Δ9-tetrahydrocannabinol (THC) is the major lipophilic cannabinoid in the cannabis plant and is responsible for many of its pharmacological effects. Cannabis and cannabinoids are often administered orally in foods or lipidic formulations. Marinol® is an oral lipidic formulation containing THC dissolved in sesame oil, and is approved to treat nausea associated with cancer chemotherapy. THC exerts its pharmacological effects by binding to cannabinoid receptors 1 and 2, both of which are involved in a large variety of downstream signalling pathways. Some of these pathways regulate cell death. In agreement with this involvement, THC has been shown to be immunosuppressive in vitro and in vivo. Previous work on synthetic cannabinoids has shown that they can be transported in the lymphatic system in significant amounts when administered orally. The lymphatic system is a passive circulatory network of vessels and lymphocyte-rich nodes that has three major functions. The first function is to maintain body fluid balance. The lymphatic system is open-ended, unidirectional, and collects excess fluid at blood capillaries and tissues. Large molecules that cannot re-enter the blood also enter the lymphatic system. The second function of the lymphatic system is to mount immune responses: after lymphocytes are produced in bone marrow and thymus, they migrate to the lymphatic system and are organised into lymph nodes, which are immunological checkpoints that screen passing fluid for antigens, where innate or adaptive immune responses can be mounted onto them. The third function of the lymphatic system is the absorption of dietary lipids. Lipids are a heterogeneous group of molecules that include lipids, triglycerides and vitamins which are found in food. Lipophilic lipids are emulsified in the gastrointestinal tract by bile and phospholipids, forming mixed micelles. The micelles provide an interface for pancreatic lipase to digest triglycerides into fatty acids and glycerol. These micelles dissociate when they approach the unstirred water layer of the intestinal wall, and the contents are absorbed into specialised enterocytes. Absorption of long-chain fatty acids triggers the production of chylomicrons (CM), a large triglyceride-rich lipoprotein which transports digested fats through the lymphatic system. Triglycerides can then be released from CM by the action of lipoprotein lipase, which is expressed in a variety of cells and tissues. Lipophilic molecules and drugs can associate with CM, and share their fate in the body. Transport through the lymphatic system associated with CM bypasses the liver, which could reduce the first-pass metabolism of a drug. Lipophilic drugs with a high association with CM are prime candidates for lymphatic transport if administered orally. The use of Marinol® by cancer patients receiving chemotherapy could target THC to the lymphatic system, further suppress the immune system of those taking it and possibly making them more vulnerable to secondary infection. Therefore, it is important to confirm whether THC is transported by the intestinal lymphatic system, and whether it is selectively targeted there at immunosuppressive concentrations. To address this research question, analytical assays needed to be developed and optimised to detect THC in a variety of different biological matrices. HPLC-UV methods were successfully developed and validated to determine concentrations of THC in plasma, CM emulsions, and simulated intestinal fluid. The first step to determine the lymphatic transport of THC is to assess the fraction available for absorption in the small intestine. As mentioned previously, Marinol® is a lipidic formulation of THC, and the emulsification and digestion of lipids are not taken into account with traditional drug dissolution techniques. However, an in vitro lipolysis model is a technique that mimics lipid digestion and can reliably predict the absorption of a drug dissolved in a lipidic formulation. After establishment and optimisation of the model, up to 45% of THC dissolved in a lipidic formulation (sesame oil) was solubilised and made readily available for absorption in simulated intestinal fluid. As the concentration of THC in formulation increased, the fraction of drug solubilised decreased. The lymphatic transport potential of THC was then assessed using three techniques – association with CM, pharmacokinetic and biodistribution studies. THC had a high association with CM (72.5%), and its absolute oral bioavailability was improved three-fold when administered in a lipidic formulation (18.5%) compared to a lipid-free formulation (5.73%). At times of peak plasma concentration, the level of THC in mesenteric lymph node homogenate was 30 times higher than plasma, and was at immunosuppressive levels shown in previous in vitro studies (8.6 µg/mL). The mesenteric lymph nodes drain the small intestine, and would be the first nodes that dietary fats pass after being absorbed from the small intestine. Finally, the antiproliferative effect of THC was assessed by exposure to peripheral blood mononuclear cells isolated from healthy individuals and cancer patients recovering from chemotherapy. THC produced a dose-dependent reduction in cell proliferation, with significant differences from vehicle above 10 µg/mL. THC had a reduced effect on cells isolated from cancer patients compared to cells isolated from healthy individuals. THC associated with CM emulsion also had a similarly reduced effect to cells isolated from healthy individuals compared to the effect of THC dissolved in organic solvent. Collectively, this work confirms that THC is transported via the intestinal lymphatic system, and can be concentrated there at extremely high levels, shown to be immunosuppressive in previous in vitro studies.

