cambridge university clinical research society first annual conference

8/3/2019 Cambridge University Clinical Research Society First Annual Conference

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PROCEEDINGS OF THE CAMBRIDGE UNIVERSITY CLINICAL RESEARCH SOCIETY FIRST NATIONAL CONFFERENCE

Cambridge Medicine Journal

FEBRUARY 2011

Journal of the University of Cambridge School of Clinical Medicine



February 2011 CUCRS 1 st Annual Conference

Cambridge University Clinical Research Society

Annual Conference 2011

Welcome

Welcome to the Cambridge University Clinical Research Society (CUCRS) National

Conference 2011.

The CUCRS is a student led initiative which attempts to foster interest in research amongst

medical students. We aim to create an environment facilitating interaction between

researchers and students, allowing the free exchange of ideas and concepts and rewarding

excellence in research.

This conference gives you an opportunity to present your own research at a national level.

Just as importantly, it provides an opportunity to meet like minded medical students from

Universities throughout the UK, offering a unique chance to discuss ideas and learn from

each others research experiences.

Please make the most of this opportunity and enjoy the conference!

Garth Funston

President CUCRS

© Cambridge University Clinical Research Society

www.cucrs.org

Published by Cambridge Medicine Journal

www.cambridgemedicine.org

ISSN: 2046-1798

Produced by:

Adam Young

Aaron D'Sa

Andreas Hadjinicolaou

CMJ 1




CONTENTS

ORAL PRESENTATIONS...............................................................................3

CAMBRIDGE U NIVERSITY A NNUAL COMPETITION AWARD WINNERS.................8

CARDIOTHORACIC MEDICINE.....................................................................11

NEUROLOGY & PSYCHIATRY.....................................................................14

E NDOCRINOLOGY & METABOLIC SCIENCE..................................................16

O NCOLOGY.............................................................................................19

GENETICS...............................................................................................22

I NFECTION, I NFLAMMATION & IMMUNOLOGY...............................................24

SURGERY...............................................................................................26

CASE R EPORTS.......................................................................................28

AUDITS..................................................................................................30

A NIMAL MODELS....................................................................................33

CMJ 2




ORAL PRESENTATIONS

Sub-optimal gluten free diet adherence in coeliac disease: reinforcing the link between gut inflammation

and irritable bowel syndrome

S. M. Barratt 1,*, A. Arnaout 1, K. E. Evans 2, J. S. Leeds 2, D. S. Sanders 2

1The University of Sheffield school of medicine and biomedial science, Sheffield, UK 2The GI & Liver Unit, Sheffield teaching hospitals NHS foundation trust, Sheffield, UK

Background: Are Coeliac Disease (CD) patients with less than full adherence to a gluten-free diet (GFD) more

likely to report Irritable Bowel Syndrome (IBS) symptoms than those with full adherence?

Methods: 224 histologically proven patients (26% male, mean disease duration 8yrs, range 0.5-52yrs)

completed the ROME II Criteria and GFD assessment. Reflux questionnaire (Type: heartburn, belching,

regurgitation, retrosternal pain, dysphagia. Severity: mild, moderate, severe) screened for upper GI dysmotility.

Full adherence (FA): GFD everyday of the previous twenty-eight. Partial/no adherence (PNA): any level below

FA.

Results: FA: 158 patients (71%). PNA: 66 patients (29%). The ROME II Criteria was fulfilled by 52 (23%) of

patients: 11% of FA patients versus 52% of PNA. RR of IBS in PNA in comparison to FA: 1.828 (95% CI 1.41-

2.36, P =<0.0001). Reflux symptoms were reported by 147 (66%) of patients: 62% of FA versus 74% of PNA

( P =>0.05). Reflux severity: Mild: FA 28%, PNA 29% ( P =>0.05); Moderate: FA 25%, PNA 23% ( P =>0.05);

Severe: FA 9%, PNA 13% ( P =>0.05). Age/sex were not confounding factors.

Conclusion: The prevalence of IBS is almost five times greater with sub-optimal adherence in comparison to

full adherence, 52% versus 11%. There is little difference in the prevalence or severity of reflux opposing an

association of widespread dysmotility and poor adherence. Our findings suggest that IBS may be attributed to

gut inflammation. This has implications in the management of IBS in CD and other chronic inflammatory GI

disorders promoting a view of IBS as inflammatory rather than functional in nature.

The effects of Urokinase on early graft function in live donor kidney transplantation

A. Mitra, J. Murphy, S. Hosgood, U. Thiyagarajan & M. Nicholson.

University of Leicester Medical School, Maurice Shock Building, PO Box 138, University Rd, Leicester, UK

Background: Thrombolytic agents have been used to enhance the preservation condition and improve graft

outcome in deceased kidney donation. However, there is no evidence for the routine use of such agents in living

donor kidney transplantation to improve early graft function.

Methods: A pilot study of 100 live donor kidney transplants was performed. Fifty cases with Urokinase added

to the preservation solution and fifty without Urokinase representing the control group. Slow graft function(SGF) was defined as a less than 10% fall in serum creatinine within the first 24 hours, and early graft function

measured by area under the curve serum creatinine (AUC Cr) over the first 7 days after transplantation.

Results: The incidence of SGF was 24% in the control group compared to 8% with the addition of urokinase

(p=0.054). However, there was no difference in AUC Cr between the urokinase and control groups (1666 ± 687

vs 1676 ± 692 μmol/L.day respectively; p=0.883). There were no incidences of intravascular thrombi and no

adverse events reported from the use of Urokinase.

Conclusion: The addition of Urokinase to the kidney preservation solution reduced the incidence of SGF in a

cohort of live donor kidney transplants. SGF has a negative impact on patient and graft survival, and occurs

more frequently in live donor kidney transplantation than is often realised. This study shows the use of

Urokinase warrants further investigation in this field.

CMJ Oral Presentations 3




Inorganic Nitrite Induces Second Window Preconditioning and Post Conditioning In Humans in-vivo

Influenced By Mitochondrial Aldehyde Dehydrogenase Polymorphism.

J. D Evans.

College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK.

Background: The nitrite anion has been shown to protect against myocardial ischaemia-reperfusion injury (IRI)in numerous animal models. Mitochondrial aldehyde dehydrogenase (ALDH2) has been implicated in

cytoprotection with a variety of stimuli. We investigated the cytoprotective effects of systemic nitrite in the

ischaemic human forearm model, with genetic and pharmacological inhibition of ALDH2.

Methods: 35 volunteers underwent 51 studies. Venous occlusion plethysmography was used to measure

changes in forearm blood flow in response to intra-arterial acetylcholine (25,50,100nmol/min), before and after

IRI (20 minutes of forearm ischemia,15 minutes reperfusion) in 3 protocols. (1) Nitrite (1mcg/ml/min for 10

minutes IV) 24 hours before IRI (2) Nitrite during ischaemia and (3) Saline (placebo). Subjects were genotyped

for the ALDH2 polymorphism after completion of all studies.

Results: In both ALDH2*1 (wild type) and ALDH2*2 (inactive polymorphism) groups, IRI with placebo

infusion caused endothelial dysfunction (n=9,p<0.0001, n=9,p=0.0006). Nitrite 24 hours before ischaemia

prevented endothelial dysfunction in ALDH2*1 volunteers (n=7,p=0.78). Nitrite during ischaemia preventedendothelial dysfunction in ALDH2*2 (n=8,p=0.63) but not ALDH2*1 (n=10,p=0.006). Nitrite during ischaemia

did not prevent endothelial dysfunction following pharmacological ALDH2 inhibition with disulfiram in

ALDH2*1 volunteers (n=6,p=0<0.0001).

Conclusion: This study provides the first evidence that systemic nitrite is cytoprotective in humans and suggests

a role for mitochondrial ALDH2, the precise nature of which is unclear.

Molecular beacon-based multi-allelic real-time PCR for fast and accurate detection of pathogenic

bacteria

A. V. HadjinicolaouUniversity of Cambridge, School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, UK

Background: Molecular beacons are synthetic nucleic acid probes that act as switches emitting fluorescence

upon hybridising to complementary targets. This study aimed at devising a molecular-beacon-based real-time

PCR diagnostic assay that improves accuracy, sensitivity and speed of microbe detection whilst discriminating

between subtypes of the same organism and determining the presence of virulence factors.

Methods: Molecular beacons and primer sets for each DNA target were designed from aligned sequences

retrieved from GenBank. Targets included DNA regions common to all Salmonella serotypes or all Bacillus

strains or specific to selected serotypes or virulence factors. Standard and melting curves for quantitative

measurements and optimal PCR temperature were created. False negative results due to PCR inhibition were

excluded by an internal amplification control. DNA from clinical samples was extracted and analysed using

combinations of beacons. Samples included 44 different Salmonella strains, 17 B.anthracis, 24 non-virulent Bacillus subtypes, 28 other bacteria and 10 human DNA specimens. Results were compared to those from

culture, serotyping and sequencing.

Results: All assays showed 100% sensitivity and specificity. The detection limit was 10 copies of target DNA

highlighting the capacity to quantify pathogen levels directly from clinical samples. Numerous samples were

analysed in just hours; much faster than conventional techniques.

Conclusion: This study shows that molecular-beacon-based multiplex PCR is rapid and accurate allowing

detection of multiple alleles for:

1) distinguishing between pathogens and subtypes in the same sample

2) enhancing precision by identifying multiple DNA target regions of a single pathogen

3) assessing multiple features of a single pathogen such as virulence genes.





Update of the HIPRO (Hypofractionated Dose Escalation utilising Intensity Modulated Radiotherapy in

Carcinoma of the Prostate) study: late toxicity and outcome at seven years

S. Merrick, A. Choudhury, D. Thomson, R. Swindell, J. Coote, J. Wylie, R. Cowan, T. Elliott, J. Logue &

J. Livsey

School of Medicine, The University of Manchester, Oxford Road, Manchester, UK

Background: Hypofractionation results in reduced treatment times, and should be biologically advantageous

given the low alpha-beta ratio for prostate cancer. However this may lead to increased toxicity. IMRT allows

dose escalation with hypofractionation, while achieving acceptable levels of toxicity. We report 7 year late

toxicity data in patients treated with two such regimens within the HIPRO study.

Methods: Sixty men, median age 75 years (50-87), with localised adenocarcinoma of prostate (T1-3NOMO)

and either Gleason score ≥7 or PSA 20-50ng/l received 57Gy in 19 fractions (n=30) or 60Gy in 20 fractions

(n=30) using 5-field inverse-planned IMRT. All patients received neoadjuvant hormone therapy, continuing for

up to 6 months. Late toxicity was assessed at 7 years follow-up using RTOG criteria and a validated

LENT/SOMA patient questionnaire. Survival was assessed at 5 years.

Results: Forty-four patients were alive at 7 years. Nine patients reported RTOG grade 1 bowel or bladder

toxicity; there was no grade 2 toxicity or above and no difference between the fractionation schedules.LENT/SOMA questionnaires were returned by 31/44 patients. Mean and median scores were less than one for

bowel and urinary symptoms. When compared with pre-treatment, the proportion of patients with significant

urinary symptoms remained similar, problems with sexual function had decreased but bowel symptoms

increased. At 5 years, overall survival was 83% and 74%, cause-specific survival 83% and 84% and bPFS 50%

and 58% in the 57Gy and 60Gy groups respectively.

Conclusions: Dose-escalated hypofractionated IMRT for prostate cancer appears well tolerated with acceptable

levels of late toxicity.

Urine Metabolic Profiling of Hepatitis B-related Liver Disease in a Nigerian Population

M. Patel

Faculty of Medicine, Imperial College London, South Kensington Campus, London UK

Background: Progressive hepatic fibrosis is common in sub-Saharan African populations, chronically infected

with hepatitis B virus. The consequent development of cirrhosis is the strongest risk factor for hepatocellular

carcinoma. Liver biopsy is the reference standard for assessing liver fibrosis, but is invasive, expensive and

repeated biopsies are impractical. The novel Enhanced Liver Fibrosis (ELF) serum test is a non-invasive

alternative to staging hepatic fibrosis. Development of urinary biomarkers for assessing the severity of liver

fibrosis would be practical and widely applicable. This study compares the urinary metabolic profiles of

Nigerian patients with a spectrum of hepatitis B-related liver fibrosis to the ELF scoring system.

Methods: Using in vitro magnetic resonance spectroscopy (MRS), urine was analysed from two subject

groups, collected in Nigeria: 24 patients with a high ELF score and 19 patients with a medium ELF score.

Multivariate factor analyses were performed using principal components analysis and partial least-squares

discriminant analysis.

Results: Nigerian patients with a high ELF score were distinguished from those with a medium ELF score

with sensitivity/specificity of 88.5%/88.9%. The metabolites, creatinine, dimethylamine, hippurate and creatine

contributed most strongly to the multivariate model.

Conclusion: Urinary 1H MRS with multivariate statistical analysis demonstrated that ELF scores correlate well

with urinary metabolic profiles of Nigerian patients with a spectrum of liver fibrosis. Discriminatory

metabolites identified could potentially serve as biomarkers for simple urinary screening tests in staging

hepatic fibrosis. This is particularly important for the developing world where there is an urgent requirement

for cheap and effective diagnostic tests.





Novel post-transcriptional regulation of aldosterone synthase expression

A Ejaz, S. Wood, S MacKenzie, J. Connell & E. Davies.

British Heart Foundation, Glasgow Cardiovascular Research Centre, 126 University Place, Glasgow, UK.

Background: Essential hypertension has a large genetic component and polymorphisms in the CYP11B2 gene

encoding aldosterone synthase enzyme are associated with hypertension. Of interest are polymorphisms present

in the 3’ untranslated region (3’-UTR), where novel regulators, microRNAs (miRNAs), bind and repress mRNA

translation. Four miRNAs, present within the adrenal glands, have putative binding sites within the 3’-UTR of

CYP11B2. Our aim was to test whether these microRNAs (125a-5p, 125b, 134 and 495a) bind to CYP11B2.

