The limited access to the brain of a large number of therapeutic actives due to the presence of the blood-brain barrier (BBB) has led to intensive research toward the development of nanotechnology-based approaches. Polysaccharides such as chitosan, guar gum, pectin and pullulan have been selected as starting materials for this study due to their biocompatibility, biodegradability, good drug carrier properties, and ease of chemical modification with short chain alkylglycerol-like moieties (expected to enhance drug permeability through the BBB). A series of butylglyceryl-modified polysaccharides were prepared and characterised using chromatographic, spectroscopic and thermal analysis techniques prior to formulation into nanoparticles (NPs) by means of a selection of methods that include reverse emulsification, nanoprecipitation, and ionotropic gelation. Dynamic Light Scattering, Nanoparticle Tracking Analysis, Electrophoretic Mobility Measurements and Electron Microscopy were employed to characterise all NPs (overall size range 120–200 nm, and zeta potential values ranging from -27 to +39 mV). Modified pullulan (PUL-OX4) and guar gum (GG-OX4) NPs were found to be most stable at physiological pH (7.4), in contrast to chitosan (CS-OX4) NPs that demonstrated an increase in size as a result of aggregation. PUL-OX4 NPs (< 145 nm) had the highest Angiotensin II model peptide loading (8.46 %), while GG-OX4 NPs showed the highest loading degree with Doxorubicin (19.11 %) and Rhodamine B (3.78 %). Drug release studies demonstrated that PUL-OX4 NPs released fastest all the model actives tested, while GG-OX4 NPs were able to retain them for the longest period of time. The in vitro interactions of NPs with mouse brain endothelial cells (bEnd3) were investigated using a Transwell permeability model, with results suggesting an increased model membrane permeability in the presence of the modified polysaccharide nanoparticles. The cytotoxicity of these NPs at physiologically-relevant concentrations was studied using MTT assays; all NPs were non-toxic at concentration below 2 mg/mL, however a decrease in cell viability was noticed at higher concentrations. PUL-OX4 nanoparticles were found to be the least toxic, having the lowest LC50 value (9.48 mg/mL; for comparison, CS-OX4 has 7.30 mg/mL). Haemolysis study demonstrated that at concentration below 12 mg/mL, all the NPs studied did not induce a haemolysis effect significantly when compared to PBS control, however an increase in the effect was observed at higher concentration. PUL-OX4 nanoparticles exhibited the highest LC30 value of 19.87 mg/mL while the lowest value was exhibited by CS-OX4 nanoparticles (13.95 mg/mL). Confocal microscopy and flow cytometry investigations confirmed that all modified polysaccharide NPs were successfully taken up by bEnd3 cells, becoming localised in the cytoplasm.

Neisseria gonorrhoeae is a public health concern due to increasing numbers of cases of gonorrhoea and the ability of the organism to develop antimicrobial resistance. N. gonorrhoeae can harbour β-lactamase plasmids that encode TEM-1 penicillinase, but do not produce extended spectrum β-lactamases (ESBLs). ESBLs are active against the last remaining option for gonorrhoea monotherapy, ceftriaxone. The aim of this research was to establish what resistance mechanisms in N. gonorrhoeae could abolish the effectiveness of ceftriaxone for the treatment of gonorrhoea. Investigations into the genetic diversity of blaTEM alleles in gonococcal isolates from 2012 detected a high proportion of blaTEM-135 alleles (27%). Only a single specific mutation near the β-lactamase active site could result in TEM-135 penicillinase evolving into an ESBL. Electroporation was established for the transformation of native gonococcal resistance plasmids into N. gonorrhoeae, and was then used in attempts to transfer the enteric plasmid pEK204 (harbouring blaCTX-M-3 and blaTEM-1) into gonococcal strains. Electroporation and natural transformation were additionally used to transform gonococci with blaCTX-M-3 and blaTEM-10 genes. Five transformants were detected using blaTEM-10 and these all showed increased minimum inhibitory concentrations of ceftriaxone. The lack of success in uptake of pEK204 and blaCTX-M-3 was probably due to large plasmid size and lack of recombination site, respectively. Nevertheless, gonococcal β-lactamase plasmids were successfully transferred into clinical strains of the multidrug-resistant clone N. gonorrhoeae ST1407, suggesting that this could also happen in natural mixed gonococcal infections. In summary, it is encouraging that no further blaTEM alleles were detected and that N. gonorrhoeae was not able to express a CTX-M-type ESBL. However, the expression of TEM-10 ESBL is concerning and this work is the first report of ESBL activity in gonococci, albeit in vitro. It is essential to continue antimicrobial susceptibility surveillance and to develop molecular surveillance to detect rapidly the emergence of an ESBL in N. gonorrhoeae.