This thesis, entitled: ‘Role of AMP-protein kinase (AMPK) in regulation of perivascular adipose tissue (PVAT) function’, has been submitted by author Tarek Ali Mohamed Almabrouk for a degree of Doctor of Philosophy (PhD) in the College of Medical, Veterinary and Life Sciences at the University of Glasgow, October 2016. Apart from the cerebral circulation, all vasculature is surrounded by layers of adipose tissue known as perivascular adipose tissue (PVAT). In health, PVAT can function as an endocrine organ to produce a wide range of adipocytokines which can attenuate vascular contraction. The exact mechanism of this anti-contractile effect is still ill-defined, although much evidence suggests that PVAT-released adipocytokines may activate K+ channels on VSMCs or eNOS on endothelial layer possibly via AMP-activated protein kinase (AMPK). However, obesity results in oxidative stress and inflammation of the PVAT leading to abnormal adipocytokine release and PVAT dysfunction. AMPK is a serine/threonine kinase with many potential physiological functions, including regulation of energy heamostasis. AMPK is expressed in the three layers of the blood vessel: smooth muscle (VSM), the endothelium and PVAT and it is known that activation of AMPK leads to vascular dilatation via both endothelium- and non-endothelium-dependent mechanisms. Although it is known that AMPK can modulate VSM and endothelial function, it is unknown whether AMPK can influence the anti-contractile activity of PVAT. Therefore, this project aimed to investigate the mechanism of the anticontractile effect of PVAT by determining the functions of AMPK within adipocytes, as well as to assess the importance of vascular AMPK to the PVAT anti-contractile function. Experiments were conducted using wild type (WT) and global AMPKα1 knockout (KO) mice aortae. The phenotypic features of the PVAT were assessed by both histological, immunohistochemical and immunofluorescent methods. Secretory function of the PVAT was tested using an immunoblotting array and ELISA, whereas the anti-contractile effect of PVAT was studied using wire myography. Immunoblotting methods were used to test AMPK activity in the PVAT and VSMCs. Aortic rings from WT and KO mice were denuded of endothelium and mounted on a wire myograph in the presence and absence of PVAT. The responses to an AMPK activator (AICAR) and the AMPK-independent vasodilator cromakalim were subsequently assessed. Relaxation responses to AICAR or cromakalim in the Sv129 (wild type) mouse were significantly enhanced in the presence of endogenous attached or unattached PVAT, an effect that was absent in vessels from KO mice. Furthermore, enhanced relaxation was observed in vessels from KO mice incubated with PVAT from Sv129 mice, whereas PVAT from KO mice had no effect on relaxation of vessels from Sv129 mice. Furthermore, conditioned medium (CM) transfer experiments demonstrated the presence of an anticontractile factor released from PVAT that was absent in KO mice. Adiponectin secretion was reduced in PVAT from KO mice and PVAT-enhanced relaxation was attenuated in the presence of adiponectin blocking peptide. Adipokine array and ELISA demonstrated that adiponectin release is significantly reduced in the KO conditioned media in comparison with wild type CM. Globular adiponectin restores the relaxation response in both wild type aortae without PVAT and in KO aortae with and without PVAT. High fat diet (HFD) fed mice showed a reduction in the relaxation response to cromakalim in wild type vessels with intact PVAT in comparison with animals fed a normal chow diet (ND). HFD animals had increased inflammatory infiltrates in the PVAT which were associated with reduced AMPK activity and adiponectin release in comparison with ND fed WT mice. In KO mice, AMPK activity was also reduced and increased inflammatory infiltration was observed in both ND and HFD mice. In conclusion, the current project demonstrates that AMPKα1 has a critical role in maintaining PVAT’s anti-contractile effect; likely mediated through altered adiponectin secretion or sensitivity, and through protection of PVAT against inflammation. Marked reduction in AMPK activity in WT PVAT, accompanied with the reduction in the release of adiponectin in HFD and KO animal may explain the impaired vascular function observed in obesity.