Methods: A pEZX reporter plasmid was used, containing a CYP11B2 3’-UTR insert coupled to the firefly

luciferase gene. Precursor miRNAs (pre-miRs) of the four miRNAs were used to test binding, while antagonists

of each miRNA (anti-miRs) confirmed binding. Scrambled pre-miRs were used as negative controls and a small

inhibitory RNA (siRNA) as positive control. HeLa cells were co-transfected with 500ng of plasmid and

50nmoles of premiR, antimiR or siRNA. Luciferase activity was measured 48 hours post-transfection.

Results: miR-125a-5p significantly decreased luciferase activity compared to negative control (36±10%,

p<0.001); while anti-miR-125a-5p increased luciferase activity (62±51%, p=0.02). miR-125b reduced luciferase

activity (24±2%, p<0.001), while anti-miR-125b increased luciferase activity (56±22%, p=0.001). miR-495a

and miR-134 showed no significant effect on luciferase activity, which was confirmed with antimiR

transfection.

Conclusion: miR-125a-5p and miR-125b bind to the 3’-UTR of CYP11B2, as confirmed by anti-miR studies,

indicating that they regulate CYP11B2 expression. miRNA regulation may underlie aberrant aldosterone

synthase expression in a subset of essentially hypertensive subjects and may become a useful therapeutic target

in the treatment of essential hypertension.

The Expression of RNA Binding Proteins in Colorectal Cancer

N.R. Hope

School of Medicine & Dentistry, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen, UK

Background: The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of RNA binding proteins

with a range of key cellular functions which are dysregulated in tumourigenesis including regulation of

translation and RNA processing. The purpose of this study was to define the hnRNP expression profile in

colorectal cancer and establish the significance of hnRNP expression.

Method: A tissue microarray containing 515 primary colorectal cancers, 224 lymph node metastasis of

colorectal cancer and 50 normal colon samples was immunostained for 6 hnRNPs.

Results: hnRNP I, hnRNP K and hnRNP L displayed the most frequent strong immunoreactivity in primary

colorectal tumour samples. hnRNP A1 (p<0.001) and hnRNP U (p=0.003) showed significant alterations in

nuclear expression in tumours compared with normal while hnRNP A1 (p=0.001), hnRNP I (p<0.001) and

hnRNP K (p<0.001) all showed significant increases in cytoplasmic immunoreactivity in tumour cells. There

were significant differences in cytoplasmic immunoreactivity between the primary tumour and the

corresponding lymph node metastasis for hnRNP A1 (p=0.001), hnRNP I (p<0.001) and hnRNP K (p=0.001).

There was a significant relationship between strong nuclear hnRNP H expression and survival (chi-squared =

14.97, p<0.001).

Conclusion: This study has defined the expression profile of hnRNPs in colorectal cancer and shown that there

are significant alterations in individual hnRNP expression in this type of tumour.





Investigation of rheumatoid arthritis susceptibility genes identifies an association between TRAF6 with

response to anti-tumour necrosis factor- therapy in a large UK cohort

R. Prajapati

School of Medicine, The University of Manchester, Oxford Road, Manchester M13 9PT, UK

Background: Anti-TNF therapy has revolutionised treatment of rheumatoid arthritis (RA); however not allindividuals respond well. The RA risk allele at the PTPRC locus has recently been reported to associate with

response to anti-TNF therapy. The aim of this study was to investigate recently confirmed RA susceptibility

variants, including PTPRC, to determine whether they predict anti-TNF response in an independent UK

population.

Methods: 1,023 UK RA patients receiving anti-TNF therapies were genotyped for 46 single-nucleotide

polymorphisms (SNPs) recently identified as RA susceptibility markers. Multivariate linear regression of

change in Disease Activity Score (DAS28) at 6 months follow-up was performed for each SNP using an

additive model. Logistic regression analyses using the European League Against Rheumatism (EULAR)

response criteria compared good (n=203) versus poor (n=161) responders.

Results: 38 SNPs were successfully genotyped. A SNP at TRAF6 (rs540386) was associated with both EULAR

response (odds ratio [OR] 1.9, 95% confidence interval [95% CI] 1.16 to 3.11, P =0.011) and change in DAS28score between baseline and 6 months (OR -0.28, CI -0.04 to -2.37, P=0.02). The effect size of the RA risk allele

at the PTPRC locus was similar to that reported previously but did not reach statistical significance in our case

series (OR 1.35, 95% CI 0.83 to 2.17, P =0.2). No convincing evidence for association was detected at the other

36 SNPs tested.

Conclusions: The RA risk allele at the TRAF6 gene may also predict response to anti-TNF treatment. Studies

are now needed to replicate this finding in additional patient cohorts.

The genetics of inflammatory bowel disease: unravelling the differences between Caucasians and South

Asians

A. S. Bancil1, D. G. Walker1, P. S. Rai1, H. R. Williams1, H. Sato2, P. Pantelidis2, J. C. Chambers3, J.

S.Kooner3, T. R. Orchard1

1.Gastroenterology, 2. Population Genetics and Gene Therapy, 3. Cardiovascular Science, Imperial College

London, London, UK

Background: Single nucleotide polymorphisms (SNP ) in autophagy-related 16-Like 1 gene ( ATG16L1;

rs2241880) and immunity-related GTPase family M gene ( IRGM ; rs13361189 & rs4958847 ) have shown strong

correlations in Caucasian Crohn’s Disease (CD) patients. Additionally, a SNP in the interleukin 23 Receptor

( IL23R; rs11209026 ) has been associated with protection in both CD and ulcerative colitis (UC) in Caucasians.

In contrast, studies from East Asia have shown no association of these SNPs with IBD1.

Methods: The aim was to investigate the prevalence of ATG16L1, IRGM and IL23R polymorphisms in a South

Asian IBD population and compare the results to a healthy South Asian control cohort. IBD patients were

recruited from the IBD clinics of five hospitals in North West London. Patients and controls were genotyped

using pyrosequencing and the results compared using Chi-squared.

Results: 232 South Asian IBD patients (66 CD: 166 UC) and 204 healthy South Asian controls were recruited.

The frequency of the ATG16L1 and IRGM mutant alleles between South Asian CD and South Asian controls

was not significant (0.576 vs 0.569, p=0.886; 0.467 vs 0.414, p=0.553; 0.55 vs 0.502, p=0.558 ). IL23R

frequencies were also not significant (CD: 0.008, UC: 0.015, controls: 0.019).

Conclusion: ATG16L1, IRGM and IL23R polymorphisms were not associated with IBD in a UK South Asian

IBD population, suggesting that this population is similar to East Asian populations that show no association

between IBD and ATG16L1/IRGM/IL23R.





CAMBRIDGE UNIVERSITY ANNUAL COMPETITION AWARD WINNERS

The Cambridge University Clinical Research Society held its 1 st Annual Competition for

students within the University. We received an enormous number of high quality entries. The

following five entrants were selected to present their work at the prize-giving event in

December 2010, and their work will be on display at the conference.

Molecular beacon-based multi-allelic real-time PCR for fast and accurate detection of pathogenic

bacteria

A. V. Hadjinicolaou

University of Cambridge, School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, UK

Background:

Molecular beacons are synthetic nucleic acid probes that act as switches emitting fluorescence upon hybridising

to complementary targets. This study aimed at devising a molecular-beacon-based real-time PCR diagnostic

assay that improves accuracy, sensitivity and speed of microbe detection whilst discriminating between subtypes

of the same organism and determining the presence of virulence factors.

Methods:

Molecular beacons and primer sets for each DNA target were designed from aligned sequences retrieved from

GenBank. Targets included DNA regions common to all Salmonella serotypes or all Bacillus strains or specific

to selected serotypes or virulence factors. Standard and melting curves for quantitative measurements and

optimal PCR temperature were created. False negative results due to PCR inhibition were excluded by aninternal amplification control. DNA from clinical samples was extracted and analysed using combinations of

beacons. Samples included 44 different Salmonella strains, 17 B.anthracis, 24 non-virulent Bacillus subtypes,

28 other bacteria and 10 human DNA specimens. Results were compared to those from culture, serotyping and

sequencing.

Results:

All assays showed 100% sensitivity and specificity. The detection limit was 10 copies of target DNA

highlighting the capacity to quantify pathogen levels directly from clinical samples. Numerous samples were

analysed in just hours; much faster than conventional techniques.

Conclusion:

This study shows that molecular-beacon-based multiplex PCR is rapid and accurate allowing detection of

multiple alleles for:

1) distinguishing between pathogens and subtypes in the same sample

2) enhancing precision by identifying multiple DNA target regions of a single pathogen

3) assessing multiple features of a single pathogen such as virulence genes.

CMJ Cambridge University Annual Competition Award Winners 8




Improvement in Glycaemic Control for Type 1 Diabetics on Insulin Pump Therapy is greater in those

with a higher starting HbA1c and may contribute to a reduced number of diabetes related inpatient days

M. Baxter & M. Evans


Background: Continuous subcutaneous insulin infusion (CSII or insulin pump therapy) is currently used totreat about 5% of patients with type 1 diabetes in UK. I examined the Addenbrookes CSII service, looking to see

whether CSII improved blood glucose control as measured by HbA1c. I hypothesised that (1) the reduction in

HbA1c would be greater in those starting with a high levels and (2) CSII use would correlate with a reduction in

diabetes-related hospital inpatient days.

Methods: Data were obtained from a clinic-based electronic database (Diamond) for 164 individuals currently

using CSII. HbA1c data were calculated as incremental change from baseline and then stratified according to

starting levels. Inpatient data were obtained from a hospital admissions database (e-MR). A catchment area was

imposed taking into account the patient’s place of residence. Data were analysed using an unpaired t-test.

Results: 3 months after starting CSII, HbA1c had fallen by 0.7 % (±0.1, p<0.001), with the improvement being

sustained over the following years. Patients with starting HbA1c >8.5% experienced a greater reduction after 3

months (1.0 % ± 0.13) vs patients with a starting HbA1c of 7.5-8.5% (0.5 % ± 0.2), p<0.05. In the first year of CSII therapy, diabetes related inpatient days per patient per annum fell from 0.19 ± 0.05 to 0.03 ± 0.003

(p<0.15).

Conclusion: CSII therapy is effective in reducing HbA1c levels in type 1 diabetes in a general UK teaching

hospital, especially in those with a starting HbA1c >8.5%. Importantly, CSII therapy may also reduce diabetes-

related emergency hospital admissions and subsequent inpatient days.

Structure Function Analysis of the Influenza A Virus Nucleoprotein

D. Liang


Background: Influenza A viruses continue to be a major healthcare threat worldwide. As well as causing

seasonal epidemics, emerging reassortant strains could potentially lead to devastating pandemics. Hence an

improved understanding of its lifecycle and virulence determinants is important in designing new

countermeasures. The influenza A viral genome consists of 8 segments which are believed to code for at least 10

proteins. Segment 5 encodes the viral nucleoprotein (NP). NP binds vRNA and forms part of the vRNP

complex.

Methods: We generated 10 mutant NP variants from wild type influenza strain A/PR8/34. Each mutant had a

single non-conservative amino acid substitution in highly conserved residues. Many of these residues map to the

‘head’ domain of NP, others were in the vRNA binding domain. We assessed the effects of these mutations oncellular NP expression, nucleo-cytoplasmic NP trafficking, and vRNP transcriptional activity.

Results: Our mutations did not have any significant effects on NP expression or nucleo-cytoplasmic NP

trafficking. However vRNP reconstitution assays showed significant defects in transcriptional activity for

mutants R150A, R204A, W207A and R208A. Correspondingly these mutations in NP failed to produce viable

viruses. Interestingly mutant variants R162K, R199A, R213A and E449A were also non-viable despite

appreciable activity in the vRNP reconstitution assays.

Conclusion: Our results suggest that NP residues (R162K, R199A, R213A, and E449A) are important to

functions other than nucleo-cytoplasmic trafficking or vRNA transcription. This could indicate new and

previously poorly characterised functions for NP. Further work should involve identifying defects in the viable

mutant viruses (E220A, K236A).





The incidence of seroma post breast surgery when using two different wound managing techniques

F. Pereira


Background: A seroma is subcutaneous collection of serous fluid, which arises as a possible complication

following breast surgery. Standard practice is to extract the serous fluid via drainage. An alternative to a draininsertion is the quilting technique, which uses sutures to anchor axillary/ mastectomy flaps to the underlying

chest wall. This study compares the incidence of seroma between the two techniques.

Methods: A group of 54 patients who underwent Wide Local Excision + Axillary Node Sampling (ANS)/

Axillary Node Clearance (ANC) or Mastectomy ± ANS/ ANC where included in this retrospective study.

Results: 26% of patients whose wound was managed by the quilting technique developed seroma. In contrast,

41% of patients who received a drain developed seroma. The mean volume of seroma aspirated in the patients

who received quilting was 118 ml compared to 813 ml for the patients who had a drain. The average no. of

aspirations for patients who underwent quilting was 1.75 and 3.25 for patients who had drains. There was no

difference in hospital stay or mean BMI.

Conclusion: There is a reduction in the incidence of seroma when the quilting technique is used and asignificant reduction in the total volume of serous fluid aspirated compared to when the patients received drains.

These findings suggest that a move towards quilting may have beneficial outcomes for both the patient (for

example, less discomfort) and the trust (reduced costs). These results support the quilting technique as a

preferable alternative to drainage for breast wound care.

Assessing the protection of H3N2 influenza vaccination against future circulating strains

S. James


Background: Annually, influenza is estimated to cause three to five million cases of severe illness and 250,000-

500,000 deaths. At-risk groups receive an influenza vaccine each year as the virus mutates to evade immune

recognition. Antigenic change is quantified using the haemagglutination inhibition (HI) assay, which measures

the cross-reactivity of different sera and antigens. The antigenic evolution of influenza is visualised by

transforming the HI data using antigenic cartography; for H3N2, this creates a 2-dimensional map. This

provides a framework for assessing the effects of vaccination.

Methods: Serum samples were collected during adult vaccine reregistration trials in 2002

(A/H3N2/Panama/2001/1999, n=92) and 2004 (A/H3N2/Wyoming/3/2003, n=92). Pre- and post-vaccination

sera were measured using HI assay against 28 antigens (1993-2004). Ferret sera were used to create an

antigenic map. The proportion of protected individuals (titre>40) was calculated at the vaccine point. The

GMT (geometric mean titre) of individuals with undetectable pre-vaccination titres was compared at all antigen points using a t-test.