This study provided epidemiological and molecular information regarding multi-resistant ESBL-producing bacteria over two years, assessed the effects of antimicrobial agents on intestinal microbiota resistance and identified potential environmental sources of ESBL contamination. The most prevalent bacterial species to express ESBL resistance was Escherichia coli [85% in 2010 and 84% in 2012]. Other species included members of Enterobacter, Klebsiella, Citrobacter and Proteus. An increase in resistance to nine antibiotics [amoxicillin 1%, coamoxiclav 14%, meropenem1%, cefotaxime 9%, ertapenem 2%, ceftazidime 7%, tigecycline 5%, cefuroxime 8% and gentamicin 6%] was observed, indicating that multi-resistant infections had increased over the sampling period. Multiplex PCR revealed that the CTX-M determinant was most prevalent [38% in 2010 and 27% in 2012], followed by TEM then SHV. Double resistance determinants were identified in 68% of the total isolates. A statistical relationship was determined between joint expression of TEM and SHV and patient age [p=0.0174], suggesting that resistance initially presents within the elderly population. ERIC-PCR analysis was used to identify clonal relationships between the isolates. Ten potential clonal groups were identified; however phylogenetic analysis revealed that antibiotic selection and conjugation was taking place simultaneously, with a turnover of isolates observed between the two year cohorts. Results obtained from the simulated colon model suggested that the receipt of antibiotics may contribute to the overall resistance of the gut microbiota. Increases in co-amoxiclav, amoxicillin and trimethoprim resistance was observed after dosing, while gentamicin remained stable. One hundred and thirteen non clinical samples [animal faeces and food products] were analysed to determine ESBL presence. ESBL producing isolates were recovered from 32% of the animal faecal and 60% of food product isolates, AmpC was also present in 15% of the faecal and 12% of the food product isolates. Sequence analysis of the SHV and CTX determinants revealed relationships between determinants associated with both animal and human infections.

Introduction: Sub-arachnoid haemorrhage (SAH) occurs when cerebral artery ruptures and blood leaks out into the sub-arachnoid space. This is often a catastrophic event for the individual and morbidity and mortality rates are significantly influenced by early intervention. This makes the role of the clinical biochemistry laboratory in early diagnosis vitally important, as delays in diagnosis can have a major clinical impact. The cerebrospinal fluid (CSF) of healthy individuals is optically clear. It has, however, been recognised for over a century that it can become coloured (xanthochromia) following a cerebrovascular incident such as a SAH. This has made the main role of the clinical biochemistry laboratory in SAH diagnosis that of detecting xanthochromia in the CSF. The majority of laboratories which offer a xanthochromia screening service use the national guidelines that are based upon ultra-violet scanning spectrophotometry (350 nm to 600 nm). This analytical technique is not without its problems: it is subjective, has a possibility of inter-operator variability and due to the specialised nature of the test can take many hours or even days for a result to be issued. This project aimed to improve the current laboratory service by investigating: turnaround times, users opinions of the current service and potential alternative analytical methods. Methods: An audit of the current analytical provision was used to assess its effectiveness and in order to elucidate the service users’ perception. This was effected by a questionnaire that was distributed to service users across three different NHS Trusts in England and Wales. In an attempt to improve the laboratory service, alternatives to scanning spectrophotometry were investigated. These were selected through consideration of the nature of SAH i.e. blood is released into the subarachnoid space and the brain is damaged. Laboratory analysis therefore needed to focus on detecting the presence of blood and/or its breakdown products, any change in CSF constituents that arise as a direct consequence of blood being introduced in to the subarachnoid space or a specific analyte which would only be present if brain damage occurred. Investigation of current research into subarachnoid haemorrhage identified the following