have been shown to have mitochondrial dysfunction including but not limited to; Multiple Sclerosis, Parkinson’s and Leber’s Hereditary Optic Neuropathy (LHON). In LHON, Complex I of the Electron Transport Chain (ETC) is affected which leads to a severe shortage of energy in the cell and eventually cell death. In particular retinal ganglion cells (RGCs) are affected, leading to retinal dysfunction and blindness. These observations have prompted interest in exploring innovative therapeutics to modulate mitochondrial disorders involving complex I deficiency. The team has explored candidate gene therapies for complex I deficiency, which could classically be delivered via Adeno Associated Viruses (AAV) such as AAV serotype 2 (AAV2), among other vectors. As such the team has developed novel in vitro methods for the analysis of complex I deficiency and the evaluation of novel candidate therapies, allowing us to monitor the efficacy of these therapeutics. Assays include a suite of methods to enable evaluation of Complex I activity and oxidative phosphorylation efficiency among other mitochondrial biomarkers. Such assays in principle would be of value for future in vitro and or in vivo studies involving therapies directed towards targeting complex I deficiencies., Background: Imprinted loci are paradigms of epigenetic regulation and are associated with a number of genetic disorders in human. A key characteristic of imprints is the presence of a gametic differentially methylated region (gDMR). Previous studies have indicated that DNA methylation lost from gDMRs could not be restored by DNMT1, or the de novo enzymes DNMT3A or 3B in stem cells, indicating that imprinted regions must instead undergo passage through the germline for reprogramming. However new putative gDMR have recently been described, along with an improved delineation of the existing gDMR locations. We therefore aimed to re-examine the dependence of methylation at gDMRs on the activities of the methyltransferases in mouse embryonic stem cells (ESCs). Method: We examined the most complete current set of imprinted gDMRs that could be assessed using quantitative pyrosequencing assays in two types of ESCs: those lacking DNMT1 (1KO) and cells lacking a combination of DNMT3A and DNMT3B (3abKO). Results: Loss of methylation was approximately equivalent in both cell types. 1KO cells rescued with a cDNA-expressing DNMT1 could not restore methylation at the imprinted gDMRs, confirming previous observations. However, nearly all gDMRs were remethylated in 3abKO cells rescued with a DNMT3A2 expression construct (3abKO + 3a2). Transcriptional activity at the H19/ Igf2 locus also tracked with the methylation pattern, confirming functional reprogramming in the latter. Conclusions: DNMT3A/B plays a vital role in methylation maintenance at imprints as the rescue with DNMT3A2 can restore imprints in these cells. This provides a useful system to explore factors influencing imprint reprogramming., SATB2, BCL11B and GATAD2A map to regions containing genomewide significant SNPs for schizophrenia and regulate key stages of neurodevelopment via epigenetic mechanisms. SATB2 mediates the projection of neurons across the cerebral hemispheres by regulating the activity of BCL11B via the NuRD nucleosome remodelling complex, which contains. GATAD2A. We hypothesized that genes within the NuRD complex and genes regulated by SATB2 in the pre- and post-natal brain may contribute to schizophrenia etiology. To test, we developed three gene-sets. 1.)Genes reported in mouse knockout studies of SATB2 during cortical development (SATB2_ Cortical). 2.)Genes mapping to SATB2 ChIP-seq peaks generated from mouse cortices at E15.5 (SATB2_Pre-natal). 3.)Genes mapping to SATB2 ChIP-seq peaks generated from mouse P0 hippocampal neurons (SATB2_Post-natal). We performed competitive gene set analysis (GSA) using MAGMA to test if genes within a gene-set were more strongly associated with schizophrenia than other genes in the genome. We applied GSA to schizophrenia GWAS (n=150,064). We also investigated these gene-sets for a genetic contribution to educational attainment (EA; proxy for cognition) using GWAS (n=405,072). After multiple test correction, we observed significant associations for (1)SATB2_Cortical with schizophrenia (P=8.65x10-05) and EA (P=0.00049), (2)SATB2_Pre-natal with EA (P=0.0068) and (3)SATB2_Post-natal with schizophrenia (P=0.0069) and EA (P=2.03x10-06). Further GSA established that effect sizes are stronger for these gene-sets when analysis is limited to genes that are highly expressed in neurons or at different key timepoints during neurodevelopment of the cortex or hippocampus. These data support a role for the NuRD complex and genes regulated by SATB2 in schizophrenia and EA, Background: Dacogen (5-aza-2’deoxycitidine) is currently used to treat Acute Myeloid Leukaemia (AML) and is in trials for myeloid dysplastic syndrome and some solid cancers. As a hypomethylating agent it is thought to act by inhibiting the enzymes which add methyl groups to DNA, chief among them DNMT1. Improved targeting has been hindered by a lack of understanding with respect to the exact mechanism of action on DNMT1 and of the gene targets affected by altered methylation following treatment. Methods: We performed a comparative treatment of the same normosomic, non-transformed fibroblast cell line hTERT1604 over three days with either pharmacological 5-aza-2’deoxycitidine (Dacogen) or with SMARTpool siRNA directly targeting DNMT1. DNA was collected for analysis of methylation levels using Illumina 450k BeadChip methylation arrays. Data was analysed in R using the tailored RnBeads pipeline and in-house scripts. Results: Both Dacogen and DNMT1 siRNA caused overall hypomethylation in the treated cells, with the latter proving more efficient at demethylation at genes in particular. Amongst the targets experiencing demethylation, some hypomethylated promoters were unique to Dacogen treatment and therefore off-target with respect to the reduction in DNMT1. However an unexpected phenomenon almost exclusively caused by 5-Aza-2’-deoxycytidine treatment was gain in methylation. Therefore we also compared our findings to an independent published 450k dataset of Dacogen treated AML cells (KG1a). Our results suggest Dacogen is also having an important effect on methylation unrelated to the inhibition of DNMT1 thus suggesting further avenues for therapeutic improvements., Disruptive, damaging ultra-rare variants (dURVs) are more abundant in schizophrenia (SZ) patients than controls and are more concentrated in neuronally-expressed genes with synaptic functions. dURVs in highly constrained genes influence educational attainment (EA; a proxy for cognition) in the general population. We used MAGMA to perform gene set analysis of the largest available GWAS datasets to investigate if association signals for SZ and EA similarly mapped to highly constrained genes and to neuronally-expressed genes with synaptic functions. We investigated if SZ and EA associations were enriched in brain regions at different timepoints from early development through to adulthood. Highly constrained genes (probability of being loss-of-function intolerant; pLI>0.9; n=3,230) are strongly enriched for association with SZ(p=3.14E-08) and EA(p=1.27E-09) in comparison to genes under less constraint (0.1, Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease affecting 45,000 people in Ireland. Prolonged joint inflammation results in tissue damage with consequent reduced functional capacity and quality of life. Damage to the joints of hands and feet, assessed by x-ray, is an important outcome measure that has genetic input of around 60%. Recent studies have identified single nucleotide polymorphisms (SNPs) in immune-related genes that are associated with severity of tissue damage in RA. One of our studies identified an association with C5orf30, a previously uncharacterized regulator of tissue damage and inflammation (1, 2). However a more comprehensive genome wide analysis is required to more fully characterize the genetic basis of RA severity. This project will identify genetic variants, and their synergistic combinations, that are associated with severity of RA. We will analyse genomewide SNP data in 1,007 RA patients using