Results: Both vaccines provided high levels of protection against the vaccine antigen (Panama/2001/1999 98%;

Wyoming/3/2003 99%). The GMT (95% CI) was 286 (209-391) for Panama/2001/1999 and 283 (165-485) for

Wyoming/3/2003. Three antigens had significant (p<0.05) differences in GMT; these were all antigenically

advanced compared to the vaccine strains. The GMT for Wyoming/3/2003 vaccines was higher against 23/27

antigens.

Conclusions: Vaccination with Wyoming/3/2003 provided equivalent or better protection against the majority

of antigens, especially those which were antigenically advanced. This could be because Wyoming/3/2003 is

closer to these strains . Alternatively, the Wyoming/3/2003 vaccine could be more immunogenic, or there might

be confounding factors such as age or sex. The range of antigenic distances needs to be extended by further

titrations. This approach has potential to aid influenza vaccine selection.





CARDIOTHORACIC MEDICINE

Assessment of image quality with iterative reconstruction (AIDR) versus filtered back projection in

cardiac computed tomography coronary angiography

F. R. Millar, M. C. Williams, N. Weir & D. E. NewbyUniversity of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, UK

Background: Computed tomography coronary angiography (CTCA) is increasingly being used to assess

patients with coronary artery disease. This is associated with significant risk due to radiation exposure. The

current average radiation dose for CTCA is 12mSv which is equivalent to 600 chest x-rays. We aimed to assess

the effect of the AIDR reconstruction algorithm on image quality in CTCA. If image quality is improved, we

can theoretically reduce our effective radiation dose while maintaining image quality.

Methods: Participants underwent contrast enhanced prospective, electrocardiogram-gated, CTCA using a 320

multidetector scanner. We assessed 59 images that were reconstructed at 2% intervals across the acquisition

window (30 to 90%) with iterative reconstruction (AIDR) and filtered back projection. These were assessed by a

blinded observer for signal-to-noise and signal-to-myocardium ratio. Medians and interquartile ranges are

presented and significance was assessed with the Wilcoxon test.

Results: Signal-to-noise ratio was significantly improved with AIDR in all vessels (left main 10.6 (9.1-11.5) vs

9.6 (8.3-10.4), left anterior descending 10.0 (8.2-11) vs 9.9 (7.1-9.9), left circumflex 7.8 (4.6-10.1) vs 7.2 (4.7-

8.3), right coronary 9.5 (8.3-11.1) vs 8.7 (7.5-10.0), p<0.001). The signal-to-myocardium ratio was not

significantly different with iterative reconstruction.

Conclusions: Iterative reconstruction improved signal-to-noise ratio in cardiac computed tomography coronary

angiography. Signal-to-myocardium ratio was not improved, perhaps due to differences in myocardial

contraction across the acquisition window. We have estimated that the implementation of this algorithm will

enable up to a 20% reduction in radiation dose while maintaining image quality.

Impulse oscillometry for the assessment of lung function deficits associated with preschool wheezing.

U. Banerjee1, S. Goldring1, J. Kirkby2, J. Stocks2, J.O. Warner1, R.J. Boyle1.

1. Department of Paediatrics, Imperial College London, London, UK

2. UCL, Institute of Child Health, London, UK

Background: There is a need for a clinical tool to evaluate lung function in preschool children. Spirometry is

the most common measurement of lung function in adults, however spirometry measurements are a challenge in

preschool children. Impulse oscillometry (IOS) is able to measure the resistance and resonant frequency of

lungs from normal breathing, and may be a suitable tool for assessing lung function in preschool children.

Methods: We recruited 66 children aged 3-4y from a paediatric outpatients department. Children underwent

lung function assessment using IOS pre and post bronchodilator, skin prick tests to assess atopy and a modified

ISAAC quesionnaire. Variables recorded were resistance across 5-25Hz and resonant frequency (Fres).

Results: 42 (64%) of 66 children successfully completed lung function assessment using IOS. Younger children

were less likely to successfully complete IOS readings (3-3.5yrs children 41% success; 3.5-4 yrs children 71%

success; p=0.03). We found a significant increase in Fres in children with a history of wheezing (mean 23.40Hz

wheeze, 19.44Hz no wheeze; p=0.01). Furthermore, significant differences were found in the Fres of children

who had previously been diagnosed with asthma by a doctor compared to non asthmatics (p=0.015); and those

with atopy and wheeze compared to those with no atopic wheeze (p=0.015).

Discussion: IOS yields high quality lung function data in most children over 3.5 years age. The technique is

able to detect group differences related to wheezing tendency in this age group.

CMJ Cardiothoracic Medicine 11




Coronary vein thrombosis may lead to LV lead displacement following CRT implantation and required

the utilisation of an alternative target vein.

P. Das1, A. Oomatia1, M. Elsik 2 & M. Virdee2

1. University of Cambridge, School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, UK 2. Cambridge Papworth Hospital NHS Foundation Trust, Papworth Everard UK

Background: In heart failure, the response to cardiac resynchronisation therapy (CRT) is improved by targeting

LV lead placement into specific coronary veins. Some patients may require revision of lead placement,

however. We aimed to study coronary vein patency in these patients and the viability of using original target

veins.

Methods: Patients requiring repeat LV lead manipulation or CRT reimplantation between 2006- 2010 were

identified from a prospectively collected database of consecutive CRT implants (n=34/442). Fluoroscopic

images were analysed to identify the target coronary vein utilised for initial lead placement. Coronary sinus

venograms performed during the repeat procedure were compared to those of the initial procedure and evaluated

for patency and thrombotic obstruction of the target vein. This was graded by an experienced cardiologist as

either >=75% (severe) or <75% (non severe) reduction of the luminal diameter.

Results: In 21/34 patients, venograms for both procedures were available for analysis. Repeat procedures wererequired for device infection (n=2), diaphragmatic pacing (n=3) and lead displacement (n=16). During the repeat

procedure, severe coronary vein stenosis due to thrombosis of the initially targeted vein, was seen in 7/21 and

non severe in 14/21. 7/7 with severe coronary venous stenosis required utilisation of a different vein, compared

to only 1/14 with non severe stenosis (p<0.0001).

Conclusion: In the majority of patients, CRT revisions were due to lead displacement, at least partly due to

coronary vein thrombosis resulting in coronary vein stenosis. Severe coronary venous stenosis requires the

utilisation of an alternative target vein at repeat procedure and may have an implication on clinical outcomes.

Severity assessment of community acquired pneumonia in Malawi; Application of the CRB-65 severity

score and derivation of a new index, the SWAT-Bp score

E. Birkhamshaw

College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK

Background: The CRB-65 score (Confusion, resp. rate>30, BP<90/60, age>65) is an effective tool for

assessing community acquired pneumonia (CAP) severity. Its validity was assessed in a Malawian hospital.

Variables predicting mortality were identified, to derive an accurate severity score for this setting.

Methods: Data for 43 variables were collated for patients admitted to Queen Elizabeth Hospital (QEH) for

management of CAP, over two months (N=240). Sensitivity and specificity of CRB-65 in predicting mortality

were calculated. Multivariate analysis identified predictor variables, to create a new algorithm.

Results: Median age 37, HIV prevalence 79.9% and overall mortality 18.3%. CRB-65 showed low sensitivity

and specificity in predicting mortality, indicated by the area under the ROC curve (AUC): 0.649. Mortality for

scores 0-4 was 6.3%, 19.7%, 27.7%, 33.3% and 100%. Independent predictors of mortality; Male sex, “S”

(AOR 2.6, p 0.035); Wasting, “W” (AOR 6.6, p <0.001); non-ambulatory, “A” (AOR 2.5, p 0.008); Temp

>38Oc or <35 Oc, “T” (AOR 3.2, p 0.022); BP<100/60, “Bp” (AOR 3.7, p 0.004). A severity index using these

factors (SWAT-Bp) has high accuracy (AUC 0.867). Mortality for scores 0-5 was 0%, 3.3%, 7.4%, 29%, 61.5%

and 87.5%. A score >2 was 84% sensitive and 77% specific for mortality.

Conclusion: Variables predicting CAP severity in QEH, Malawi, were identified. CRB-65 lacks efficacy in this

population. A score combining ‘male sex’, ‘wasting’, ‘non-ambulatory’, ‘high/low temperature’ and

‘hypotension’ accurately stratifies patients; ≤2 indicates non-severe pneumonia (mortality 4.4%) and ≥3 severe

illness (mortality 45%). This tool should be tested to determine accuracy in settings throughout sub-SaharanAfrica.





Testing repeatability and reproducibility of Structured Light Plethysmography (SLP) as a method of

measuring lung function

K. Prosser, C.K. Weerasuriya & S. Alimohamed.


Abstract: Structured Light Plethysmography (SLP) is a recently developed technology for non-invasivemonitoring of lung (respiratory) function. The system projects structured light onto the thoraco-abdominal

surface of the subject, which is imaged in two cameras giving a dynamic 3D reconstruction of the surface as the

subject breathes. From this data we can infer changes in chest/abdomen volume over time giving a non-invasive

alternative to spirometry. The hope is that SLP will provide an inexpensive replacement for conventional

spirometry, which is an invasive methodology unusable in a number of patient classes (e.g. neonates). This

study aims to test the reproducibility and repeatability of measurements derived from SLP.

Methods: An experimental protocol was designed and executed, capturing 72 datasets in total from 12

randomly chosen adult subjects. User-dependence (reproducibility) was tested by collecting data sets from each

subject using 3 different experimenters. Repeatability was tested by collecting the data from each subject once,

and again after a 40 minute break.

Results: To assess variability, we extract tidal volume (TV), forced vital capacity (FVC) and forced expiratoryvolume in 1 second (FEV1) from each of the SLP datasets. Statistical measures of similarity between datasets

taken by different operators and between datasets on the same person at different times are given. Additionally

we validate this against simultaneously recorded Pneumatach spirometry data.

Conclusions: Given the statistical measures of similarity, we will discuss the SLP and spirometer measurements

both in terms of operator dependence and individual repeatability. We also discuss the consequences of these

results for future usage of the SLP system.

Cardiovascular Effects of Apelin Interaction with the Renin-Angiotensin System

K. Lee

University of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, UK

Background: The newly discovered apelin-APJ system has been widely implicated in cardiovascular

homeostasis and the pathophysiology of heart failure. Increasing evidence suggests interaction between the

apelin-APJ and renin-angiotensin system (RAS) with largely counter-regulatory effects. We assessed the

cardiovascular effects in the presence of increased angiotensin II, analogous to heart failure activity, to provide

insight to the potential therapeutic benefit of APJ agonism.

Methods: 12 healthy male volunteers participated in a single-blinded, randomised, placebo controlled crossover study. Prior to each visit, subjects were randomised a normal diet (ND) or a salt-depleted diet (SDD) for three

days, with a single dose of furosemide. During each visit, volunteers received three incremental doses of apelin

and matched saline placebo. Cardiac index and stroke systemic vascular resistance index were measured

throughout by thoracic electrical bioimpedance, and heart rate and mean arterial pressure were assessed using a

semi-automatic sphygmomanometer. Results were analysed using a two-way ANOVA.

Results: Sodium urine concentrations in response to salt restriction, measured by 24 hour urine collection were

significant (P<0.0001). Apelin caused a significant increase in cardiac index under both dietary conditions

(P<0.0001) for both. There was a trend to reduced apelin efficacy under SDD (p=0.056).

Conclusion: Apelin mediated inotropy was demonstrated to be maintained after a period of increased RAS

activity. As such its actions may be preserved in conditions characterised by RAS overactivity. Apelin may

therefore represent a potential novel future therapeutic target for the use in heart failure.





NEUROLOGY & PSYCHIATRY

Effects of a Single Dose of Pramipexole on components of human decision-making

P. Scollo


Background: Despite increasing rates of pathological gambling in Britain, little is currently known about the

neural substrates of the decision-making patterns that play a central role in the development and persistence of

problem-gambling. Research has consistently indicated the importance of dopamine systems in the processing

of reward stimuli and reinforcement and now preliminary evidence indicates abnormalities in dopamine activity

in pathological gambling specifically. The drug most strongly associated with the development of pathological

gambling in Parkinson’s disease is the D2/D3 dopamine agonist Pramipexole.

Methods: 16 healthy volunteers ingested a single oral 0.176 mg dose of Pramipexole while 16 matched controls

ingested a placebo. Subjective changes and mood were assessed using visual analogue scales, and self reported

positive and negative ratings. Systolic and diastolic blood pressure was also monitored. Two hours after

treatment, all volunteers completed a risky-decision making task where they were asked to make choices

between two simultaneously presented gambles.

Results: Pramipexole produced a selective change in volunteer decision-making in the form of significantly

reduced discrimination between magnitudes of expected gains when the expected losses were low. However,

further analyses of these data suggest that the selective effect of Pramipexole upon decision-making is lost when

including positive emotional ratings (significantly reduced following Pramipexole treatment) as a control

variable.

Conclusions: Mildly increased D2/D3 dopamine activity appears to influence risky decision making under

conditions of uncertainty, though elucidating the mechanisms by which this occur requires further assessment.

The present study provides a much-needed pilot to guide future research.

Pain catastrophizing in major abdominal surgery

N. Sultan


Background: Pain Catastrophizing is defined as an exaggerated mental set brought to bear during an actual or

anticipated pain experience. No study has looked at preoperative catastrophizing levels and outcomes in major

abdominal procedures.

Methods: Thirty-one patients scheduled for major abdominal procedures were selected. Participants completed

‘Pain Catastrophising Scale (PCS)’,‘Hospital Anxiety and Depression Scale(HADS)’,‘Short Form-12 Quality of

Life Questionnaire(SF-12)’, and ‘Verbal Rating Scale(VRS) for Pain’ pre-operatively, at day 3 and 6 months

post-operatively. Total analgesic consumption(Bupivacine epidural and morphine) were documented over 48

hours post-operatively.