Background Previous studies have demonstrated suboptimal management of care for osteoarthritis (OA). The objectives of this study were to (i) identify indicators of quality of care for OA in general practice, (ii) measure quality of care using routine general practice records and through an enhanced recording template (iii) estimate the effect of the template introduction on quality of care, and (iv) assess the feasibility of quality indicators as trial outcome measures. Methods A systematic review and narrative synthesis of quality indicators was undertaken. An iterative process of development resulted in an electronic template to record management of OA in consultations, based on identified quality indicators. This was triggered by a case definition of clinical OA derived through consensus. An assessment of coding, diagnostic misclassification using consultation narrative, and baseline recorded quality of care before template installation in eight practices was undertaken. Measurement after template installation facilitated a before-and-after comparison of care. The indicators were used as secondary outcomes in a cluster-randomised trial of a model OA consultation. Results There were fifteen valid, feasible quality indicators. Consultation prevalence of clinical OA was comparable to other estimates but up to one-third of cases may not represent true OA. Prescribing and referral data were well-captured in the routine record; assessment and core treatment indicators (such as education and advice) were not and so were included in the recording template. The template had small-to-moderate effects on weight recording, and paracetamol and topical anti-inflammatory prescription. Assessment of the effect of the model consultation was limited by high baseline quality achievement and variation between trial arms, practices and clinicians. Conclusion Assessment of quality of care for OA in general practice through quality indicators is feasible but comprehensive assessment requires enhanced recording approaches. Inter-clinician variability requires further understanding and reduction, and triangulation with patient-experienced quality is needed.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC), is the deadliest type of cancerous malignancy with a survival rate of only 3.7 % after 5 years. The first line current treatment is a drug called gemcitabine, which has been shown to display effectiveness in only 23.8 % of patients. Hybrid nanoparticles (HNPs) comprised of an iron oxide core and outer gold coat have shown great potential for anti-cancer therapies. The magnetic iron oxide cores and the surface plasmon resonance (SPR) properties of the gold surface provide the HNPs with the capabilities of diagnostic imaging and drug delivery, making them true theranostic agents. A novel thiolated thermally labile drug (TTLD) analogue of gemcitabine was successfully synthesized and attached to the surface of HNPs forming a novel drug formulation called TTLD+HNP. This TTLD compound is comprised of a gemcitabine molecule with a Diels Alder cycloadduct. Gem-Mal, a maleimide derivative of gemcitabine is released during retro Diels Alder (rDA). The mode of release involves heat-activation of the rDA reaction facilitated by the SPR of the gold shell. TTLD was characterised with mass spectrometry, nuclear magnetic spectroscopy and IR spectroscopy. Preliminary studies determined that the TTLD compound doesn’t undergo rDA at 20 °C and a subsequent 4 week study displayed no rDA occurring at 20 °C. Afterwards in vitro experiments including the MTT and trypan blue assay determined that Gem-Mal is 4.6 times less cytotoxic than gemcitabine but is taken up by cells 11 fold faster when attached to the HNPs. Upon heat-activation at 44 °C, the TTLD+HNP formulations cytotoxicity increased by 56 % outperforming gemcitabine by 26 %, confirming its temperature driven activity. The TTLD+HNP drug formulation is the first of its kind and has displayed superior anti-cancer activity to the current first line drug gemcitabine after heat mediated controlled release.