state-of-the-art genetic epidemiology and computational techniques, including negative binomial modelling, to identify variants linked with joint damage severity. The study population is uniquely large and detailed clinical and genetic datasets will be used for validation studies using five European early RA cohorts. Simulations for statistical power indicate excellent power will be achieved for moderately frequent alleles, for effect sizes (IRR) over 1.4. The aim is to develop both a genetic prognostic score for RA, and to identify novel mediators of tissue destruction. The earlier identification of RA patients at risk of poorer outcome would facilitate patient stratification and inform therapeutic targeting with more aggressive regimes whilst avoiding such treatment in patients likely to have a better outcome, Bloodstream infection and sepsis are often instigated by the bacterium Staphylococcus aureus. Upon accessing the bloodstream, S. aureus binds to the endothelium triggering vascular leakage, inflammation and oedema. These characteristics are difficult to treat pharmacologically as the nature of signalling guiding this host response remains unclear. microRNAs (miRNAs) regulate ~60% of the human genome through post-transcriptional silencing/ degradation of target genes. Previously, bacteria were shown to profoundly affect miRNA expression via up-regulation of dendritic miR-99b elicited by M. tuberculosis infection. This study investigates contributions of S. aureus induced endothelial miRNA dysregulation to sustained and excessive host responses in sepsis. Sheared (10dynes/cm2) human endothelial cells were treated with plasma and TNFα to mimic sepsis conditions. Infection induced miRNA alterations were uncovered using Taqman cards to generate miRNA profiles of uninfected and infected cells (RQ = 2-ΔΔCt). Potential mRNA targets were established bioinformatically and confirmed by RNAseq, western blots and qPCR. Following infection, 58 endothelial miRNA were significantly downand 35 significantly up-regulated, including miR-330 (p, DNA methylation is a critical mechanism for regulating gene expression and ensuring genomic stability. However, loss of function mutations of methyltransferase enzymes such as DNMT1 in normal differentiated cells result in a lethal phenotype. Consequently, existing investigations have only assessed DNMT1 knockdowns in embryonic stem cells or cancer cell lines. Here, isogenic lines of hypomorphic, normal, immortalised fibroblasts have instead been generated via stable integration with short hairpin RNA. Enrichment analysis of epigenome-wide methylation arrays indicated widespread demethylation within promoter and gene body regions. In addition, four specific gene categories were highlighted as most affected; protocadherins, genes regulating body mass, olfactory receptors and cancer/testis antigens. Comparison of short-term siRNA and long-term shRNA-mediated depletion of DNMT1 indicated that many regions recover methylation as shRNA-containing cell lines adapt to lowered levels of DNMT1. Interestingly, polycomb-regulated genes are refractory to de novo DNA methylation in these cells following recovery, reinforcing the concept of mutually-exclusive domains that are regulated by these two major epigenetic mechanisms., Background: The MTHFR C677T is a common polymorphism of the folate metabolising enzyme methylene tetrahydrofolate reductase (MTHFR) associated with hypertension. Riboflavin is a cofactor to MTHFR in the one-carbon cycle for generating methyl groups important for biological reactions such as DNA methylation. Supplementation with riboflavin has previously been shown to reduce blood pressure specifically in individuals with the homozygous MTHFR 677TT genotype. The mechanisms underlying the blood pressure lowering effect of riboflavin are currently unknown however aberrant DNA methylation has been implicated in the development of hypertension. The aims of this study were to examine global DNA methylation on hypertension in adults stratified by MTHFR genotype and in response to intervention with 1.6mg/ day of riboflavin in individuals with the MTHFR 677TT genotype. Methods: Stored peripheral blood leukocyte samples from participants who had consented and participated in targeted RCTs at Ulster University’s Nutrition Innovation Centre for food and HEalth (NICHE) and previously screened for the MTHFR C677T polymorphism were accessed for this study. Bisulphite conversion and pyrosequencing was used to analyse global and gene-specific DNA methylation. Results: Preliminary results show that methylation at the repeat element, LINE-1, and imprinted gene, IGF2 was not significantly different between the MTHFR C677T genotypes at baseline. However, subsequent supplementation with riboflavin resulted in a decrease in global methylation and an increase in IGF2 methylation in MTHFR 677TT participants. Conclusion: This is the largest study to date examining the interaction between the MTHFR C677T genotypes, riboflavin supplementation and DNA methylation. Riboflavin supplementation influenced repeat element and imprinted gene methylation in MTHFR 677TT genotype individuals. Further work will provide insights into the mechanism of riboflavin action in lowering blood pressure in these genetically at risk adults., Background: microRNAs are small, non-coding RNAs which are potentially valuable markers of cardiovascular disease (CVD) risk, including hypertension. This novel investigation aims to profile circulating serum concentrations of microRNAs in premature CVD patients to identify microRNAs that correlate best with hypertension. Methods: Serum samples from an existing cohort of 75 premature CVD patients were analysed for expression of 68 CVD-related microRNAs. Patients had been screened for the methylenetetrahydrofolate reductase (MTHFR) gene polymorphism C677T, a risk factor for hypertension. Samples had been collected at baseline and following intervention with riboflavin, co-factor for the MTHFR enzyme, as part of a placebo-controlled double-blind, randomized trial. The associations between miRNA expression and blood pressure at baseline and post-intervention were investigated. Comparisons of data between CC and TT MTHFR genotype groups, and in response to intervention, were assessed using ANOVA, Pearson’s correlation and corrected t-test statistical analyses. Results: microRNA expression was successfully detected and quantified in all samples. At baseline miR-199a-5p expression was inversely correlated (r=-0.51;p, Background: Hypoxia in prostate tumours has been associated with disease progression and metastasis. MicroRNAs are short non-coding RNA molecules which are important in several cell processes, but their role in hypoxic signalling is still poorly understood. miR-210 has been linked with hypoxic mechanisms, but this relationship has not been extensively studied in a prostate cancer setting. Therefore, in this study, we investigate the link between hypoxia and miR-210 in prostate cancer cells. Methods: In this study we have used prostate cancer models of hypoxia to investigate the functionality of miR-210. Expression levels of miR-210 have been measured by qPCR in in vitro and in vivo samples. Functional bioassays were used to examine its effect on prostate cancer cell behaviour. Target genes have been identified and bioinformatic analysis has been employed to investigate a clinical significance for miR-210 in prostate cancer. Results: miR-210 is induced by hypoxia in prostate cancer cells. Over-expression of miR-210 impacts upon target genes which in turn may affect cell proliferation. Data-mining of online repositories of clinical prostate sample data shows that miR-210 is significantly correlated with Gleason grade and other clinical markers of prostate cancer progression. Further in silico analysis of miR-210 cellular networks reveal that miR-210 plays a key role in a number of important cell processes, the dysregulation of which can promote the development of prostate cancer. Conclusions: We propose that miR-210 is an important regulator of cell response to hypoxic stress and may play an important role in the pathogenesis of prostate cancer. Further study will focus on determining its function in prostate cancer and its potential as a biomarker in this disease.