Results: A high pre-operative PCS was significantly associated with a high chronic HADS score

(r=478,p=0.29); and thus the development of chronic anxiety and depression. Pre-operative anxiety and

depression were predictive of epidural usage within the first 12 hours post-operatively and the development of

chronic pain (VRS)(r-0.433,p=0.21and r-0.458;p=0.37respectively). High PCS scores were not associated with

post-operative analgesia consumption; although the total48 hour morphine consumption was predictive for

eventual chronic pain development (r=0.588, p=0.006). High levels of catastrophising, depression and anxiety

were additionally associated with a poorer mental function at six months (r=0.496, p=0.22and r=0.645,p=0.02).

Conclusion: Those with high pre-operative PCS scores are at increased risk of developing chronic anxiety,

depression and pain. Targeted psychological management and optimizing analgesia use may potentially reduce

the risk for these chronic conditions.

CMJ Neurology & Psychiatry 14




Bipolar affective disorder in the perinatal period: Risk factors for postpartum relapse

K. Doyle 1, J. Heron 2, G. Berrisford 2.

1. School of Medicine, University of Birmingham, Birmingham, UK

2. Perinatal Research Programme, Birmingham & Solihull Mental Health Foundation Trust, Birmingham, UK

Background: Bipolar affective disorder (BPAD) is a severe mental illness with a chronic relapsing nature andis estimated to affect around 1% of individuals. Women with BPAD have a 25 to 50% risk of relapse in the

postpartum. In this study the existing management of women with BPAD in pregnancy is described and risk

factors for postpartum relapse are identified.

Methods: A retrospective case-note review was conducted of women with BPAD referred to the Birmingham

Perinatal Mental Health Services between the years 1998 and 2009. Of the women who were referred during

pregnancy, those who relapsed in the postpartum were compared with those who remained well.

Results: 78 women with a history of BPAD were referred for perinatal management. 47% of those referred in

pregnancy suffered postpartum relapse. The following risk factors for postpartum relapse were identified: being

unwell at referral; younger age; unplanned pregnancy; previous perinatal episodes and a family history of

BPAD.

Conclusion: Identifying such risk factors for postpartum relapse will enable clinicians to provide a more

accurate and individual estimation of a woman’s risk and modify care plans accordingly; in doing so childbirth

may become a safer time for all women with BPAD.

Neuropsychological and Neuropsychiatric Personality Profiles in Developmental Synaesthesia

J. CassellSt George's Medical School, University of London, Cranmer Terrace, London, UK

Background: Synaesthesia is a condition in which one source of sensory input triggers a response in another

sensory modality typically unrelated to the first. For example, some synaesthetes experience colours whilst

looking at letters or numbers, and for others music evokes colour.

The aim is to assess whether people with developmental synaesthesia have personalities with more schizotypal

elements (personality traits associated with psychosis and schizophrenia) than a control group of non-

synaesthetes. This may allow synaesthesia to be used as a non-pathological research model for schizophrenia.

Methods: A questionnaire, O-LIFE (The Oxford-Liverpool Inventory of Feelings and Experiences), was set up

to measure schizotypic personality traits (n = 30 matched controls; n = 30 synaesthetes).

Results: A significant difference on the questionnaire (at 95% CI) between controls and synaesthetes wasfound, particularly in positive (‘Unusual Experiences’) and disorganised (‘Cognitive Disorganisation’)

schizotypy subscales.

Conclusion: The results show that synaesthetes exhibit trends towards increased schizotypal personality traits,

however they do not display the cognitive impairments typically associated with schizophrenia. Therefore they

should not be considered a patient group. Synaesthetes therefore may offer a way to study these abnormal

perceptions but in the absence of the cognitive impairments and other problems seen in psychotic disorders

CMJ Neurology & Psychiatry 15




ENDOCRINOLOGY & METABOLIC SCIENCE

An epidemic of vitamin D deficiency in the central Manchester population:Relationship to diabetes

complications and ethnicity.

Z. HussainSchool of Medicine, The University of Manchester, Oxford Road, Manchester, UK

Background: Whilst Vitamin D levels have traditionally been associated with bone and muscle health, an

increasing body of evidence suggests that they may affect a range of metabolic, cardiovascular and cancer

outcomes. We assessed the prevalence and management of vitamin D deficiency in relation to ethnicity,

diabetes, cardiac and microvascular complications.

Methods: Data was collected in 1582/5822 patients who had undergone assessment of vitamin D between

2005-2010 from a primary care practice in central Manchester.

Results: Mean vitamin D ~ 16.4ng/ml. 87% of patients were sub-optimal (<30ng/ml), 72% insufficient

(<20ng/ml), and 38% markedly deficient (<10ng/ml). South-Asians had the lowest level (10.5±7.9ng/ml),

followed by African-Caribbean’s (15.7±11.9ng/ml) and Caucasians (22.5±13.6ng/ml). There was no significantdifference in vitamin D levels between diabetic (16.6±11.4) and non-diabetic (16.4±12.3) patients (p=0.79).

Only 10-15% of patients with vitamin D <20ng/ml had musculoskeletal symptoms. Hypertension (36%), asthma

(19%), arthritis (15%) and depression (11%) were the most prevalent co-morbidities in patients with low

vitamin D. Cardiac history was present in 15% of patients with a vitamin D <10ng/ml compared to 3% in those

with a vitamin D >30ng/ml. Both neuropathy and retinopathy was increased 2 fold in patients with a vitamin D

<20ng/ml. Treatment only increased Vitamin D by 5.41ng/ml.

Conclusion: Our data demonstrates an epidemic of vitamin D deficiency which cannot be detected by

symptoms alone and which is associated with cardiovascular and microvascular complications. This urges the

need to develop a consensus on assessment and treatment of vitamin D deficiency both locally and nationally.

Dysglycaemia screening in Type 2 Diabetes Mellitus

B. Fisher & Y. Ang


Background: Type 2 diabetes mellitus (T2DM) is a cause of significant morbidity and mortality, but half of

cases remain undiagnosed. Screening for dysglycaemia is universally advocated, but there is currently no

consensus on which population to screen or which test to use.

Methods: We audited a record-based screening programme in a 9700-patient general practice, whereby patients

attending a healthcare assistant-led Annual Review Clinic for hypertension, cardiovascular disease, or chronic

kidney disease were screened for dysglycaemia by random venous plasma glucose (RVPG) measurement. Those

with a RVPG ?6.1 mmol/l were recalled for a fasting measurement, allowing diagnosis of T2DM or impaired

fasting glucose (IFG). We audited the programme over a one-year period by searching the practice's database.

Results: 786 patients were eligible for T2DM screening. 544 were screened, of whom 120 had a RVPG ?6.1

mmol/l. Only 40 of these had a subsequent fasting measurement, leading to 5 diagnoses of T2DM and 11 of

IFG. The positive predictive value (PPV) of the test for T2DM was 13%, and the laboratory cost was £155 per

patient diagnosed. Increasing the RVPG cut-off would increase the PPV but also the number of false negatives

and cost. 80% of newly-diagnosed diabetics had hypertension. The programme accounted for 41% of all T2DM

diagnoses at the practice.

Conclusion: Integration of this simple T2DM screening protocol with an Annual Review Clinic is a logical andefficient use of scarce primary care resources. Limitations include a low uptake of diagnostic testing and an

inability to detect impaired glucose tolerance.

CMJ Endocrinology & Metabolic Science 16




The effect of glucosylsphingosin on actin dynamics.

C St.John-Green1, Dr N.J Smith2 & Professor T.M Cox2

1. Stage 3 Medical Student, University of Cambridge School of Clinical Medicine, Hills Rd, CB20SP 2. Addenbrooke's Hospital, Box 1112. Lysosomal Diseases Research Group, University Department of

Medicine, Cambridge University Hospitals, Cambridge, UK

Background: Gaucher disease is characterized by a pathological increase of sphingolipids.

Glucosylsphingosine (GlcSph) is considered the predominant toxic metabolite. Recent work in immortalised

lines suggests a pathological effect due to disruption of normal cellular actin dynamics causing impaired

cytokinesis and cytoskeletal function. The concentration at which pathological effects occur, and the nature and

degree of morphological and functional impairment of GlcSph on cells is uncertain.

Methods: We treated primary human fibroblast monolayers with quasi-pathological concentrations of GlcSph

in culture medium (0.625µM - 20 µM). A wound assay was performed to assess cellular migration, a process

dependant on actin function. Sample coverslips were fixed and stained with TRITC-conjugated phalloidin for

visualisation with epifluorescent bright field microscopy. In addition morphological and cell viability analyses

were performed using image J software.

Results: Cell viability was inversely related to GlcSph concentration, and was significantly reduced at 10µM

GlcSph in comparison to controls (p<0.05) (t-test). A concentration dependent decrease in wound healing rate

and percentage of wounds achieving total closure was seen up to 5µM GlcSph concentrations. At 5µM and

above cells demonstrated blunting with decreased average cell size; decreased lamellopodia and a thickened,

disorganised stress fibre network.

Conclusion: This work supports the previously recognised cytotoxic effect of GlcSph and the morphological

observations are consistent with its purported effect on the actin cytoskeleton.

Refining the management of Non-functioning Pituitary Adenomas

J. Joshi


Background: Non-functioning Pituitary Adenomas (NFAs) are the most common tumours of the pituitary

gland. They usually present late, typically with symptoms of visual field defects and headache. Currently, there

are no consensus guidelines for their treatment but different management options exist. This study aimed to

investigate the effect of different treatment modalities (a Conservative approach without treatment, Surgery and

Surgery and radiotherapy (RT) combined) in patients with a NFA on tumour growth, clinical features and

pituitary function.

Methods: One-hundred and seventy-six patients (84 males, 92 females) with a NFA were identified from a

database of all the endocrine patients seen at the Hammersmith and Charing Cross hospitals over the last 12

years. Data was collected for their clinical features, pituitary function tests and tumour size.

Results: Conservatively managed NFAs remained the same or decreased in size (78%), and 22% increased in

size. Visual field defects were present in 78% of patients pre-operatively and improved in 70% of patients with

further deterioration prevented in 20%. Adjuvant RT increased the percentage of patients that were

hypopituitary (63%) and surgery alone increased hypopituitarism (24%).

Conclusion: Conservative management with close radiological and endocrine follow-up is safe for those with

normal visual fields. Surgery is mandatory for large tumours causing visual defects as it results in immediate

decompression of the optic chiasm. However, it increases the incidence of hypopituitarism. Similarly Adjuvant

RT causes a significant increase in hypo- and panhypopituitarism which have associations with increased rates

of mortality. This study may thus influence NFA management.





The relationship between physical function and the metabolic syndrome in an older Chinese population

L Li1, CQ Jiang2, WS Zhang2, KK Cheng2, TH Lam2, GN Thomas3,1College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK 2Guangzhou Occupational Disease Prevention and Treatment Centre, Guangzhou No. 12 Hospital, Guangzhou

510620, China3 Public Health, Epidemiology snd Biostatistics, University of Birmingham, Birmingham, UK

Background: The metabolic syndrome (MS), a collection of related risk factors for cardiovascular disease and

diabetes, has increased in prevalence over recent years. A relationship between walking speed and elements of

cardiovascular disease and diabetes has been indicated. We aimed to determine if an association exists between

a stand and walk test (SWT), a physical function measure, and the MS.

Methods: 992 male and 1004 female older residents of Guangzhou, China, were investigated within the

Cardiovascular Subcohort study, part of the larger Guangzhou Biobank Cohort Study. Physical function and

metabolic factors were measured. Mann-Whitney tests compared SWT time between those with and without the

MS. ANOVA compared how metabolic syndrome components varied within each tertile of SWT time taken.

Regression analysis measured the strength of the association between the MS and physical function after

controlling for confounding factors.

Results: The median SWT time for those with and without the metabolic syndrome was 4.91s and 4.68s

respectively ( P < 0.001). The proportion with hypertension significantly increased as SWT time increased, the

proportion of participants with hypertension in SWT tertiles 1, 2, 3 were 39.2%, 42.9%, 59.4% respectively ( P <

0.001). Central obesity, hyperglycaemia and high triglyceride prevalence also increased significantly as SWT

time increased ( P < 0.05 for all). After adjusting for confounding factors, SWT performance still significantly

predicted the MS for women only ( P = 0.01).

Conclusion: Those with the MS are slower at performing the SWT than those without in an older, urban

Chinese population.





ONCOLOGY

Survey of epidermal growth factor receptor (EGFR) mutation in patients with non-small-cell-lung cancer

Y.K. Huong

1

& F. Blackhall

2

1. School of Medicine, The University of Manchester, Oxford Road, Manchester, UK

2. Christie Hospital NHS Trust, Manchester, UK

Introduction: EGFR mutation testing was recently launched in the North West following the IPASS trial,

which showed the superiority of gefitinib over chemotherapy among EGFR mutated NSCLC patients. The

mutation occurs more frequently in females, East Asians, non-smokers and those with adenocarcinoma

histology.

Methods: Data were collected from EGFR mutation referral forms and medical notes using a proforma for each

patient. Data were then tabulated and analyzed using Excel software.

Results: 52% and 48% of 148 patients screened were females and males, respectively. Patients selected tend to

be over 50 years old (81.1%), Caucasians (97.3%), current or ex-smokers (70.3%), Stage IV (53.4%)adenocarcinomas (81.8%) and those with 1 and 2 WHO performance status (48.0%). Majority of the specimens

have unspecified origins and methods of biopsy. With a turnaround time of <11 days (81.4%) and a failure rate

of 4.7%, exon 19 in-frame deletions (50%) were detected the most, followed by exon 21 point mutations

(33.3%). Mutations were detected equally between genders, more frequently in the age group of 70 – 79

(58.8%), ex-smokers (58.8%) and Stage IV (94.1%) adenocarcinomas (88.2%). 58.8% of EGFR positive

patients were treated with TK-Is. The response rate was 60.0%, with the commonest side effects being Grade I

rash (30.0%) and diarrhoea (30.0%).

Conclusion: EGFR mutation screening is feasible in NSCLC patients, with smoking status as the strongest

predictor. The treatment with TK-Is is well tolerated with better clinical outcome.