analytes as potential alternatives: CSF diazo bilirubin, CSF Ferritin, CSF protein S100 and serum protein S100. Results: The audit revealed the average turnaround time for reporting xanthochromia results to be 26 hours, with almost 20% of samples being reported as equivocal. The service user’s questionnaire revealed a general lack of awareness of current United Kingdom National External Quality Assurance Scheme (UKNEQAS) guidelines for the ‘Analysis of cerebrospinal fluid for bilirubin in suspected Subarachnoid haemorrhage’ and a lack of understanding regarding the timing of lumbar punctures. Additionally, one third of users felt that the turnaround time for results was inadequate. CSF protein S100 was found to be unsuitable due to the difficulty in achieving a suitable balance between sensitivity and specificity; at a cut-off of 0.40 μg/l sensitivity is 80% and specificity is 4%, at a cut-off of 1.60 μg/l sensitivity is 40% and specificity is 94%. Serum protein S100 was found to be unsuitable due to the difficulty in achieving a suitable balance between sensitivity and specificity at appropriate cut-offs (66 % and 73%, respectively, at a cut-off of 0.09 μg/l). When the CSF diazo bilirubin and CSF ferritin were compared to current laboratory practises using pre-defined criteria then CSF diazo bilirubin was found to be the analyte of choice to base new guidelines upon. CSF diazo bilirubin was then used as an initial ‘rule-out’ step in a new set of guidelines for the determination of SAH utilising CSF analysis. Conclusion: The new guidelines employ CSF diazo bilirubin analysis as a ‘rule-out’ step with all samples that are above the cut-off (300 nmol/l) being processed through the UKNEQAS guidelines. In order for the guidelines to be introduced and accepted, local training and education programmes for laboratory and clinical staff will need to be developed and implemented and they will need to be disseminated through publication of articles in journals relevant to both the clinical biochemistry community and requesting clinicians.

Meticillin-resistant Staphylococcus aureus (MRSA) is globally recognised as a major causative organism of hospital acquired infection (HAI) and continues to present many challenges for infection prevention and control. Once established within hospitals and healthcare centers, the control of spread of MRSA and therapy is difficult due to resistance to otherwise effective antimicrobials. Government initiatives in the United Kingdom (UK) have led to considerable investments in improving infection control practices, with emphasis on improving hand hygiene compliance of healthcare professionals and hospital environmental cleanliness to control the spread and limit the source of MRSA and other HAIs. This has resulted in the subsequent increase in disinfectant and antiseptic usage containing, quaternary ammonium compounds (QACs), cationic biocides such as chlorhexidine and the bisphenol ether, triclosan, for decontamination of surfaces and disinfection of skin. Thus, there is serious concern that as with antibiotic resistance, continual and intensive exposure of MRSA (and other hospital pathogens) to biocides, may result in the emergence of resistance to these agents with further detrimental consequences and substantial burden for prevention, treatment and control of hospital infections. MRSA carry a number of plasmid-borne qac genes, predominantly qacA, qacB and smr that encode resistance to commonly used antiseptics and disinfectants in hospitals, nursing homes and other healthcare establishments. The proteins encoded by qacA and qacB mediate efflux via active transport; QacA multidrug exporter mediates resistance to monovalent, divalent cationic and lipophilic antimicrobial compounds, whilst the closely related export protein QacB mediates lower levels of resistance to divalent cations. In this research a “snapshot” study of hospital strains of MRSA stored at the Hospital Infection Research Laboratory (HIRL), City Hospital, Birmingham, was carried out to determine the prevalence and distribution of qacAB in these isolates and determine a possible association between presence of these genes and biocide resistance. The intercalating dye, ethidium bromide (EtBr) is a substrate for many S. aureus multi-drug resistant (MDR) efflux pumps and was used in the present study as a marker for detection of efflux pump activity. Previous studies have reported that MRSA strains with an MIC of ≥ 64 mg/L to EtBr have qacAB, however, the present study used a lower baseline value of ≥ 32 mg/L resistance to EtBr to capture any isolates with low MICs that may have qacAB and may be missed. Initially 3,400 MRSA strains collected between October 2002 and October 2006 were screened to identify and select isolates with ≥ 32 mg/L resistance to EtBr. A second MRSA collection stored at the Antimicrobial Chemotherapy Laboratory, City Hospital, Birmingham, comprised 63 isolates that showed MICs of ≥ 64mg/L, were also included in the study. At this stage the study set (Set A) comprised 112 isolates with varying MIC to EtBr ranging from ≥ 32 mg/L to 256 mg/L. At a later date an additional 400 strains were screened from the same stored collection to include strains with lower MICs, i.e. < 32 mg/L. Thus a total of 336 isolates with varying levels of resistance to EtBr were studied. PCR was carried out on all 336 isolates for detection o qacAB, smr, qacG, qacH and qacJ to determine the presence and prevalence of the genes. Set A isolates positive for qacAB were further investigated to differentiate between qacA and qacB. Restriction digestion using the restriction enzyme Rsa1 was carried out on PCR products followed by PCR using specific primers for detection of the two genes. Urease activity and neomycin sensitivity were used as a means of basic characterization applied to all the study isolates. A select number of samples negative for qacA and qacB were typed using spa typing. Transfer studies involving, conjugation, plate mating and transformation on selected strains were carried out to attempt transfer of qacAB using the marker EtBr from a strain of MRSA with an MIC of ≥ 256 mg/L to EtBr and qacAB positive to a strain with < 32mg/L MIC to EtBr and lacking qacAB. Unfortunately, conjugation experiments were not successful in this study. Plasmid curing experiments were also carried out to demonstrate loss of plasmid through continual passaging onto selective plates. A variety of antiseptics and disinfectants are used in hospitals for prevention of HAIs. The present study was limited to carrying out minimum bactericidal concentration (MBC) determinations and MIC of four commonly used hospital biocides against randomly selected strains. The strains reflected ranges of MICs to EtBr and presence or absence of qacAB. These experiments, determined the efficacy of the biocides tested, to effectively destroy MRSA on skin and environment when used in healthcare settings. The results suggest that in the majority of strains showing high MICs to EtBr i.e. ≥ 64 mg/L, qacAB is present and thus, the mechanism of resistance to biocides may be attributed to an efflux protein pump encoded by these genes. Following restriction digestion of qacAB positive strains, with the restriction enzyme Rsa1, 81 of the 112 qacAB positive strains tested positive for qacA, i.e. 90% and 9 (11%) for qacB. The predominant prevalence of the qacA gene indicates that most of these strains are likely to be resistant to organic cationic biocides and intercalating dyes such as EtBr and acriflavine. However, the results of the MIC and MBC determinations carried out on a selection of biocides commonly used in the healthcare environment implies that the four biocides tested are likely to be 99.9% effective at killing the majority of isolates in this study set. However, five isolates demonstrated MBCs to chlorhexidine of > 32 mg/L. Chlorhexidine is a compound that is widely used in hand hygiene and surgical antisepsis products, and the results suggest that solutions containing this compound would be ineffective in removing MRSA from the hands of healthcare workers and skin sites if used. Molecular spa typing of selected samples negative for qacAB revealed that Endemic-MRSA (EMRSA) type 15 was the most frequent spa type identified in this study, followed by EMRSA-16 and EMRSA-1. Three strains identified jointly as EMRSA-3 and EMRSA-1. One strain identified as the Berlin clone. With regards to the challenges presented to infection prevention and control, MRSA has the potential to develop increased tolerance to biocides commonly used in the hospital environment, due to expression of efflux pumps, although currently there is little evidence of this. Further research is required to understand and learn of the various mechanisms of resistance, supported by adherence to control of infection strategies for prevention and spread of infections in healthcare facilities.