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- 2018
12. (113) Improved ex Vivo Lung Perfusion (EVLP) with Dialysis and Nutrition to Achieve Successful 36h EVLP and Lung Transplantation
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Hough, O., Mariscal, A., Yamamoto, H., Mangat, H., Taniguchi, D., Gokhale, H., Chen, M., Shan, H., Bojic, D., Aulja, T., Ali, A., Main, K., Yoshiyasu, N., Chan, C., Cypel, M., Keshavjee, S., and Liu, M.
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- 2023
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13. (22) A Computational Approach to Breath-By-Breath Ventilator Waveform Data Extraction and Analysis During Ex Vivo Lung Perfusion Enables Enhanced Physiological Lung Assessment
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Zhou, X., Sorbo, L. Del, Hough, O., Chao, B.T., Ali, A., Brambate, E., Ribeiro, R.V., Gomes, B., Nardo, M. Di, Yeung, J.C., Liu, M., Cypel, M., Wang, B., Keshavjee, S., and Sage, A.T.
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- 2023
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14. Revisiting an Old Concept in a New Era: 36 Hour Lung Preservation Using 10ºC Static Cold Storage
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Ali, A., primary, Gomes, B., additional, Wang, A., additional, Ribeiro, R., additional, Galasso, M., additional, Hough, O., additional, Beronical, E.L., additional, Waddell, T.K., additional, Liu, M., additional, Andreazza, A.C., additional, Keshavjee, S., additional, and Cypel, M., additional
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- 2020
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15. Addition of Dialysis to Ex-Vivo Lung Perfusion Maintains Homeostasis and Stability of Donor Lungs: A Pilot Study
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Hough, O., primary, Gao, X., additional, Yang, C., additional, Takahashi, M., additional, Mariscal, A., additional, Nykanen, A., additional, Gomes, B., additional, Ali, A., additional, Cypel, M., additional, Chan, C., additional, Keshavjee, S., additional, and Liu, M., additional
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- 2020
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16. Pulmonary Stress Index During Ex Vivo Lung Perfusion is Associated with Evlp and Lung Transplant Recipient Outcomes.
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Hough, O., Zhou, X., Nardo, M. Di, Ali, A., Brambate, E., Ribeiro, R., Gomes, B., Cypel, M., Slutsky, A., Sage, A., Keshavjee, S., and Sorbo, L. Del
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LUNG transplantation , *ARTIFICIAL respiration , *STRAINS & stresses (Mechanics) , *PERFUSION , *LUNGS , *LUNG injuries - Abstract
During ex vivo lung perfusion (EVLP), hourly assessment of ventilator parameters, such as compliance, are used to assess suitability of lungs for transplant. Previous analysis of the pressure-time curve during EVLP suggested the stress index (SI) as an additional marker of tidal recruitment (SI <0.95) and tidal hyperinflation (SI>1.05), which are mechanisms of lung injury caused by mechanical ventilation. Herein, we sought to validate the change of the SI over time during EVLP and its association with EVLP and lung transplant recipient outcome. Flow, volume and airway pressures were recorded continuously from a Servo-i ventilator during the entire duration of n=50 clinical EVLP cases, of which 32 were accepted for transplant. Breath-by-breath measurements of the SI were extracted during standard EVLP assessment settings (V T = 10 ml/kg donor predicted body weight, delivered with squared flow waveform at 10 breaths/min). We evaluated whether the SI during EVLP and the difference of SI (Δ-SI) above or below its safe threshold (0.95-1.05) predicted EVLP outcome and post-transplant recipient duration of mechanical ventilation. Median [min-max] SI during EVLP changed from 1.06 [0.68-1.63] to 1.07 [0.78-1.6] in accepted cases and from 1.2 [0.7-1.7] to 1.3 [0.6-1.6] in declined cases. Δ-SI was significantly higher in lungs declined for transplant compared to those accepted (p<0.01) (Figure a). In EVLP cases accepted for transplant, SI was lower in lungs whose recipients had post-transplant duration of mechanical ventilation of ≤72h compared to those with mechanical ventilation >72h (1.05 [0.8-1.2] vs 1.14 [1-1.6];p<0.05) (Figure b). Stress index is a novel indicator of lung injury during EVLP that can predict EVLP outcome and short-term outcome of lung transplant recipients. Use of this parameter may guide organ-specific ventilation settings to minimize lung injury from mechanical stress and enhance clinical lung management and decision making during EVLP. [ABSTRACT FROM AUTHOR]
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- 2023
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17. (318) - Towards the Digital Lung: A Deep Learning Approach to Simulating Physiological Lung Function During Ex Vivo Lung Perfusion.
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Zhou, X., Del Sorbo, L., Hough, O., Borrillo, T., Grubert Van Iderstine, M., Chao, B.T., Orsini, L., Valero, J., Cypel, M., Wang, B., Keshavjee, S., and Sage, A.T.
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DEEP learning , *LUNGS , *PERFUSION - Published
- 2024
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18. Pearl Harbor to Guadalcanal.
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Smith, Robert Ross, primary, Hough, O., additional, Ludwig, Verle E., additional, and Shaw, Henry I., additional
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- 1959
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19. Development of Mini-Circuit Ex-Vivo Lung Perfusion to Accelerate Human Lung Translational Research.