Cell cycle dependent association of CTCF with papillomavirus genomes

D. Roberts


Background: Papillomaviruses are known aetiological agents of benign and malignant disease in animals and

humans. The human papillomavirus (HPV) has been found to be the principal causative factor in cervical

cancer. Furthermore, HPV is increasingly being implicated in other disease processes in humans such as

oropharyngeal and perineal cancers.Recent studies have shown that the Kaposi's sarcoma-associated herpes

virus (KSHV) associates with the ubiquitous cellular protein CTCF in maintaining infection within cells and that

this occurs in a cell cycle dependent manner. Since it is known that the DNA viruses exhibit similar behavioursin maintaining episomal infections, we postulated that HPV may associate with CTCF in a similar manner.

Methods: Using conventional cell synchronisation techniques, association of CTCF with the bovine

papillomavirus (BPV) genome, which shows a close homology to the HPV genome, was analysed using the

technique of chromatin immunoprecipitation (ChIP).

Results: Experiments showed that the association between CTCF and the BPV genome is strongest between 3

and 5 hours after release, corresponding to entry into G2 phase of the cell cycle. We postulate that this indicates

that the BPV genome recruits CTCF during S phase as genomes are replicated. Following on from this,

immunoprecipitation experiments were conducted which showed that levels of CTCF expression remain

constant throughout the cell cycle and that CTCF and the E2 papillomavirus protein bind to each other.

Conclusion: We conclude that CTCF is indeed recruited to the viral genome in a cell cycle dependent manner and that this may have important implications for the viral lifecycle.

CMJ Oncology 19




Comparison of prognostic scores for preoperative prediction of survival for spinal metastases

M.M. Hou


Background: Spinal surgery for metastatic tumours is associated with significant morbidity and mortality. A

patient’s predicted survival is one of the main factors considered when deciding between surgery or radiotherapy treatment. Many scoring systems have been developed to predict prognosis in patients with spinal

metastases, which differ in the parameters assessed. This study evaluated the prognostic value of scoring

systems proposed by Bauer, Tomita, Tokuhashi, Sioutus and van der Linden.

Methods: 60 patients who underwent surgery for spinal metastases since 2005 were included in this study.

Pre-operative prognostic parameters were recorded retrospectively for each patient, from which their prognostic

scores were calculated. The prognostic value of each scoring system was assessed by correlating prognostic

scores to observed survival.

Results: Median survival was 13 months. Out of the prognostic scoring systems tested, the Tomita score

correlated best with observed survival (Pearson’s r=-0.389, p=0.002) and had the best sensitivity and specificity

for determining which patients will survive for less than 3 months compared to more than 3 months (ROC curve

area=0.767, p=0.004).

Conclusion: The Tomita score correlated best with survival and was also best at discriminating those patients

with a survival of less than 3 months, for whom spinal surgery is usually not advocated. This score is simple

and includes the type of primary tumour, presence of visceral metastases and bone metastases as prognostic

factors. The Tomita score could be used as an adjunct for determining which patients have a good enough

prognosis to benefit from spinal surgery.

A Retrospective Study on the Demographic Trends of Gallbladder Cancer Patients in an Indian Hospital

S. Ganguli

St George's Medical School, University of London, Cranmer Terrace, London, UK

Background: Gallbladder cancer is a relatively rare malignancy, and one that displays an unusual geographical

distribution. The purpose of this study was to compare the prevalence of different malignancies in the UK with

those of a north Indian hospital. The study also compared the demographic trends of gallbladder cancer patients

between the two places.

Methods: Data was retrospectively collected from medical notes and referral letters of 300 inpatients at ESI

Hospital Sealdah, Kolkata, oncology department during a six year period. Information documented included sex,

age and type of malignancy. For patients with gallbladder cancer, the presence or history of gallstones andevidence of metastases, were recorded. This information was then compared with data from the UK.

Results: Of the 300 patients, the most prevalent malignancies were oral (13%), breast (11%), lung (11%) and

gallbladder cancers (10%). The prevalence of breast and lung malignancies remained similar between ESI

Hospital Sealdah and the UK; however there was a significant increase in the prevalence of oral (13 times) and

gallbladder cancers (10 times) in Sealdah. The mean age and male-to-female ratio of gallbladder cancer patients

remained constant in both the UK and Sealdah. Of the gallbladder cancer patients, 21 (70%) had evidence of

previous gallstones and 23 (77%) showed signs of metastases.

Conclusion: There is a strong correlation between gallstones and the development of gallbladder cancer,

suggesting a possible role for prophylactic cholecystectomy following the diagnosis of gallstones in high risk

patients.

CMJ Oncology 20




Characterisation of Basal K + Conductances Responsible for Setting the Resting Membrane Potential in

the Human Erythroleukaemic (HEL) Cell Line

A. Teo


Background: Potassium channels play a variety of roles in both excitable and non-excitable cells, by virtue of their significant involvement in the setting of the resting membrane potential. Cancer cells of various origins

have been shown to lack conventional K + channels. For instance, the loss of voltage-activated K +-channel

activity has been reported in megakaryocytes from patients with acute myelogenous leukaemia (AML) and in

numerous leukaemic cell lines used as models for many megakaryocyte functions including the human

erythroleukemia (HEL) cell line.

Methods: Whole cell patch-clamp experiments were undertaken to define the basal K + conductance(s) in HEL

cells and their contribution to the setting of resting membrane potential.

Results: Two distinct Cs+- sensitive inwardly-rectifying K + currents were found; a voltage-independent, non-

inactivating current, and a voltage-dependent, inactivating current displaying similar characteristics to the

human eag-related gene (HERG) – a gene encoding a family of K +-channels that have been found to be

upregulated in primary tumours and cancer cell lines of various neural origins including AML. Furthermore, theformer current was found to be Mg 2+- modulated while the HERG-like current was found to be sensitive to

inhibition by HERG-specific inhibitor E-4031.

Conclusion: The discovery of these either or both these currents in all cells studied suggest a role for these in

the setting of their resting potential, which may in turn contribute to their ability to survive. Further work will

have to be undertaken in primary leukaemias to determine their potential role in leukaemic pathology, and more

generally their existence in other forms of cancer.

CMJ Oncology 21




GENETICS

Genes associated with adult cerebral venous thrombosis

T. Marjot, S. Yadav, N. Hasan, P. Bentley & P. Sharma

Faculty of Medicine, Imperial College London, South Kensington Campus, London, UK

Background: Quantitative predictions of risk of cerebral venous thrombosis (CVT) conferred by certain

genotypes have yet to be reliably established. We conducted a comprehensive meta-analysis of all candidate

genes studied to assess their genetic contribution to its aetiology. We compared our findings against equivalent

analyses for paediatric CVT and adult ischaemic stroke.

Methods: Databases were searched to August 2010 for all genes investigated in adult CVT and Odds Ratios

(OR) for each gene-disease association calculated. A mendelian randomiszation strategy was also undertaken to

determine whether a causal relationship to one gene could be ascertained.

Results: We identified 26 case-control studies, investigating 6 polymorphisms in 6 genes and included 1183

CVT cases and 5189 controls. Statistically significant associations with CVT were found for Factor V/G1691A

(OR 2.40, 95% CI: 1.75-3.30, p<0.00001) and prothrombin/G20210A (5.37, 95% CI: 3.78–7.63, p<0.00001).

After iterative analysis controlling for inter-study heterogeneity, MTHFR/C677T was also found to be

significantly associated (OR 2.30, 95% CI: 1.20–4.42, p=0.02). Variants in the remaining 3 genes (JAK2, PAI-1

and Protein Z) were not significantly associated. Pooled odds ratios for CVT risk in adults for Factor V Leiden

and Prothrombin were significantly greater when compared against childhood CVT and adult arterial ischaemic

stroke. A causal relationship with MTHFR may be exist.

Conclusions : CVT has a genetic basis. Genes involved in the clotting cascade provide a greater level of

thrombosis risk in the cerebral venous circulation compared to its arterial circulation, and; a greater level of risk

for adults compared to children.

Genetic Screening of Patients with Thoracic Aortic Aneurysm for Mutations in ACTA2

S. Harris1, J. A. Aragon-Martin1 & G. Arno1

1. Cardiac & Vascular Sciences, St George's, University of London, London, UK

Background: Thoracic aortic aneurysms (AAT) are a growing cause of mortality and morbidity in the

developed world. There are several genetic conditions that are clearly linked to AAT, such as Marfan, Loeys-

Dietz and Turner syndromes. In patients without one of these syndromes, up to 21 % have been found to have a

strong family history of AAT. Recently studies have implicated mutations in ACTA2 (MIM#102620) as a cause

of familial AAT.

Method: A total of 78 patients with known AAT and not fulfilling the Ghent criteria for Marfan syndrome and

with no demonstrable mutations in their FBN1 or TGFBR2 genes were recruited to this study. They were

screened for mutations in all exons in ACTA2 including intron/exon boundaries.

Results: In 1/78 (1.3%) a mutation was found in exon 3, p.R64K (c.G191A). This mutation was not found in

100 control chromosomes.

Conclusion: This study supports data from previous studies that links mutations in ACTA2 with AAT. In our

cohort of probands, the detection rate is lower than previously published. This may be due to the relatively

heterogeneous population studied. We included all non-Marfan patients with AAT and no FBN1 mutation.

CMJ Genetics 22




Assessing the Role of SEMA5A in Neurodevelopmental Disorders

T.X.C.B. Diaz & A. Gawthrope


Background: Genetic studies have associated various semaphorins, their receptors and related signalling

components with several neurological and neurodevelopmental disorders. In particular, two independent

genome wide association studies have identified the semaphorin 5a (SEMA5A) gene as a candidate in the

aetiology of Parkinson’s disease and more recently Autism, suggesting this gene may have pleiotropic effects.

This study aims to investigate whether common polymorphisms within the SEMA5A gene affect its level of

expression.

Methods: SNP markers within the promoter of the SEMA5A gene were selected on their ability to tag variation

in the region. RNA extracted from post-mortem amygdala brain tissue was used as an estimate for gene

expression. Utilising TaqMan genotyping and expression assays, statistical correlation of these two variables

was investigated.

Results: Statistical analysis did not reveal any significant relationship between SNP genotype and relative geneexpression of the SEMA5A gene.

Conclusions: There was no observed effect between SNP genotype of the SEMA5A gene and its level of

expression, suggesting that the polymorphisms investigated have no functional consequence. However, as not

all of the common variation within this gene was explored, further research is required to fully determine

whether SNP markers within the gene have a role in the aetiology of neurodevelopmental disorders.

Genotype-phenotype correlations in FBN1 and ADAMTSL4 Ectopia Lentis

K. Hughes1, A. Chandra2, G. Arno1, D. Charteris2, A. Child1

1. Cardiac and Vascular Sciences, St. George’s, University of London, London, UK 2. Vitreoretinal Surgery, Moorfields Eye Hospital, London, UK

Background: Ectopia lentis (EL) is a heterogeneous condition with autosomal-dominant and autosomal-

recessive forms caused by mutations in FBN1 and ADAMTSL4, respectively. The aim of this study was to

determine if the inheritance pattern and ocular presentation could differentiate two major genetic subgroups of

EL.

Methods: Of 33 non Marfan syndrome EL patients studied, 21 had FBN1 mutations with no cardiac

involvement (Ghent negative); 6 had causative ADAMTSL4 mutations; 6 had neither. Clinical notes were

reviewed.

Results: Of the 21 FBN1-mutation patients, 19 had bilateral lens dislocation compared to four patients in the

ADAMTSL4 group. Lens movement was in no specific direction in either group. Fifteen (71%) of the FBN1 had

dominant family history. Three out of the six ADAMTSL4 group had an affected sibling in the absence of an

affected parent, and two had first cousin parents: demonstrating recessive inheritance. No FBN1 mutation

patients had cataracts compared to three out of six ADAMTSL4 patients.

Of the remaining six patients where no mutations have been found, four out of six demonstrated dominant

inheritance, and two had a possible recessive inheritance, suggesting at least two further genes (1 dominant, 1

recessive) causing EL.

Conclusion: Two major EL subgroups can be distinguished clinically by trends in inheritance pattern and

incidence of cataract. Further work is required to increase the cohort size and thus identify other genes

predicted to cause EL, their possible interactions, and further clarify genotype-phenotype relationships.

CMJ Genetics 23




INFECTION, INFLAMMATION & IMMUNOLOGY

Screening models for Barrett’s oesophagus

R. GunasekeraThe University of Sheffield school of medicine and biomedial science, Sheffield, UK

Background: The broad objective of this study is to determine whether a screening programme for Barrett’s

oesophagus would be cost effective in the Sheffield population using systems thinking as a tool. Barrett’s is a

condition in which the stratified squamous epithelium at the lower end of the oesophagus becomes replaced with

columnar epithelium due to acid reflux. Its predisposition to adenocarcinoma and its asymptomatic behaviour

make it an ideal disease for screening. However the viability of such a screening programme has come under

much scrutiny in literature with many contradictory studies. This project includes a screening model which runs

over 40 years, which allows an insight how it performs over a substantial time period.

Methods: Three methods are used: cognitive mapping; causal loop diagrams and systems dynamics. This

systems approach produced two models representing a screening programme for the Sheffield population.

Results: In Model I the screened individuals in the population are over 40 years and those turning 40 every year

whilst Model II describes a model that screens individuals just turning 40 years. The different parameters in

each model were varied using sensitivity testing in the system dynamics software (iThink). From this we are

able to establish the optimum criteria for carrying out a Barrett’s oesophagus screening programme.

Conclusion: We came to the conclusion that model II appears to be the viable option, as its costs and

implementation into the community seem more feasible when compared with model I. The future of Barrett’s

oesophagus screening may lie in the recent development of a technique, cyotosponge, which would replace

endoscopy as a primary screening modality.

The prevalence of Klebsiella pneumoniae infections caused by strains with a hypermucoviscosity

phenotype isolated from blood culture specimens between 2008 and 2010

N. Begum, M.J. Pond, P.D. Butcher & P. Riley

Division of Cellular and Molecular Medicine & Microbiology St. Georges University of London, London, UK

Background: Klebsiella pneumoniaeis a gram-negative, facultative anaerobic, non-motile bacillus in the

family Enterobacteriaceae. Recently an association between K.pneumoniae strains possessing a

hypermucoviscosity (HMV) phenotype and development of a specific invasive clinical syndrome has been

demonstrated. The rmpA gene is associated with expression of the HMV phenotype and the presence of K1 or

K2 capsular antigen has been commonly found within HMV isolates (ref.1). Infections with the HMV

phenotype have mostly been described in Asia. The prevalence of this organism has not been widely

investigated in the UK.