The Breakthrough Generations Study and Laboratory was set up in 2007 to study the epigenetics of breast cancer in women and men in the United Kingdom over a potential forty year prospective period from 2006 to 2046. The remit was to collect blood samples, lifestyle and medical information from volunteers and then to compare the genetic analysis of the samples to the data collected to look for trends and patterns. The blood samples were stored in a relatively unique format, being in 500 μl buffy coat samples in micro-capillary tubes and at the time of writing 4 million aliquots were in storage. This project had two main aims. Firstly to develop a modified micro-column genomic DNA extraction method that could process the frozen buffy coat samples, and process a high throughput of sample numbers in any given extraction batch. Secondly to use this validated method to extract DNA from male breast cancer patient samples and thereafter genotype these for the FGFR2 single nucleotide polymorphism which has been shown to have a high association with breast cancer in women. Commercially available genomic deoxyribonucleic acid (gDNA) extraction micro-column kits were modified, by altering incubation times and reagent volumes in stages and tested to validate yield and reproducibility, to allow for a greater sample through put than normally processed at one time. In addition, a purpose-designed centrifuge rack dual-level multi tube holder was developed and tested to allow for 96 samples to be processed in one batch. This allowed for the uniquely stored Generations Study samples to be processed, and for the high sample throughput required of such a large study, where batches of 20,000 were often processed at any given time. Male breast cancer blood samples and controls, to a total of 900 samples, were processed using the validated DNA extraction method and the resultant gDNA was genotyped by Taqman™ analysis for the FGFR2 point mutation or SNP at position 116,968,271 in chromosome 10q26. Relative Odds Ratios (OR) for the heterozygote mutant allele were 1.012 and for the homozygote allele were 1.289 – the p-values for these results, however, showed a lack of statistical significance. To ensure this was not due to lack of sample size or power, an additional 1,148 samples were acquired from UK Genetic Lung Cancer Predisposition Study and genotyped with statistically significant OR of 1.06 and 1.43 for homozygote and heterozygote mutations respectively, where Fisher exact test gave p-value of 0.03. This indicated that the FGFR2 SNP has a positive risk association in men, as it has been shown to have in women. The developed extraction method will allow the processing of further samples in high numbers, to genotype further SNPs in male cases. It may provide a direct comparison to the genetic risk association of breast cancer in men to women. This initial set of results for the FGFR2 SNP is the first step in the epigenetic analysis of breast cancer in men in the UK, and the start of the comparison of the epigenetics of breast cancer across the sexes.

Over the past forty years concerns over the presence of pharmaceuticals in the environment have grown considerably. Some pharmaceuticals can be effectively biodegraded in wastewater treatment plants but others can be sorbed onto sludges that are often subsequently used as fertilisers or disposed of to landfill. This work aimed to understand how a given pharmaceutical will be distributed between aqueous and solid phases (characterised by the sorbed:dissolved partition coefficient, Kd) within a treatment plant, which is important to be able to make accurate risk assessments. An official guideline test to measure the partitioning of a pharmaceutical in sewage sludge is available, but it is time consuming and fastidious. As activated sewage sludge is a complex matrix, commercially available solid-phase extraction (SPE) cartridges with different chemistries were used to characterise the pharmaceutical-sludge binding processes. As part of this work a new solid-phase extraction screening method has been developed to provide rapid measurements of Kd and its performance was evaluated against partition coefficients obtained with the official guideline method with a correlation coefficient of 0.93 and a r2 of 0.94. In addition, this rapid method allowed the measurement of partition coefficients for pharmaceuticals for which values were not available in the literature and these have been used to further validate new predictive models. Predictive models based on the octanol-water partition coefficient have been developed to calculate partitioning properties of compounds in soil, and these have been extended for application to sewage sludge. These models are optimised mainly for neutral organic chemicals, and only a few consider ionic substances. The work described in this thesis compared the performance of these soil-based models for a range of pharmaceuticals, including ionisable compounds, and assessed their application for the binding of ionic and non-ionic pharmaceuticals in sewage sludge. It also explored other predictive models based on molecular descriptors obtained from chemical structure. These models provided improved predictions over previous models based solely on the octanol-water partition coefficient. In this thesis, partial least squares (PLS) and Bayesian artificial neural network (ANN) models were evaluated for their accuracy in predicting the partition coefficient for neutral, basic, acidic and zwitterionic pharmaceuticals. Literature values were used to develop the models based on a range of molecular descriptors, and their predictive ability was assessed on an external test set of compounds excluded from the model-building process. The performance of the linear PLS and non-linear ANN models were discussed, and their predictive performance and interpretability were compared. Attempts to apply the method for rapid measurement of the sorption of pharmaceuticals to soils were also made to investigate potential read-across from one environmental matrix to another but the two matrices were too dissimilar to achieve this.

Towards the development of drug carriers that are capable of crossing the Blood Brain Barrier, the techniques of emulsion polymerisation and nanoprecipitation have been utilised to produce nanoparticulate carriers from a systematic series of alkylglyceryl dextrans (of two different average molecular weights, 6 kDa and 100 kDa) that had been functionalised with ethyl and butyl cyanoacrylates. Also, zero length grafting of polylactic acid to butyl, octyl and hexadecylglyceryl dextrans has allowed the preparation of polylactic acid-functionalised nanoparticles. All materials and derived nanoparticles have been characterised by a combination of spectroscopic and analytical techniques. The average size of nanoparticles has been found to be in the range 100-500 nm. Tagging or loading of the nanoparticles with fluorophores or model drugs allowed the preliminary investigation of their capability to act as controlled-release devices. The effects of an esterase on the degradation of one such nanoparticulate carrier have been studied. Testing against bend3 cells revealed that all materials display dose-dependent cytotoxicity profiles, and allowed the selection of nanocarriers that may be potentially useful for further testing as therapeutic delivery vehicles for conditions of the brain.