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Yamamoto, H., Mariscal, A., Hough, O., Mesaki, K., Taniguchi, D., Gokhale, H., Chen, M., Shan, H., Suzuki, Y., Yoshiyasu, N., Yamanashi, K., Aujla, T., Bojic, D., Sorbo, L. Del, Yeung, J., Liu, M., Cypel, M., and Keshavjee, S.
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LUNG diseases , *OXYGENATORS , *TRANSLATIONAL research , *LUNGS , *PERFUSION , *LUNG transplantation - Abstract
Ex-vivo lung perfusion (EVLP) has significantly advanced translational research. Studies using human donor lungs declined for clinical transplantation or diseased lungs explanted from the recipient, create a unique opportunity to evaluate new therapies in human lungs prior to clinical trials. Considerable and heterogeneous injuries present in these human lungs, however, and this has been a challenge in performing reliable EVLP assessments. We hypothesized that developing a mini-circuit EVLP could allow for effective selective ex vivo perfusion of lobes with lung protective low-flow EVLP and protective ventilation to enable prolonged evaluation of treatment response in injured lungs. In this study, we developed a mini-circuit to perform lobar EVLP and evaluated its feasibility. To accurately perfuse a lobe or single lung at low-flow, we developed a novel mini circuit using a pump, oxygenator, and ventilator which are used for pediatric surgery (Fig A, B). As a proof of concept, we first tested the Mini-EVLP using a pig left lung. Next, we adapted the low-flow EVLP to perfuse a lobe or single lung of the declined human donor lungs and diseased human lungs explanted from a recipient. The group of pig lung Mini-EVLPs showed stable peak airway pressure, lung compliance and delta pO 2 , comparable to the conventional EVLP group (Fig C). In the human lung study, we successfully established lobe-selective EVLP of declined severely injured human donor lungs up to 21h (Fig D). Furthermore, with this protective low-flow Mini-EVLP system, we maintained within physiologic targets diseased lungs, which include lungs explanted from ILD, COPD, and CF patients, up to 24h (Fig E). We have developed a novel mini EVLP circuit and successfully perfused porcine lungs, declined human donor lungs, and diseased human lungs. Our Mini-EVLP protective perfusion protocol provides a new platform for efficient drug screening and high-quality preclinical evaluation of novel therapies in the human lung. [ABSTRACT FROM AUTHOR]
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- 2023
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20. Human Pulmonary Endothelial Function is Improved by Nutrient Supplementation in Ex Vivo Lung Perfusion Perfusates.
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Bojic, D., Vellanki, R., Hough, O., Wouters, B., Andreazza, A., Keshavjee, S., and Liu, M.
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HUMAN cell culture , *CELL physiology , *DIETARY supplements , *PERFUSION , *LUNGS - Abstract
Ex Vivo Lung Perfusion (EVLP) uses a circulating perfusate to restore organ metabolism at normothermia to allow for donor lung assessment and treatment prior to transplant. Supplementing the lung perfusate with nutritional additives such as total parental nutrition (TPN) or glutamine (GlutaMAX) has extended porcine EVLP. However, to further prolong EVLP duration, which is required for advanced donor lung repairs, high throughput evaluation of the role of specific nutrients is required. We worked to optimize nutrient supplementation in the perfusate to address metabolic demands and maintain basic cell function of human endothelial cells in culture. Mathematical models were developed to estimate nutrient and metabolic waste accumulation in the perfusate during EVLP. TPN, Intralipid and glutamine additives were assessed in different combinations based on a dose response curve in our high-throughput screening pipeline. Important cellular characteristics such as confluence, apoptosis, and migration, were evaluated in human pulmonary microvascular endothelial cells (HPMECs) and monitored on the Incucyte SX5 system. In total, 21 experimental perfusates were studied, with the current clinically used Steen solution serving as a control. HPMECs exposed to perfusates supplemented with TPN showed improved cell confluence and migration, and decreased apoptosis only up to 24h relative to the control (p<0.05). However, cells cultured in perfusates containing glutamine at 4 mM showed improved results in all three parameters at 24h and 48h (p<0.05). Intralipid supplementation alone did not differ from control. The combination of TPN, glutamine and Intralipid was similar to glutamine alone. The addition of TPN and Intralipid to control EVLP perfusate showed benefits only until 24h, however the addition of glutamine demonstrated significant improvement in human endothelial cell function until 48h. This suggests that in addition to basic supplementation with TPN and Intralipid, further improvement in cell function can be achieved with the addition of glutamine. This cell culture model provides an important screening capability to develop enhancements of ex vivo lung perfusion solutions to better support donor lungs ex vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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21. A Computational Approach to Breath-By-Breath Ventilator Waveform Data Extraction and Analysis During Ex Vivo Lung Perfusion Enables Enhanced Physiological Lung Assessment.
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Zhou, X., Sorbo, L. Del, Hough, O., Chao, B.T., Ali, A., Brambate, E., Ribeiro, R.V., Gomes, B., Nardo, M. Di, Yeung, J.C., Liu, M., Cypel, M., Wang, B., Keshavjee, S., and Sage, A.T.
- Subjects
- *
DATA extraction , *DATA analysis , *LUNGS , *WAVE analysis , *PERFUSION - Abstract
During clinical ex vivo lung perfusion (EVLP), extended criteria donor lungs are supported by an ICU-grade ventilator and hourly assessments of lung physiology are recorded. While the monitoring of breath-by-breath data is possible, manual data acquisition and analysis is impractical which precludes its use in clinical decision-making. Herein, we describe the development of a computational approach to ventilator waveform analysis and report novel physiological ex vivo lung features associated with patient outcomes post-transplantation. Flow and pressure data were recorded at a resolution of 100Hz using the Servo-i ventilator (Maquet, USA) during n=50 clinical EVLP cases. An algorithm for the extraction of waveform data was developed using R programming language and designed to analyze individual breaths for lung compliance (static, dynamic) and airway pressure (peak, mean, plateau) during all breaths recorded during EVLP (n≥1200 breaths per case). Waveform features were used to predict recipient time to extubation post-transplantation. Automated breath-by-breath data processing was successfully achieved for airway pressures and lung compliance. Analysis of the dynamic compliance waveform yielded novel features associated with transplant outcomes. We observed that a subtle decreasing trend in dynamic compliance during assessment (-0.01mL/cmH 2 O/breath, p=0.05) was associated with prolonged time to extubation. In addition, the magnitude of the difference in compliance from assessment to baseline tidal volume (10 to 7cc/kg) was significantly associated with the duration of mechanical ventilation post-transplant (+14.9mL/cmH 2 O, p<0.01). Donor lungs that demonstrated an improvement in baseline, non-assessment, dynamic compliance during EVLP were strongly associated with good recipient outcomes (+7.85mL/cmH 2 O, p<0.001). Lastly, a multivariate logistic regression model combining dynamic compliance waveform features had an AUROC of 79% for predicting a time to extubation of <72h. This study presents a novel computation approach to analyze EVLP ventilator waveform data in detail. Breath-by-breath features extracted during ex vivo lung ventilation enables the discovery of novel predictors of post lung transplant outcomes. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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22. (555) - CRISPR Genome Engineering in Donor Lung During Cyclic EVLP with 10°C Preservation: Usage of TorEx Lung Perfusion System.