Methods: K.pneumoniae strains isolated from blood culture specimens where stored at -70oC until required.

Subsequently each isolate was investigated using the ‘String Test’ for HMV. A standard bacteriological loop

was passed through the colonies and if the colony formed a mucoviscous “string” that is greater than 5 mm, the

isolate was defined as possessing the HMV phenotype.

Results: 7 % of isolates possessed the HMV phenotype. Furthermore, 56% of patients admitted into hospital for

K.pneumonia were aged >60. HMV phenotype was present in males and females equally.

Conclusion: This preliminary study has demonstrated that 7% of K.pneumoniae isolated at St George’s posses a

HMV phenotype. We hope to determine the presence of specific serotypes (K1 & K2) by investigating the

presence of magA and K2wzy genes. Investigation of rmpA gene may prove as a link between HMV and the

invasive symptoms. Following characterization of our collection we intend to correlate the phenotypic andgenotypic strain characteristics with patient clinical data.

CMJ Infection, Inflammation & Immunology 24




The safety and acceptability of ultrasound guided biopsy in early arthritis patients.

I. Parwaiz, H. Kurunadalingam, K. Kumar, K. Howlett, C. Buckley, K. Raza, S. Kelly & A. Filer

Rheumatology Research Group, School of Immunity and Infection, The University of Birmingham, UK Barts and the Royal London and Queen Mary University of London, UK

Background: There is strong evidence that early detection and intervention in rheumatoid arthritis patients cangreatly improve treatment outcomes, especially within the first three months of the disease. US guided biopsy is

a less invasive alternative to formal arthroscopy used as a procedure to obtain research samples in patients with

early arthritis.

Methods: 123 participants gave written informed consent to be recruited from hospitals in London and

Birmingham. The main entry criteria for the study were patients with undifferentiated early arthritis or RA

affecting joints in either the upper or lower limb. The patients were followed-up post procedure for at least 6

months.

Results: 123 participants were included with a mean age of 53 (19-83) years. There were no instances of joint

infection, DVT, neurological damage, or thrombophlebitis seen in our participants. However, there was a wound

infection in an early London biopsy (0.8% of the US biopsied cohort), which was used as a critical event to

inform practice in the collaborative network. In addition, the incidence of haemarthrosis was similar to that seenwith arthroscopies, 0.8% compared to 0.9%. Most of the patients felt no pain or mild to moderate discomfort

during the procedure (90% of patients in Birmingham and 99% of patients in London) with small joint biopsies

causing less discomfort. The majority of patients felt the same after the biopsy with many feeling ‘better’ or

‘much better’ post procedure (39% of patients in Birmingham and 15% of patients in London). A significant

number of patients stated they would be likely to repeat the procedure in the future, (81% in Birmingham and

72% in London).

Conclusion: From this early cohort, US guided biopsies may have fewer complications than formal arthroscopy

as a research tool, particularly when small joints are biopsied. This novel technique could replace arthroscopy as

an investigator led research procedure or within clinical trials as it is well tolerated, with fewer side effects

related to the procedure.

The Aetiology and Epidemiology of Bacterial and Viral Maternal Infections in the Developing World

P.P. Velu, H. Campbell & I. Rudan


Background: Bacterial and viral maternal infections are important contributors to high maternal morbidity and

mortality in low and middle income countries. However, information on the precise nature of these infections is

sparse and poorly characterised, hampering the implementation of policies to improve maternal health. This

review aims to identify the aetiology and epidemiology of bacterial and viral maternal infections in the

developing world.

Methods: A comprehensive search of published literature (MEDLINE, EMBASE and Global Health) wasconducted and data on aetiology and epidemiology of maternal infections was extracted from relevant studies.

Results: 1565 relevant studies were identified following initial screening of 8580 titles. The application of strict

inclusion and exclusion criteria selected 158 high quality studies from which data was extracted and analysed to

characterise the epidemiology of 10 most extensively reported maternal infections and their median prevalence

rates - Treponema pallidum (2.6%), Neisseria gonorrhoeae (1.5%), Chlamydia trachomatis (5.8%), Group B

streptococci (8.6%), Bacterial Vaginosis (20.9%), Hepatitis B virus (4.3%), Hepatitis C virus (1.4%),

Cytomegalovirus (95.7% past infection), Rubella (8.9% susceptible) and Herpes simplex (20.7%). Data on study

characteristics, diagnostic tests used and variations in the prevalence of these infections between countries and

regions was also extracted and reported.

Conclusions: This review confirms and quantifies the suspected high prevalence of maternal bacterial and viral

infections in the developing world, identifying particular diseases and regions requiring urgent attention. Thisinformation will influence public health policy planning, research priorities and donor funding towards reducing

infection-related maternal morbidity and mortality in developing countries.

CMJ Infection, Inflammation & Immunology 25




SURGERY

Reviewing the management of thoracolumbar burst fractures

J. Rowton

School of Medicine & Dentistry, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen, UK

Background: Management of thoracolumbar burst fractures is a controversial topic. It is generally accepted that

unstable fractures should be managed operatively, but there still remains no general consensus as to what

represents a stable or unstable fracture. Current indications for operative treatment entail the use of radiological

findings including measuring the loss of vertebral body height and local kyphosis angle.

Methods: A retrospective analysis of thirty-six patients identified as having suffered an isolated thoracolumbar

burst fractures and treated at The Alfred Hospital from 2007 to 2010 was performed. The Alfred’s orthopaedic

database was used to identify eligible patients and data obtained from medical records and radiological imaging,

including radiographs and computed tomography. In addition, all patients were contacted and asked to complete

a questionnaire to assess functional outcome, pain, and satisfaction with treatment.

Results: Operative and conservative treatment displayed similar functional outcomes. Average low back pain

scores were lower in the non-operative cohort compared to the operative. Greater disability was also reported inthe operative cohort. Mental scores were also assessed with the non-operative cohort reporting higher levels of

mental functioning.

Conclusions: This review has added to the current popular opinion that non-operative treatment results has

outcomes comparable with operative for a thoracolumbar burst fracture, lending further weight to the body of

evidence suggesting equivalence between the two. Nevertheless, clinical equipoise will remain until higher

quality prospective studies are conducted.

Improving the Quality of Colon Cancer Surgery through a Multidisciplinary Education Programme:Early Results

K. Sutton1, N.P. West1, P. Ingeholm2, W. Hohenberger3 & P. Quirke1

1 Pathology & Tumour Biology, Leeds Institute of Molecular Medicine, Leeds, UK 2 Department of Pathology, Hillerød Hospital, Copenhagen, Denmark 3 Department of Surgery, University Hospital of Erlangen, Erlangen, Germany

Background: The importance of the plane of rectal cancer surgery is well established, however, the evidence

for a similar effect in colon cancer is limited. We have previously reported better outcomes with mesocolic

plane surgery and shown that complete mesocolic excision with central vascular ligation (CME & CVL)

produces oncologically superior specimens.

Methods: We received specimen photographs and clinicopathological data from a series of 263 primaryresections for colon cancer; 93 from surgeons trained in CME & CVL and 170 from surgeons prior to training.

The plane of dissection was graded using a method described previously. Tissue morphometry was performed

using ImageScope v10 (Aperio, CA).

Results: CME & CVL surgeons were more likely to operate in the mesocolic plane (75% vs. 48%, p<0 .0001)

and remove more lymph nodes per specimen (median 28 vs. 18, p<0.0001). 123 fresh and 145 fixed specimen

photographs were suitable for morphometry. CME & CVL surgeons removed more tissue longitudinally in both

the fresh (median 315 vs. 247mm, p<0.0001) and fixed (269 vs. 207mm, p<0.0001) specimens, and centrally

between the tumour and the high vascular tie in both fresh (105 vs. 84mm, p=0.006) and fixed (82 vs. 67 mm,

p=0.002).

Conclusion: We have shown that surgeons trained in CME & CVL are more likely to operate in the mesocolic

plane, remove more tissue centrally and longitudinally, and achieve greater lymph node yields. This providesfurther evidence for the oncological superiority of CME & CVL, demonstrating that surgical education can

directly influence the quality of specimens produced.

CMJ Surgery 26




Intra-Medullary fixation of 5th Metacarpal shaft fractures: A retrospective study to determine outcomes

in the utilization of standard K-wiring vs. the Small Bone Fixation System

H. F. Kassam


Introduction: Metacarpal shaft fractures are common, yet, potentially devastating injuries to hand functionality

if not treated in a prompt and effective manner. These injuries are commonly managed surgically and several

options exist for obtaining adequate fixation. Intra-medullary (IM) fixation has emerged as a valid and highly

utilized method. This study compares the benefits of K-wiring fixation (£3.15 per unit) vs. the small bone

fixation system (£197.20 per unit).

Methods: 21 patients who had undergone surgical fixation of 5th (along with any concurrent 4th) metacarpal

shaft fractures in a Northwest District General Hospital were subdivided into two groups; Group A: patients

who had undergone fixation with standard K-wires with an external endpoint (n=10) and a subcutaneous

endpoint (n=3) and Group B: patients who had undergone fixation with the Small Bone Fixation System (n=8).

Patients who had undergone transverse pinning or ORIF’s were excluded from the data set. Patients were sex

and age matched (n=13,8; p=0.59).

Results: The use of K-Wires significantly reduced the time in situ being removed on average 36.6+/- 13.0 daysafter fixation in comparison with 50.3+/-4.0 days in Group B (p=0.01). No significant difference was

demonstrated for the tourniquet time between groups, taking on average 34.2+/ -11.3 minutes and 37.6+/-12.2

minutes in Group A & B respectively (p=0.52). Interestingly, 92.3% of K-wires in Group A were removed using

local anaesthetic +/- Entonox. Group B IM devices required general anaesthetic.

Conclusions: The use of K-wiring significantly reduced the time of device in situ, with no detriment to

tourniquet time, infection rate and at a significantly reduced cost to the Small Bone Fixation System.

Development of a virtual reality colonoscopy training curriculum

A. Banerjee,

1

C. Sugden,

1

R. Aggarwal,

1

A. Haycock,

2

S. Thomas-Gibson,

2

C. Williams,

2

A. Darzi

1

1. Department of Surgery and Cancer, St Mary’s Campus, Imperial College Healthcare NHS Trust, London,UK.

2. Wolfson Unit for Endoscopy, St. Mark’s Hospital, Imperial College Healthcare NHS Trust, London, UK.

Background: Colonoscopy requires detailed knowledge and technical skill. However, recent changes to

working practices have lead to a reduction in traditional training opportunities. Much might, therefore, be

achieved by applying novel technologies such as virtual reality (VR) simulation to colonoscopy. Scientifically

developed device-specific curricula aim to maximize the yield of laboratory training by providing instructions

for efficient and effective use of the simulator.

Methods: The objective was to design a proficiency-based curriculum for a high fidelity VR colonoscopy

simulator. 30 novices (<10 colonoscopies), 10 intermediates (100-500 colonoscopies) and 10 experienced

(>500 colonoscopies) participants were recruited. Construct validity was demonstrated for 14 tasks, bycomparison of performance between groups on various simulator-derived metrics. The performance

improvements of novice participants over 10 repetitions were used for learning curve analysis. Performance

benchmarks were based on the experienced group.

Results: Excellent construct validity and significant learning curves were demonstrated for 8 tasks and were

therefore included in the curriculum. The tasks dissociated into 3 categories: 3 abstract skills, 4 part procedures

and 1 complete case. The complete case was construct valid for 8 metrics including: total time (701, 352 and

252s; P<0.001) and insertion length with obscured lens (0.017, 0.003 and <0.001m; P=0.007). Learning curves

plateaued between the sixth and ninth attempts.

Conclusion: This is the first study to develop a hierarchical, benchmarked, proficiency-based VR training

curriculum for colonoscopy. By applying this validated curriculum to the training of novice practitioners, it

may be possible to enhance the safety and efficiency of patient-based colonoscopy training.

CMJ Surgery 27




CASE R EPORTS

Non-specific histopathology in a patient with livedo reticularis associated with violaceous papules on the

forearms.

S. ZafarUniversity of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, UK

Background: A 68-year-old Caucasian female presented with a one-month history of skin changes over her feet

and flexor forearms bilaterally. The patient complained of cough, nasal congestion, wheeze and dysponea on

mild exertion, refractory to repeat antibiotic therapy.

Results: Examination revealed non-blanching, reticulate erythema(livedo reticularis) with violaceous papules.

Punch excision of dermal-based-papules showed evidence of a single, medium-sized vessel vasculitis. Her past

history included pulmonary infiltrates and chronic sinusitis. Bronchial brushings showed caseating,

granulomatous infiltrates with changes suggestive of chronic bronchitis. Work-up revealed positive peri-

nuclear-anti-neutrophil-cytoplasmic antibodies(p-ANCA), anti-myeloperoxidase antibodies, elevated

erythrocyte-sedimentation-rate and c-reactive-protein. She was subsequently diagnosed with an ANCA-

associated-vasculitis, with pulmonary and cutaneous involvement, suggestive of Wegener’Granulomatosis(WG). Intravenous steroids and oral immunosuppressive therapy improved cutaneous and

pulmonary lesions, also normalizing p-ANCA levels.

Conclusions: Histopathology specimen of cutaneous polyarteritis nodosa(CPAN) lesions, like WG, demonstrate

a leukocytoclasitc vasculitis or perivascular granulomatous inflammation of small-to-medium vessels.

Histologic features of this patient were inconspicuous and in this case, the diagnosis of cutaneous WG was

entirely conditional on identifying the extramural and transmural granulomatous inflammation in a solitary

vessel. Clinical presentation was also atypical of WG, more closely resembling CPAN. This case therefore

illustrates a potential for misdiagnosis and consequent mismanagement of a patient with non-specific

histopathology and subtle clinical changes. It also suggests that diagnostic yield must be improved by selecting

multiple sections for biopsy, and necessitates the consideration of clinical and histological data in context

(clinicopathological correlation.)