Infection with Coxiella burnetii in humans is associated with severe disease, known as Q fever. Acute infection, that can be sub-clinical in nature, can lead to the development of a chronic life-threatening cardiac condition. The largest reported human outbreak of Q fever recently occurred in the Netherlands between 2007 and 2010. This outbreak gave rise to over 4,000 reported human cases and efforts such as the culling of 50,000 pregnant goats in an attempt to control the outbreak. While an effective vaccine exists for protection against infection, it has an unsatisfactory safety profile that has led to it being licenced in only a single country and in a defined group at risk of occupational exposure to infection. In addition, this vaccine involves culturing the organisms at high levels of biological containment; this makes the vaccine expensive to produce and presents a challenge in attaining reproducible batch-to-batch variation. In this work, two species of laboratory animals were infected by the aerosol route to closely mimic the likely natural route of infection in humans. The species investigated were guinea pigs (Dunkin Hartley strain) and mice (BALB/c and A/J strains). The infection in guinea pigs more closely mimicked human acute disease than that in the two different strains of mice: In terms of a more acute febrile response and greater clearance of the organism from the tissues. After clinical recovery from disease, the antibodies from the guinea pigs’ sera were used to isolate immune-reactive proteins found in axenically-grown C. burnetii organisms. Isolation of protein was achieved by picking spots from 2D-PAGE gels corresponding to reactive spots on parallel Western blots as well as immunoprecipitation (pull down) of bacterial protein extracts, a method not previously applied to this type of analysis. These isolated proteins were then identified using nanoLC-electrospray ionisation tandem mass-spectrometry (Q Exactive™ instrument). More than 100 proteins were identified, 71 of which had not been previously described in the literature. The identified proteins were considered for their suitability for inclusion in a future subunit vaccine to protect against Q fever. The most promising candidates were an OmpA-like surface protein similar to a molecule that has already been described as an invasin, two surface-exposed components of the bacterial type IV secretion system (DotA and IcmX), and two hypothetical proteins that warrant further study.

Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome and is strongly linked with obesity and type 2 diabetes. The role of gut-liver interaction is increasingly recognised in the development of NAFLD. Modification of gut microbiota may lower cardiovascular risk and reduce liver injury beyond existing treatment in those with NAFLD. This study tests the hypothesis that probiotic supplementation may improve endothelial function and insulin sensitivity; and reduce oxidative stress, inflammation and liver injury in subjects with NAFLD. Methods: This is a randomised, double-blinded, placebo-controlled, proof-of-concept trial in which subjects with NAFLD are allocated to take either two sachets VSL#3 probiotic twice daily or the placebo equivalent for 10 weeks. Biophysical markers for endothelial function, oxidative stress, vascular inflammation, insulin resistance and liver injury were undertaken before and after the intervention period. Results Forty-two patients participated and 35 of them completed the study. There were 28 males and 7 females; and 74% had type 2 diabetes or impaired fasting glycaemia. Mean age was 57 ± 8 years, body mass index 32.6 ± 5.0 kg/m2, blood pressure 134/82 ± 13/7 mmHg, HbA1c 53 ± 14 mmol/mol (7.0 ± 3.4%), total cholesterol 4.42 ± 1.15mmol/l, HDL 1.06 ± 0.29mmol/l, LDL 2.43 ± 1.06 mmol/l, triglycerides 2.00 ± 0.88 mmol/l, ALT 53 ± 26 iu/l and AST 40 ± 15 iu/l. Median duration of NAFLD was 0.3 ± IQR 2.0 years. No significant difference was seen in markers of cardiovascular risk and liver injury following VSL#3 probiotic supplementation. Conclusion: There was no significant improvement in the markers of endothelial function, oxidative stress, inflammation, insulin resistance, liver fibrosis scores, liver transaminases or liver imaging in this group of patients with NAFLD treated with 10 weeks of VSL#3 probiotic supplementation. The results may be due to a number of factors such as a small sample size, subjects with relatively good metabolic control and possibly less severe liver disease, and the lack of consensus on an effective dose and duration of probiotic supplementation.