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Yoshiyasu, N., Mariscal, A., Salvador, A., Hough, O., Yamamoto, H., Gokhale, H., Shan, H., Chen, M., Guan, Z., Suzuki, Y., Yao, Y., Mesaki, K., Nagoya, A., Juvet, S., Waddell, T.K., Liu, M., Cypel, M., and Keshavjee, S.
- Subjects
- *
GENOME editing , *CRISPRS , *LUNGS , *PERFUSION , *CYCLIC voltammetry - Published
- 2024
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23. Alpha-1-antitrypsin safely promotes rapid recovery of pigs after lung transplantation.
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Mariscal A, Tikkanen J, Calderone L, Hough O, Chen M, Martinu T, Juvet S, Cypel M, Liu M, and Keshavjee S
- Subjects
- Animals, Swine, Neutrophils, Leukocyte Elastase, Lung, alpha 1-Antitrypsin therapeutic use, Lung Transplantation adverse effects
- Published
- 2023
- Full Text
- View/download PDF
24. L-alanyl-L-glutamine modified perfusate improves human lung cell functions and extend porcine ex vivo lung perfusion.
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Huang L, Hough O, Vellanki RN, Takahashi M, Zhu Z, Xiang YY, Chen M, Gokhale H, Shan H, Soltanieh S, Jing L, Gao X, Wouters BG, Cypel M, Keshavjee S, and Liu M
- Subjects
- Animals, Humans, Endothelial Cells, Interleukin-8 pharmacology, Organ Preservation, Perfusion, Swine, Lung blood supply, Lung physiology, Lung Transplantation
- Abstract
Background: The clinical application of normothermic ex vivo lung perfusion (EVLP) has increased donor lung utilization for transplantation through functional assessment. To develop it as a platform for donor lung repair, reconditioning and regeneration, the perfusate should be modified to support the lung during extended EVLP., Methods: Human lung epithelial cells and pulmonary microvascular endothelial cells were cultured, and the effects of Steen solution (commonly used EVLP perfusate) on basic cellular function were tested. Steen solution was modified based on screening tests in cell culture, and further tested with an EVLP cell culture model, on apoptosis, GSH, HSP70, and IL-8 expression. Finally, a modified formula was tested on porcine EVLP. Physiological parameters of lung function, histology of lung tissue, and amino acid concentrations in EVLP perfusate were measured., Results: Steen solution reduced cell confluence, induced apoptosis, and inhibited cell migration, compared to regular cell culture media. Adding L-alanyl-L-glutamine to Steen solution improved cell migration and decreased apoptosis. It also reduced cold preservation and warm perfusion-induced apoptosis, enhanced GSH and HSP70 production, and inhibited IL-8 expression on an EVLP cell culture model. L-alanyl-L-glutamine modified Steen solution supported porcine lungs on EVLP with significantly improved lung function, well-preserved histological structure, and significantly higher levels of multiple amino acids in EVLP perfusate., Conclusions: Adding L-alanyl-L-glutamine to perfusate may provide additional energy support, antioxidant, and cytoprotective effects to lung tissue. The pipeline developed herein, with cell culture, cell EVLP, and porcine EVLP models, can be used to further optimize perfusates to improve EVLP outcomes., (Copyright © 2022 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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- View/download PDF
25. Evaluation of 10°C as the optimal storage temperature for aspiration-injured donor lungs in a large animal transplant model.
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Abdelnour-Berchtold E, Ali A, Baciu C, Beroncal EL, Wang A, Hough O, Kawashima M, Chen M, Zhang Y, Liu M, Waddell T, Andreazza AC, Keshavjee S, and Cypel M
- Subjects
- Animals, Disease Models, Animal, Lung metabolism, Organ Preservation, Swine, Temperature, Lung Transplantation, Reperfusion Injury metabolism
- Abstract
Background: Our recent work has challenged 4°C as an optimal lung preservation temperature by showing storage at 10°C to allow for the extension of preservation periods. Despite these findings, the impact of 10°C storage has not been evaluated in the setting of injured donor lungs., Methods: Aspiration injury was created through bronchoscopic delivery of gastric juice (pH: 1.8). Injured donor lungs (n = 5/group) were then procured and blindly randomized to storage at 4°C (on ice) or at 10°C (in a thermoelectric cooler) for 12 hours. A third group included immediate transplantation. A left lung transplant was performed thereafter followed by 4 hours of graft evaluation., Results: After transplantation, lungs stored at 10°C showed significantly better oxygenation when compared to 4°C group (343 ± 43 mm Hg vs 128 ± 76 mm Hg, p = 0.03). Active metabolism occurred during the 12 hours storage period at 10°C, producing cytoprotective metabolites within the graft. When compared to lungs undergoing immediate transplant, lungs preserved at 10°C tended to have lower peak airway pressures (p = 0.15) and higher dynamic lung compliances (p = 0.09). Circulating cell-free mitochondrial DNA within the recipient plasma was significantly lower for lungs stored at 10°C in comparison to those underwent immediate transplant (p = 0.048), alongside a tendency of lower levels of tissue apoptotic cell death (p = 0.075)., Conclusions: We demonstrate 10°C as a potentially superior storage temperature for injured donor lungs in a pig model when compared to the current clinical standard (4°C) and immediate transplantation. Continuing protective metabolism at 10°C for donor lungs may result in better transplant outcomes., Competing Interests: Disclosure statement MC, TW, SK are shareholders of Traferox Technologies Inc, Traferox devices were not used in any part of the study. MC, TW and SK are consultants for Lung Bioengineering Inc. None of the other authors have conflict of interest to disclose. This work was supported by the UHN Foundation #5790-6833-0776 (MC). We thank Paul Chartrand (Latner Thoracic Surgery Laboratories) for supplies and logistics management throughout the study., (Copyright © 2022 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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26. Near-infrared fluorescence imaging during ex vivo lung perfusion: Noninvasive real-time evaluation of regional lung perfusion and edema.