False Positives and False Negatives: A difficult diagnosis of SLE

M. Rodziewicz


Background: A 47 year-old Nigerian female was admitted with a history of right-sided abdominal pain, cough

and fatigue. She had returned 2 weeks previously from a trip to Nigeria.

Results: Blood results were abnormal. Chest X-ray showed shadowing in the left upper and right lower zones.

Autoantibody, TB, malaria, sickle cell and HIV screens were all negative. She was treated for presumed TB

given her ethnic background, but returned 6 months later with fatigue and pyrexia of unknown origin. Her

respiratory and renal function then rapidly deteriorated such that she required intensive care for ventilation and

haemofiltration. A Pneumocystis jirovecii polymerase chain reaction was positive and she was commenced on

antifungal therapy to treat pneumocystis pneumonia (PCP) but her condition continued to worsen. After 21 days

on ICU, a repeat autoantibody screen showed positive antinuclear (ANA) and Ro antibodies and a low C4 level.

Scarring alopecia was also discovered under the headscarf the patient always wore.

Conclusion: 2-3% of patients with SLE are negative for ANA and SLE should therefore always remain a

diagnostic possibility if clinical features are present. The patient was also HIV negative and had a CD4 count

within normal limits, giving a low likelihood of PCP.

CMJ Case Reports 28




Primary Cerebral Lymphoma Causing Remitting and Relapsing Neurological Symptoms

T. B. Stoker1, A.M.H. Young1, T.F. Massoud2, R. Patani3, & M. Manford3

1. School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, CB2 0SP, England, U.K.2. Department of Radiology, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, England, U.K.

3. Department of Neurology, Addenbrooke’s Hospital, Cambridge, CB2 0SP, England, U.K.

Background: Primary central nervous system lymphoma (PCNSL) accounts for 3-5% of brain tumours.

Incidence of PCNSL has risen over the past 30 years, particularly in immunocompetent individuals. We present

an unusual case of PCNSL, characterised by remitting-relapsing neurological symptoms.

Methods: A 44-year old man with no significant past medical history presented to his GP with numbness in his

left arm and leg. A brain MRI scan revealed a right hemisphere lesion, which was considered to be

inflammatory. Within four months the patient experienced a spontaneous near-full recovery. After one month,

the patient deteriorated with increasing left hemiparesis involving the limbs and face. After a further seven

months, there was an abrupt loss of sensation and coordination in the left arm and leg respectively. Routine

haematological examination was grossly normal, and there was no evidence of infectious or inflammatory

causes. After five months the patient complained of lethargy and epistaxis. Full blood count demonstrated a

pancytopenia with 8.4g/dL haemoglobin (13-18g/dL), 86x109/L platelets (150-400x109/L) and 4.8 x109/L

leukocytes (4-11x109/L).

Results: Trephine biopsy revealed an abnormal cellular infiltrate in keeping with large cell lymphoma. R-CHOP

therapy was initiated for treatment of PCNSL with bone marrow involvement. Unfortunately, the patient’s

symptoms progressed and therapy was complicated by febrile neutropenia. The patient died 2 months later.

Conclusion: Spontaneous remission of symptoms in PCNSL can occur, and should not discourage the diagnosis

if consistent with clinical and radiological findings. Extra-neuronal disease is increasingly recognised, and

patients should undergo thorough staging to rule out systemic disease.

Trigeminal herpes zoster and Ramsay Hunt Syndrome with a lesion in the spinal CN V nucleus and tract

A. Khajuria


Background: Varicella Zoster Virus,a HHV-3,double-stranded DNA virus can cause both RHS and Trigeminal

herpes Zoster.Virus latency,in the Gasserian and geniculate ganglion,is maintained via episomes in host cell

nucleus.Upon reactivation,VZV causes perineural and intraneural inflammation along the nerve to the particular

dermatome. This rare case involved a 77-year-old immunocompetent man,with acute onset of right facial

pain,who subsequently developed herpes zoster in V2 and V3 trigeminal divisions. Within 3 weeks,he developed

ipsilateral peripheral facial palsy,hearing loss,vesicles over the external auditory canal, pain in face and ear,and

allodynia with decreased sensory responses to pain,temperature,touch,and vibration, throughout all three right

trigeminal branches.

Methods: T2-weighted brain MRI was used to reveal any lesions. Gadolinium-based agents further supported/confirmed the abnormal brain area.One patient was tested and compared with 2 subjects’ results from

two different studies.

Results: A hyper-intense lesion in the right spinal trigeminal nucleus and tract(STNT)was observed.

Gadolinium enhancement was seen over the right facial nerve. STNT involvement and infection of two separate

nuclei suggests transaxonal spread; anterograde transaxonal VZV spread, along trigeminal nerve fibers to the

STNT, might occur following reactivation of VZV in the gasserian ganglion, with subsequent spread to the

adjacent facial nerve nucleus and fibers. No anti-VZV antibodies, in CSF, suggested interneuron transmission

rather than via CSF. Clinical presentation sequence, for all three patients, differed, suggesting transaxonal

spread may occur in either direction.

Conclusion: Only two other patients,with both these conditions, have been reported in literature. Transaxonal

VZV spread may account for association between RHS and Trigeminal neuralgia, and understanding this iscrucial to help physicians diagnose more promptly and manage the conditions more effectively, if/when they

arise together within primary healthcare.

CMJ Case Reports 29




AUDITS

Re-audit into the management and documentation of third and fourth degree perineal tears

E. Badger

University of Manchester, UK

Background: Third and fourth degree tears have a substantial effect on women’s continence with 20-50% of

women suffering with incontinence. In 2007 an audit was carried out in the ELHT on the documentation and

management of third and fourth degree tears.

Aim: to determine the management of third and fourth degree tears in ELHT and compare the findings against

the previous audit and the recommendations of the Royal Obstetrics and Gynaecology guideline number 29 to

establish if the previous recommendations have been achieved.

Method: A retrospective audit of patient’s notes was performed on 94 women who had given birth within the

ELHT between 1st July 2009 and 31st March 2010. Data collected included where the procedure was carried out,

the surgical techniques used and follow up appointments.

Results: This showed a decreased compliance with the original audit and the RCOG guidelines. In the originalaudit carried out in 2007 the overlap repair technique was 82% of the time in comparisons to 36% in this re-

audit. There was a decreased compliance with the recommendation that repairs should be performed in theatre

from 93% to 78%, and an increase of the procedure being carried out in the delivery rooms.

Conclusions: Overall there has been an improvement of the recommendations of the previous audit.

Documentation in nearly all aspects have improved except in the follow up appointment in the perineal clinic.

Eight recommendations have been put forward and seven are currently being put into practice.

Local audit of inpatient prescription charts on the medical wards at Glenfield General Hospital (GGH):implications for clinical practice

F. Frame, R.K. Stansfield, M.I. Smith, T. MacCarrick, S.R. Knight, A.S. Patel, L.D. Wright, M. Afzal,

C.R. Thomas & A. Stanley

University of Leicester Medical School, Maurice Shock Building, PO Box 138, University Rd, Leicester, UK

Background: Adverse drug events cost the NHS £750 million a year, with a large number of events attributed

directly to prescription errors1. A recent systematic review demonstrated that errors will potentially occur in

over half of all hospital admissions1,2. Prescription errors can occur at any stage of the prescribing process, with

the majority (61%) at the writing stage 2. The aim of this audit was to assess the standard of drug prescription

charts to ensure they are written in accordance with local guidelines3.

Methods: The Leicestershire Medicines Code3

was utilised to formulate the audit criteria and standards. Allmedical wards in GGH were included in the one day audit. Findings were coded and recorded against the

criteria, and data were entered into a spreadsheet for analysis. Recommendations for change were made,

targeting key professional groups, and re-audited three months later.

Results: 195 drug charts were analysed with 2418 drugs and 4718 errors. Each drug chart contained 24.2 errors,

an average of 1.9 errors per drug – with no drug chart error free. Rates and types of error were similar between

each ward audited. Of particular concern, patient allergies were often ignored (17%), and prescriber contact

details were repeatedly not left (70%), meaning that errors could not subsequently be rectified.

Recommendations for change included education and visual cues, however re-audit failed to establish any

significant improvement.

Conclusion: Errors in prescription writing can have serious consequences. This audit aims to raise awareness

and improve prescribing in the hope of preventing adverse events.

CMJ Audits 30




The efficacy of Mycophenolate Mofetil in renal transplant patients: A Clinical Audit

S. Qureshi


Background: Mycophenolate Mofetil (MMF) is an immunosuppressant for renal transplant recipients used in

combination with Tacrolimus. The British National Formulary advocates the use of MMF at a continued fixeddose of 1g twice daily. However, the dosing regimen at St. George’s Transplant Unit is 1g MMF twice daily for

the first 30 days and 500mg twice daily thereafter. This is based on the FDCC3 and APOMYGRE4 studies,

which suggested higher doses of MMF were required to achieve target blood concentrations during the first 30

days post-transplant than were required subsequently.

Aim: To investigate the efficacy of the use of a lower dose of MMF than the recommended licensed dose in

renal transplant recipients.

Methods: This audit included all renal transplant recipients between 2006 and 2009, started on a combination of

MMF and Tacrolimus with at least 90 days follow-up. Primary end points at day 90 include biopsy-confirmed

acute rejection rates, graft loss and estimated GFR.

Results: The graft survival rate at day 90 was 100% and there were only 13% biopsy-confirmed acute rejectionepisodes. Median GFR was 56 mL/min/1.73m² at 3 months. Additionally, the Kaplan-Meier function curve

shows that at day 31 when the dose of MMF was reduced from 1g twice daily to 500mg twice daily there was

no immediate increase in acute rejection episodes.

Conclusion: Using a reduced dose of MMF did not result in unacceptable rates of acute rejection and was

associated with satisfactory renal function. Therefore, this regimen should be advocated.

Are paediatric asthma attendees to A&E being followed up according to the BTS guidelines?

J. Hayter


Background: BTS guidelines state that a comprehensive discharge plan, including a written reducing

medication plan, checking of inhaler technique and follow up by a GP within 48 hours of an asthma

exacerbation, helps to reduce A&E presentations and subsequent re-admissions to hospital 1. The aim of the

study was identify whether children seen at a London hospital A&E department were being followed up

accordingly.

Methods: Paediatric A&E notes from 1 st-31st January 2009 inclusive, were reviewed retrospectively. For each

patient, aged ≤16 with asthma or viral-induced wheeze, age, gender, outcome (admitted, discharged) and any

asthma prophylaxis medication taken, was recorded. For those discharged, documented follow up arrangements

and prescribed medications were also recorded.

Results: 84 children, aged between 3.5 months and 14.5 years, presented to A&E with asthma or wheeze. 3

children presented more than once, making 88 presentations in total. This group represented 88/1994 (4.4%) of

the total population of children aged ≤16 years presenting to A&E. Of those with asthma or wheeze 49/88

(55.7%) were taking asthma prophylaxis. 69/88 (78.4%) were discharged and 42/69 (60.9%) of these had no

follow up advice recorded in the notes. 34/42 (81%) of this group were discharged with Salbutamol ±

Prednisolone.

Conclusion: Most children presenting with asthma or wheeze were discharged. However more than half

appeared not to be receiving appropriate advice about follow up, even though 81% were discharged with new

medications. In an effort to better implement the guideline recommendations regular asthma teaching for new

A&E staff has been implemented.

CMJ Audits 31




Lymph node yield in open and laparoscopic colorectal surgery

T. Davies,


Background: Colorectal cancer represents a significant burden of disease with around 100 new cases diagnosed

each day in the UK. Aside from timely surgery, long--‐term survival is predicated by accurate staging of

regional lymph nodes, with large yields presented to Pathology shown to be independently associated with

longer survival for patients. We performed an audit of the surgical technique for treatment of colorectal cancer

to measure a significant difference in lymph node yield between the established open and relatively recently

adopted minimally invasive laparoscopic technique.

Methods: Retrospective study of 271 patients treated by colorectal surgeons at a London hospital during a 30

month period. Operating technique, lymph node yield and possible confounding factors were retrieved from

associated pathology reports. 171 Open and 100 laparoscopic procedures where included in the analysis.

Results: Overall, there was no statistical difference in lymph node yield reported between the operating

techniques (mean 18 nodes each). Subgrouping by operation type showed the laparoscopic approach yielded 4.5

fewer lymph nodes for lower rectal cancers though this was not statistically significant.

Conclusions: Surgeons and their patients will be reassured by the overall finding that the laparascopic technique

did not compromise quality of oncologic staging in terms of lymph node yield. However, further study of lower

rectal cancers is required to obtain a complete picture.

Assessing management delay in myocardial ischaemia

D. RasoulSchool of Medicine, The University of Manchester, Oxford Road, Manchester, UK

Background: As part of the Myocardial Ischaemia National Audit Project (MINAP) set out by the National

Service Framework for coronary heart disease (NSF) the ‘call to door time’, ‘door to needle time’ and combined

‘call to needle time’ in 49 patients presenting with ST-elevation myocardial infarction (STEMI) to

Wrightington, Wigan & Leigh NHS Foundation Trust was assessed to identify areas for improvement in care.

Method: Management of 49 admitted cases were analysed and as advised by the NSF the time limits for

each criteria was calculated in order to further pinpoint areas where there is a possibility in improving timely

management of STEMI. In cases there had been a breech to either time frame, a reason was sought and

discussed in collaboration with the North West Ambulance Service (NWAS).

Results:8% admissions to A&E were within the time limit with an average of 47 ±13 minutes exceeding well

above the 30 minute limit. However 84% of patients, exceeding the internally outlined trust target, were

thrombolysed within 30 minutes, average 23 ±9 minutes. The combination of these, equalling the CTNT time

was on average 71 ± 13 minutes totalling significantly over the 60 minute limit.

Conclusions: It can be concluded from this audit that the internal acute coronary syndrome clinical pathway at

WWL NHS Foundation Trust, have failed to meet the national guidelines for thrombolysis after admission in the

majority of occasions. The underpinning issue seems to be the delay by the NWAS in transporting patients to

RAEI, contributing to the total CTNT time. Effective teamwork between WWL and NWAS needs to be put in

place to reduce the CTDT time to the national 30 minute limit.