- Author
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Nykänen AI, Mariscal A, Ali A, Hough O, Michaelsen VS, Liu M, Cypel M, and Keshavjee S
- Subjects
- Animals, Edema, Humans, Lung blood supply, Lung diagnostic imaging, Optical Imaging, Perfusion methods, Prospective Studies, Swine, Indocyanine Green, Lung Transplantation adverse effects, Lung Transplantation methods
- Abstract
Objective: Ex vivo lung perfusion (EVLP) is an excellent platform to evaluate donor lung function before transplantation, but novel methods are needed to accurately confirm transplant quality. Near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) has been used in various clinical perioperative applications to evaluate tissue perfusion. We used NIRF imaging during pig and human EVLP to evaluate donor lung perfusion and edema., Methods: Pig lungs with various degrees of lung injury (n = 10) and human lungs rejected from clinical transplantation (n = 3) were imaged during EVLP using intravascular ICG and a SPY Elite (Stryker) NIRF imaging unit. Optimal ICG and imaging conditions, and perfusion and edema quantification methods, were established. Pig lung transplants with extended graft preservation (n = 5) and control native lungs (n = 6) were also imaged., Results: A single ICG dose resulted in sustained donor lung NIRF throughout the EVLP. Even and homogenous ICG signal was demonstrated in areas of normal lung. Low NIRF was present in regions with poor tissue perfusion, and rapid, intense ICG accumulation occurred in damaged and edematous areas. Segmental perfusion defects were common in the peripheral and elevated regions of the lungs, and serial imaging showed gradual perfusion recovery during EVLP. Impaired microvascular reperfusion, indicated by a decreased NIRF ingress rate, was detected in transplanted pig lungs early after reperfusion., Conclusions: NIRF imaging enables noninvasive real-time evaluation of lung perfusion and edema during EVLP. Prospective clinical studies are needed to determine the role of NIRF imaging in donor lung assessment and selection, and prediction of posttransplant outcomes., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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- View/download PDF
27. Cell death and ischemia-reperfusion injury in lung transplantation.
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Capuzzimati M, Hough O, and Liu M
- Subjects
- Cell Death, Humans, Inflammation etiology, Lung, Perfusion adverse effects, Lung Transplantation adverse effects, Reperfusion Injury prevention & control
- Abstract
Lung transplantation is the most effective therapy for patients with end-stage lung disease. However, concern of donor lung damage and ischemia-reperfusion induced lung injury limits the use of "marginal" donor lungs. Recent transcriptomic studies have demonstrated that the enrichment of gene-clusters related to cell death and inflammation are the most profound signals during ischemia-reperfusion in human lung transplants. Herein, we focus on the relationship between inflammation and programmed cell death, especially necroptosis, mitochondrial permeability transition-initiated regulated necrosis, pyroptosis, ferroptosis, and autophagic cell death. Cell death-related molecules have been tested as potential biomarkers for donor lung assessment. Inhibitors for various types of cell death have been explored as therapeutics for ischemia reperfusion injury in lung transplantation. A deeper understanding of these mechanisms may help to improve donor management, organ preservation, prevention and treatment of primary graft dysfunction during and post transplantation. Moreover, evaluation and treatment of cell death and inflammation during ex vivo lung perfusion may be a game changer in donor organ management, assessment, repair, and reconditioning., (Copyright © 2022 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
28. Precocious White Matter Inflammation and Behavioural Inflexibility Precede Learning and Memory Impairment in the TgAPP21 Rat Model of Alzheimer Disease.
- Author
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Levit A, Gibson A, Hough O, Jung Y, Agca Y, Agca C, Hachinski V, Allman BL, and Whitehead SN
- Subjects
- Aging genetics, Aging pathology, Alzheimer Disease genetics, Alzheimer Disease pathology, Animals, Corpus Callosum pathology, Disease Models, Animal, Male, Maze Learning physiology, Memory Disorders genetics, Memory Disorders pathology, Rats, Rats, Inbred F344, Rats, Transgenic, White Matter pathology, Aging metabolism, Alzheimer Disease metabolism, Corpus Callosum metabolism, Discrimination Learning physiology, Memory Disorders metabolism, White Matter metabolism
- Abstract
Neuroinflammation and behavioural inflexibility are both common in late adulthood but far more profound in Alzheimer disease (AD). To investigate the relationship between ageing, AD, neuroinflammation, and behavioural flexibility, male wild-type Fischer 344 (Wt) and the transgenic APP21 (TgAPP21) rats were aged to 4, 8, 13, and 22 months and evaluated for neuroinflammation and cognitive impairment. TgAPP21 rats overexpress a pathogenic variant of the human amyloid precursor protein (hAPP; Swedish and Indiana mutations) but do not spontaneously develop overt pathology related to AD. In both genotypes, learning and memory were similarly impaired in older rats. However, at 8 months of age, TgAPP21 rats demonstrated behavioural inflexibility in set shifting, reversal, and the Morris water maze, while Wt rats showed inflexibility at 13 and 22 months of age. This early inflexibility in TgAPP21 rats was accompanied by a precocious increase in microglia activation within the corpus callosum; 8- and 13-month-old TgAPP21 rats had similar levels of microglia activation as 13- and 22-month-old Wt rats, respectively. However, while neuroinflammation within the white matter continued to progress with age, behavioural inflexibility peaked in 8-month-old TgAPP21 rats; in older TgAPP21 rats, memory and learning impairments masked inflexibility. These findings suggest that the behavioural inflexibility and white matter inflammation seen in normal ageing are accelerated in AD and may precede impairments of learning and memory., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2021
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29. Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment.
- Author
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Nykänen AI, Mariscal A, Duong A, Estrada C, Ali A, Hough O, Sage A, Chao BT, Chen M, Gokhale H, Shan H, Bai X, Zehong G, Yeung J, Waddell T, Martinu T, Juvet S, Cypel M, Liu M, Davies JE, and Keshavjee S
- Abstract
Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSC
IL-10 ) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSCIL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies., Competing Interests: C.E. is an employee of Tissue Regeneration Therapeutics Inc.; J.E.D is the CEO of Tissue Regeneration Therapeutics Inc.; S.J. and T.M. have received research support from Sanofi; T.W., M.C., M.L., and S.K. are owners of Perfusix Canada and XOR Labs Toronto. T.W., M.C., and S.K. are scientific medical advisors for Lung Bioengineering. The remaining authors declare no competing interests., (© 2021 The Author(s).)- Published
- 2021
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30. Static lung storage at 10°C maintains mitochondrial health and preserves donor organ function.
- Author
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Ali A, Wang A, Ribeiro RVP, Beroncal EL, Baciu C, Galasso M, Gomes B, Mariscal A, Hough O, Brambate E, Abdelnour-Berchtold E, Michaelsen V, Zhang Y, Gazzalle A, Fan E, Brochard L, Yeung J, Waddell T, Liu M, Andreazza AC, Keshavjee S, and Cypel M
- Subjects
- Mitochondria, Lung, Lung Transplantation
- Abstract
Cold static preservation on ice (~4°C) remains the clinical standard of donor organ preservation. However, mitochondrial injury develops during prolonged storage, which limits the extent of time that organs can maintain viability. We explored the feasibility of prolonged donor lung storage at 10°C using a large animal model and investigated mechanisms related to mitochondrial protection. Functional assessments performed during ex vivo lung perfusion demonstrated that porcine lungs stored for 36 hours at 10°C had lower airway pressures, higher lung compliances, and better oxygenation capabilities, indicative of better pulmonary physiology, as compared to lungs stored conventionally at 4°C. Mitochondrial protective metabolites including itaconate, glutamine, and N -acetylglutamine were present in greater intensities in lungs stored at 10°C than at 4°C. Analysis of mitochondrial injury markers further confirmed that 10°C storage resulted in greater protection of mitochondrial health. We applied this strategy clinically to prolong preservation of human donor lungs beyond the currently accepted clinical preservation limit of about 6 to 8 hours. Five patients received donor lung transplants after a median preservation time of 10.4 hours (9.92 to 14.8 hours) for the first implanted lung and 12.1 hours (10.9 to 16.5 hours) for the second. All have survived the first 30 days after transplantation. There was no grade 3 primary graft dysfunction at 72 hours after transplantation, and median post-transplant mechanical ventilation time was 1.73 days (0.24 to 6.71 days). Preservation at 10°C could become the standard of care for prolonged pulmonary preservation, providing benefits to both patients and health care teams.
- Published
- 2021
- Full Text
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31. Transcriptomic investigation reveals donor-specific gene signatures in human lung transplants.
- Author
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Baciu C, Sage A, Zamel R, Shin J, Bai XH, Hough O, Bhat M, Yeung JC, Cypel M, Keshavjee S, and Liu M
- Subjects
- Extracorporeal Circulation, Humans, Lung, Perfusion, Tissue Donors, Lung Transplantation, Transcriptome
- Abstract
Introduction: Transplantation of lungs from donation after circulatory death (DCD) in addition to donation after brain death (DBD) became routine worldwide to address the global organ shortage. The development of ex vivo lung perfusion (EVLP) for donor lung assessment and repair contributed to the increased use of DCD lungs. We hypothesise that a better understanding of the differences between lungs from DBD and DCD donors, and between EVLP and directly transplanted (non-EVLP) lungs, will lead to the discovery of the injury-specific targets for donor lung repair and reconditioning., Methods: Tissue biopsies from human DBD (n=177) and DCD (n=65) donor lungs, assessed with or without EVLP, were collected at the end of cold ischaemic time. All samples were processed with microarray assays. Gene expression, network and pathway analyses were performed using R, Ingenuity Pathway Analysis and STRING. Results were validated with protein assays, multiple logistic regression and 10-fold cross-validation., Results: Our analyses showed that lungs from DBD donors have upregulation of inflammatory cytokines and pathways. In contrast, DCD lungs display a transcriptome signature of pathways associated with cell death, apoptosis and necrosis. Network centrality revealed specific drug targets to rehabilitate DBD lungs. Moreover, in DBD lungs, tumour necrosis factor receptor-1/2 signalling pathways and macrophage migration inhibitory factor-associated pathways were activated in the EVLP group . A panel of genes that differentiate the EVLP from the non-EVLP group in DBD lungs was identified., Conclusion: The examination of gene expression profiling indicates that DBD and DCD lungs have distinguishable biological transcriptome signatures., Competing Interests: Conflict of interest: C. Baciu has nothing to disclose. Conflict of interest: A. Sage has nothing to disclose. Conflict of interest: R. Zamel has nothing to disclose. Conflict of interest: J. Shin has nothing to disclose. Conflict of interest: X-H. Bai has nothing to disclose. Conflict of interest: O. Hough has nothing to disclose. Conflict of interest: M. Bhat has nothing to disclose. Conflict of interest: J.C. Yeung has nothing to disclose. Conflict of interest: M. Cypel has nothing to disclose. Conflict of interest: S. Keshavjee reports grants from Canadian Institutes of Health Research (operating grants MOP-31227, MOP-119514 and PJT-148847) and Genome Canada (Genomic Application Partnership Program grant 6427), during the conduct of the study. Conflict of interest: M. Liu reports grants from Canadian Institutes of Health Research (operating grants MOP-31227, MOP-119514 and PJT-148847) and Genome Canada (Genomic Application Partnership Program grant 6427), during the conduct of the study., (Copyright ©ERS 2021.)
- Published
- 2021
- Full Text
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32. Hypertension and Pathogenic hAPP Independently Induce White Matter Astrocytosis and Cognitive Impairment in the Rat.
- Author
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Levit A, Cheng S, Hough O, Liu Q, Agca Y, Agca C, Hachinski V, and Whitehead SN
- Abstract
Hypertension is recognized as a risk factor for Alzheimer disease, but the causal link remains undetermined. Although astrocytes and microglia play an important role in maintaining the neurovascular unit, astrocytes and microglia have been understudied in comorbid models of hypertension and Alzheimer disease. In this study, male transgenic Fischer 344 rats (TgAPP21) overexpressing a pathogenic human amyloid precursor protein received 8 weeks of Angiotensin II infusion to increase blood pressure, and the rats were evaluated for astrocytosis, microgliosis, and cognitive function. A linear relationship between astrocytosis and blood pressure was observed in the corpus callosum and cingulum of wildtype rats, with hypertensive wildtype rats matching the elevated baseline astrocytosis seen in normotensive transgenic rats. In contrast, hypertensive transgenic rats did not demonstrate a further increase of astrocytosis, suggesting a deficient response. Angiotensin II infusion did not affect activation of microglia, which were elevated in the white matter and hippocampus of transgenic rats. Angiotensin II infusion did impair both wildtype and transgenic rats' executive functions in the Morris Water Maze. These results present important implications for the interaction between hypertension and pathogenic human amyloid precursor protein expression, as Angiotensin II infusion produced cognitive impairments in both genotypes, but transgenic rats were additionally impaired in developing a normal astrocytic response to elevated blood pressure., (Copyright © 2020 Levit, Cheng, Hough, Liu, Agca, Agca, Hachinski and Whitehead.)