CMJ Audits 32




ANIMAL MODELS

The role of astrocytes in an animal model of multiple sclerosis grey matter demyelination

D. Bargiela

Faculty of Medicine, Imperial College London, South Kensington Campus, London, UK

Background: We previously described an association between glia limitans damage and cortical grey matter

demyelination in human postmortem multiple sclerosis cases. Here we characterise astrocyte changes at the pial

surface in a rat model of grey matter demyelination to evaluate the consequence of decreased barrier function in

the propagation of meningeal inflammation.

Methods: Rats were immunised with a sub-clinical dose of myelin oligodendrogyte glycoprotein (MOG)

followed by injection of pro-inflammatory cytokines (TNF-α & IFN-γ) into the subarachnoid space. Using

immunofluorescence labelling of the glia limitans (GFAP) and basal lamina (Laminin) layers on coronal

cryosections of the cerebrum, analysis of disruption was carried out using both quantitative and semi-

quantitative methods. Leakage of serum protein across a disrupted glia limitans and consequent demyelination

and inflammatory activation in the subpial region were further assessed.

Results: A significant early disruption of the glia limitans was present in rats receiving cytokine injections asopposed to non-cytokine controls (p < 0.05). Extravasated fibrinogen within the subarachnoid space showed

leakage into the parenchyma across sites of glia limitans damage. The number of Iba-1 positive

microglia/macrophages within the parenchyma was associated with sites of glia limitans disruption. Extensive

demyelination was associated with sites of glia limitans damage and inflammation.

Discussion: Our results demonstrate an early disruption in the pial glia limitans barrier and we suggest a

cytokine-mediated mechanism of glial retraction as a cause of such damage. Resulting leakage of CSF proteins

and subsequent immune cell activation within the parenchyma may contribute to the creation of an

inflammatory milieu within the grey matter. This may encourage lesion formation and immune cell infiltration

within this region supporting the hypothesis that meningeal inflammation is involved in multiple sclerosis grey

matter pathology.

Expression of ion channels in the atrioventricular ring tissue in the rat heart

1A. Sinha, 1A. Atkinson, 1R.H. Anderson, 2D. Henderson, 3D. Buckley, 1M. R. Boyett, 1H. Dobrzynski1University of Manchester 2University of Newcastle 3University of Leeds, UK

Introduction: The cardiac conduction system (CCS) consists of the sinus node(SN), atrioventricular

node(AVN), His bundle and Purkinje fibres. Further areas of specialised tissue - atrioventricular ring

tissue(AVRT) also exists around the tricuspid, mitral and aortic valves forming the left, right and aortic

rings(LR/RR/AR), which unite to form the retroaortic node(RAN). The function of the AVRT is not known, but

catheter ablation of these areas has been shown to terminate atrial tachycardias(Kistler et al 2004). Therefore the

aim of this study was to investigate the expression of ion channels in the AVRT and compare their expression to

that in the CCS/working myocardium(WM).

Methods: 12 hearts from rats were frozen and cryosectioned. Immunohistochemistry was used to label serial

sections to visualise the: atrial muscle(AM), ventricular muscle(VM), SN, AVN, RR and RAN which were then

collected via laser assisted microdissection. qPCR using 18 ion channels and markers was used to analyse the

level of mRNAs in the tissue samples.

Results: As expected, there was a classical distribution of ion channels in the AM/VM/SN/AVN tissues. In the

RR/RAN, there was significantly lower expression of Tbx3(positive CCS marker) and HCN4(responsible for

the pacemaker current If ) than in the CCS and when compared to the WM, there was lower expression of

K ir 2.1(responsible for resting potential IK1) but higher expression of HCN4.

Conclusion: The RR/RAN have a unique profile in the expression of ion channels, but more importantly the

molecular properties of this tissue are pacemaker like and therefore could in fact contribute to ectopic pacemaker activity.

CMJ Animal Models 33




Evidence that granule cells are a source of glutamate for mGluR2 mediated inhibition in golgi cells in

vivo

Vanessa Sivam, Tahl Holtzman, Steve Edgley

Departments of Physiology, University of Cambridge, Cambridge, UK

Background: Golgi cells appear to be a vital component of the cerebellar circuitry that has been so intricatelydesigned for information processing. These inhibitory interneurons appear to be capable of modulating entry of

information into the cerebellum. They show long lasting depressions in firing in response to sensory inputs that

decreases their inhibition on the transfer of information across the mossy fibre to granule cell relay.

Methods: In vitro studies have shown that glutamate can act on mGluR2 receptors on Golgi cells and generate

these long lasting depressions of firing in Golgi cells. We have taken the leap from in vitro to in vivo to show

this same phenomenon and to determine what the source of this glutamate is.

Results: We have made extracellular recordings of Golgi cells in anaesthetised rats and have showed that

LY341495, an antagonist at mGluR2 receptors, attenuated their long lasting depressions. These results have

helped to confirm previously obtained data in our lab, using alternative methods of drug application, which

showed an inhibitory role for glutamate acting on Golgi cells. We have also used 4,5,6,7-

tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a super-agonist at extrasynaptic GABA-A receptors on granulecells, to silence granule cell firing and determine whether these cells are a possible source of ‘inhibitory

glutamate’. THIP also attenuated the long lasting depressions of Golgi cells, confirming that granule cells

contribute glutamate to act on mGluR2 receptors and generate long lasting depressions.

Conclusions: These results take us one step closer to understanding how Golgi cells regulate information

processing within the cerebellum.

The Role of CpG Oligonucleotides in the Immunopathogenesis of Experimental Cerebral Malaria

S. Salam


Background: Cerebral malaria is a life-threatening complication of malaria, for which therapies are lacking.

Previous studies suggest that early non-specific innate immune responses may limit parasitaemia and prevent

progression to severe disease. There has been growing interest in the role of Toll-like receptor (TLR)-mediated

inflammatory cytokine release during malaria. However the precise role of TLR activation during experimental

cerebral malaria (ECM) is uncertain. We investigated the effects of the TLR-9 agonist, CpG on blood-stage

Plasmodium berghei ANKA (PbA)-induced ECM in C57BL/6 mice.

Methods: Mice were pre-treated with CpG, 24 hours before infection with 104 PbA parasitised erythrocytes.

Parasitaemias were recorded and the animals were monitored for signs of ECM. Flow cytometric analysis of

brain and spleen cells from CpG-treated and untreated mice was performed to examine cellular activation.Circulating plasma Tumour Necrosis Factor-α (TNF-α) levels were determined by ELISA.

Results: CpG pre-treatment significantly reduced parasitaemias on days 4 to 6 post-infection; (p<0.05). Pre-

treatment enhanced activation of splenic macrophages as judged by elevated surface expression of MHC-II and

CD40; (p<0.05). TNF-α levels were also increased. CpG pre-treatment appeared to provide protection against

PbA-induced ECM with greatly reduced homing of pathogenic CD8+ T cells to the brain; (p<0.05). In addition,

levels of the early surface activation marker CD69 were reduced on CD8+ T and CD4+ T cells after CpG pre-

treatment; (p<0.01).

Conclusion: Our study shows that priming TLR-9-mediated immune responses limit PbA blood-stage infection

with resultant control of ECM. These findings suggest a possible role for CpG in adjunctive therapies against

malaria.





Adiponectin plays a vital role in mediating the anti-contractile properties of perivascular adipose tissue in

mice mesenteric arteries

Z. Yao, F.M. Lynch & A.M Heagerty


Background: Adiponectin is an adipokine produced by the perivascular adipose tissue (PVAT). The bioavailability of adiponectin is impaired in obesity and type 2 diabetes mellitus. Evidence suggests that PVAT

has anti-contractile effect on the vascular wall via activation of the large calcium sensitive potassium (BK Ca)

channel at least in part. This study explores the involvement of adiponectin in the mediation of the anti-

contractile effect of PVAT in isolated mesenteric mice arteries.

Method: Male and female C57BL/6 wild type mice were killed by stunning and cervical dislocation. The

mesenteric arteries were dissected with and without PVAT. Vessels were mounted on a wire myograph and

constricted with 60mM KPSS. Following washout cumulative concentration responses (10 -9 – 10-5M) to

norepinephrine (NE) were performed before and after 45 minutes incubation of adiponectin receptor blocking

peptide (ARBP) (5g/mL). A second series of experiments examined the effects of adiponectin (3g/mL) on

pre-constricted arteries (NE 10-5M). Responses are expressed as mean (±SEM) % of KPSS constriction and

analysed using 2-way ANOVA.

Results: NE produced a concentration dependent constriction of arteries. PVAT (n=11) significantly (p<0.05)

reduced vasoconstriction compared to arteries lacking PVAT (n=12). The anti-contractile effect of PVAT is

significantly (p<0.05) diminished in vessels incubated with ARBP (n=8). With addition of adiponectin, a

vasodilator response was observed in pre-constricted vessels (n=3) with PVAT (7.5±3.5%) or (n=5) without

PVAT (12.3±5%).

Conclusion: This suggests adiponectin mediates the anti-contractile properties of PVAT in small mesenteric

arteries from C57BL/6 mice. Future work will examine the effects of adiponectin on BK Ca channel knockout

models.

Localising vasopressin within the rat retina

C. Saunders


Background: The presence of vasopressin within the eye, particularly the retina, has been known for many

years yet the origin of intraocular vasopressin remains unknown. From previous data, both intraocular and

extraocular sources have been proposed yet no strong evidence has been put forward in support of either. In the

present study, we aimed to determine the presence, location and cellular identity of vasopressin-expressing cells

within the retina.

Methods: Using rats which express enhanced green fluorescent protein (eGFP) under the control of thevasopressin gene promoter, retinal sections were collected and fluorescence immunohistochemistry used to

identify vasopressin cells. Blood vessels, glial cells and cell nuclei were also identified to isolate the

vasopressin cells to a particular retinal layer. Furthermore, immunoperoxidase techniques involving 3,3-

diaminobenzidine (DAB) staining of vasopressin cells were also conducted in retinas from wildtype rats.

Results: The existence of these cells has been verified and localised principally to the inner nuclear layer of the

retina. The cellular structure and distribution appear to be similar to that of amacrine cells, suggesting that these

cells may be an amacrine sub-population and are well placed to both modulate ganglion cell input and control

vascular changes.

Conclusion: For the first time, vasopressin cells have been identified and localised within the retina. With this

insight, further research can target the precise cellular physiology and the role of vasopressin expressed by these

cells in both normal and pathological disease states such as glaucoma, opening up new possibilities for therapeutic targets and interventions.





Administration of Bone Marrow Stromal Cells facilitate axonal regereneration in the hemisected adult

rat spinal cord

K. Bhatt, M. Enomoto, T. Hirai & K. Shinomiya

1. Faculty of Medicine, Imperial College London, South Kensington Campus, London, UK 2. Tokyo Medical and Dental University, Tokyo, Japan

Background: Spinal Cord Injury causes a huge burden for patients, with a lack of effective surgical treatment &

continuous cost of care. Bone Marrow Stromal Cells (BMSC) and artificial extracellular matrices have been

shown to aid recovery in various models. We proposed the combined use of BMSCs with a Honeycomb

Collagen (HC) matrix to aid axonal regeneration.

Methods: In-vitro study of explanted rat Dorsal Root Ganglia onto BMSC infused HC scaffolds compared

against the HC control were evaluated. Maximum neurite length after 10 days was calculated. The study

continued to an in-vivo study of spinal cord hemisection in rat models, with the injury site being surgically

implanted with HC or BMSC+HC scaffolds. 4 weeks post injury the cords were evaluated for injury site volume

compared to total cord volume. Basso Beattie Bresnahan score and sub score was used to analyse motor

recovery.

Results: Explants showed a significant difference in neurite length, with BMSC + HC group producing 3xgreater growth (p value 0.0004). The SCI Injury model showed a tendency of BMSC+HC to have a smaller

injury site volume. Motor recovery was significantly higher in the BMSC+HC group in both the BBB score and

BBB subscore (p = 0.03 & p = 0.005 respectively).

Conclusion: We successfully showed that BMSCs have efficacy compared to HC controls in both in-vitro and

in-vivo axonal regeneration, with a functional recovery being greater in the BMSC group. Therefore both

structural and cellular support is needed for a surgical intervention to aid recovery.

The anticontractile function of perivascular adipose tissue is attenuated in obesity: Results from a diet-induced obese rat model

A. Mastan, I. Boon, R. Aghamohammadzadeh, A.S Greenstein & A.M. Heagerty


Background: Historically, when studying the function of small arteries, the surrounding adipose tissue has

always been removed. We now know that, in health, this perivascular adipose tissue (PVAT) reduces vessel

constrictions in response to vasoconstrictors.

The aim of this study was to assess whether diet-induced obese rats show a reduction in the anticontractile

function of their PVAT, thus potentially contributing to higher blood pressures.

Methods: 16 Sprague Dawley rats were fed a high fat diet (HF) over 15-18 weeks. 7 control animals received a

normal diet. Weight and blood pressure were monitored. Mesenteric arteries were studied using wire myographywith construction of cumulative dose responses to noradrenaline, with and without PVAT intact.

Results: The HF rats showed a significant increase in weight (HF: 622.3 ± 14 g, control: 511 ± 14 g; P =0.0002),

systolic BP (HF: 144 ± 3 mmHg, control: 127 ± 3 mmHg; P =0.0051), diastolic BP (118 ± 3 mmHg, control: 100

± 5 mmHg; P = 0.0098) and blood glucose (HF: 9.7 ± 0.6 mmol/l, control: 7.1 ± 0.9 mmol/l; P =0.0351).

In skeletonised vessels (PVAT removed), there was no statistically significant difference between the healthy

and control groups (ANOVA, p=0.776). In vessels with intact PVAT, there was a significant difference between

the response to cumulative doses of noradrenaline (ANOVA, p=0.0004).

Conclusion: We have shown that in animals fed a high fat diet, the vessels with intact PVAT show an increased

sensitivity to cumulative doses of noradrenaline indicating a reduction in the anticontractile function of PVAT.

cambridge university clinical research society first annual conference

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