- Published
- 2020
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33. Blood-Brain Barrier Closure Time After Controlled Ultrasound-Induced Opening Is Independent of Opening Volume.
- Author
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O'Reilly MA, Hough O, and Hynynen K
- Subjects
- Animals, Contrast Media, Image Enhancement, Magnetic Resonance Imaging, Male, Microbubbles, Rats, Rats, Sprague-Dawley, Time Factors, Blood-Brain Barrier diagnostic imaging, Blood-Brain Barrier surgery, Ultrasonic Surgical Procedures methods, Wound Healing physiology
- Abstract
Objectives: Microbubble-mediated focused ultrasound (US) opening of the blood-brain barrier (BBB) has shown promising results for the treatment of brain tumors and conditions such as Alzheimer disease. Practical clinical implementation of focused US treatments would aim to treat a substantial portion of the brain; thus, the safety of opening large volumes must be investigated. This study investigated whether the opened volume affects the time for the BBB to be restored after treatment., Methods: Sprague Dawley rats (n = 5) received bilateral focused US treatments. One hemisphere received a single sonication, and the contralateral hemisphere was targeted with 4 overlapping foci. Contrast-enhanced T1-weighted magnetic resonance imaging was used to assess the integrity of the BBB at 0, 6, and 24 hours after focused US., Results: At time 0, there was no significant difference in the mean enhancement between the single- and multi-point sonications (mean ± SD, 29.7% ± 18.4% versus 29.7% ± 24.1%; P = .9975). The mean cross-sectional area of the BBB opening resulting from the multi-point sonication was approximately 3.5-fold larger than that of the single-point case (14.2 ± 4.7 versus 4.1 ± 3.3 mm
2 ; P < .0001). The opened volumes in 9 of 10 hemispheres were closed by 6 hours after focused US. The remaining treatment location had substantially reduced enhancement at 6 hours and was closed by 24 hours. Histologic analysis revealed small morphologic changes associated with this location. T2-weighted images at 6 and 24 hours showed no signs of edema. T2*-weighted images obtained at 6 hours also showed no signs hemorrhage in any animal., Conclusions: The time for the BBB to close after focused US was independent of the opening volume on the time scale investigated. No differences in treatment effects were observable by magnetic resonance imaging follow-up between larger- and smaller-volume sonications, suggesting that larger-volume BBB opening can be performed safely., (© 2017 by the American Institute of Ultrasound in Medicine.)- Published
- 2017
- Full Text
- View/download PDF
34. Focused ultrasound-mediated drug delivery through the blood-brain barrier.
- Author
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Burgess A, Shah K, Hough O, and Hynynen K
- Subjects
- Animals, Humans, Magnetic Resonance Imaging methods, Blood-Brain Barrier pathology, Brain pathology, Drug Delivery Systems methods, Microbubbles, Ultrasonic Waves
- Abstract
Despite recent advances in blood-brain barrier (BBB) research, it remains a significant hurdle for the pharmaceutical treatment of brain diseases. Focused ultrasound (FUS) is one method to transiently increase permeability of the BBB to promote drug delivery to specific brain regions. An introduction to the BBB and a brief overview of the methods, which can be used to circumvent the BBB to promote drug delivery, is provided. In particular, we discuss the advantages and limitations of FUS technology and the efficacy of FUS-mediated drug delivery in models of disease. MRI for targeting and evaluating FUS treatments, combined with administration of microbubbles, allows for transient, reproducible BBB opening. The integration of a real-time acoustic feedback controller has improved treatment safety. Successful clinical translation of FUS has the potential to transform the treatment of brain disease worldwide without requiring the development of new pharmaceutical agents.
- Published
- 2015
- Full Text
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35. Alzheimer disease in a mouse model: MR imaging-guided focused ultrasound targeted to the hippocampus opens the blood-brain barrier and improves pathologic abnormalities and behavior.
- Author
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Burgess A, Dubey S, Yeung S, Hough O, Eterman N, Aubert I, and Hynynen K
- Subjects
- Algorithms, Alzheimer Disease pathology, Animals, Contrast Media administration & dosage, Disease Models, Animal, Fluorocarbons administration & dosage, Gadolinium DTPA administration & dosage, Mice, Mice, Transgenic, Alzheimer Disease therapy, Blood-Brain Barrier, Hippocampus, Magnetic Resonance Imaging, Interventional, Ultrasonic Therapy
- Abstract
Purpose: To validate whether repeated magnetic resonance (MR) imaging-guided focused ultrasound treatments targeted to the hippocampus, a brain structure relevant for Alzheimer disease ( AD Alzheimer disease ), could modulate pathologic abnormalities, plasticity, and behavior in a mouse model., Materials and Methods: All animal procedures were approved by the Animal Care Committee and are in accordance with the Canadian Council on Animal Care. Seven-month-old transgenic (TgCRND8) (Tg) mice and their nontransgenic (non-Tg) littermates were entered in the study. Mice were treated weekly with MR imaging-guided focused ultrasound in the bilateral hippocampus (1.68 MHz, 10-msec bursts, 1-Hz burst repetition frequency, 120-second total duration). After 1 month, spatial memory was tested in the Y maze with the novel arm prior to sacrifice and immunohistochemical analysis. The data were compared by using unpaired t tests and analysis of variance with Tukey post hoc analysis., Results: Untreated Tg mice spent 61% less time than untreated non-Tg mice exploring the novel arm of the Y maze because of spatial memory impairments (P < .05). Following MR imaging-guided focused ultrasound, Tg mice spent 99% more time exploring the novel arm, performing as well as their non-Tg littermates. Changes in behavior were correlated with a reduction of the number and size of amyloid plaques in the MR imaging-guided focused ultrasound-treated animals (P < .01). Further, after MR imaging-guided focused ultrasound treatment, there was a 250% increase in the number of newborn neurons in the hippocampus (P < .01). The newborn neurons had longer dendrites and more arborization after MR imaging-guided focused ultrasound, as well (P < .01)., Conclusion: Repeated MR imaging-guided focused ultrasound treatments led to spatial memory improvement in a Tg mouse model of AD Alzheimer disease . The behavior changes may be mediated by decreased amyloid pathologic abnormalities and increased neuronal plasticity., (© RSNA, 2014.)
- Published
- 2014
- Full Text
- View/download PDF
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