Over half of the United Kingdom (UK) population, aged 75 or over, experience chronic pain. Chronic pain can negatively affect activities of daily living, quality of life and an individual’s ability to maintain an independent lifestyle. UK rural populations comprise a disproportionate number of older adults; however, a paucity of research exists on the experiences of older adults living with chronic pain in these environments. The aim of this thesis is to explore and understand experiences of older adults with chronic pain living in rural environments to inform healthcare policy and service provision. The study is framed by an interpretivist paradigm informed by narrative and ethnographic inquiry to capture storied representations and situated natures of lived experience. A mixed-methods exploratory design supported data generation and analysis through narrative interviews, life-grids, a quality of life measure, photo-elicitation, and ‘go-alongs’. Participants were recruited using snowball and spectrum sampling through third sector organisations. Eight participants (four females; 67-90 years) contributed to 14 interviews (spouses were present during four interviews). Findings focus on the multi-dimensionality of chronic pain, explicitly the relational nature of the rural environmental dimension, and factors that support the maintenance of quality of life domains and ageing well. Quality of life and common beliefs were critically examined: chronic pain as a normal part of ageing, “carrying on”, community spirit, and rurality. Implications for methodology and policy and service provision are described.

Overweight and obesity in Oman have increased dramatically in recent years and are associated with increases in cardiovascular disease and diabetes. This project explored the food choices, dietary intake and physical activity of Omani adults. The effectiveness of smartphone apps on physical activity behaviours in Omani adults was also investigated. In study 1, a survey was conducted on 500 healthy adults living in Oman during the period from December 2013 to April 2014. Eligible participants completed questionnaires, specifically designed for this study and administrated by trained health educators in all governorates in Oman. 55% of the population studied were either overweight or obese (particularly those over 30y of age). BMI was significantly (p < 0.05) affected by gender and age group but not by residential area (urban vs rural). Dietary analysis indicated a high consumption of ‘fast food’ but total energy intakes failed to explain the high incidence of obesity. However, the questionnaire indicated a high level of physical inactivity within the population. Study 2, implemented in two phases, aimed to investigate the efficacy of three smartphones apps (MapMyFitness, Lose it, and Pacer) on improving physical activity in Omani male and female adults. Phase1 studied male and female Omani citizens living in the UK while phase 2 specifically investigated females living in Oman. Both phases showed significant increases in physical activity amongst the participants though, due to the short period of intervention (4 weeks), this was associated with only modest changes in BMI. The prevalence of overweight and obesity amongst adults highlights the need for intervention strategies, targeting unhealthy lifestyle behaviours, to curb the prevalence of NCDs in Oman. Smartphone apps, represent potential tool to facilitate changes in lifestyle factors associated with the high prevalence obesity in Omani adults. Key words: Obesity, physical activity, energy intake, MapMyFitness, Lose it, Pacer, Oman.

The reliability of data obtained from the existing United States Pharmacopeia (USP) method, USP <231> for elemental impurities (EI) have been questioned in the literature. New regulations regarding EI in pharmaceutical products were recently implemented on the 1st January 2018. The new regulations are USP <232>/<233> and the International Council for Harmonisation equivalent guidelines (ICH Q3D). The new regulations include the use of instrumentation such as inductively coupled plasma optical emission spectroscopy (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS). The aim of this work was to develop, optimise and validate analytical methods for the determination of Class 1 and Class 2A elements, arsenic (As), cadmium (Cd), mercury (Hg),lead (Pb), cobalt (Co), nickel (Ni) and vanadium (V) simultaneously in pharmaceutical products in compliance with the new guidelines. The developed ICP-OES and ICP-MS methods were validated using the only available solid standard reference material (SRM) NIST 3280 Multivitamin/Multielement tablets. It was found that relying solely on spiked addition technique as suggested by USP<233> is inefficient as it may not reflect clearly the method’s accuracy particularly when the sample preparation involves the use of microwave (MW)-assisted acid digestion step, which is very common for pharmaceutical samples. Sample preparation was performed using a developed MW-assisted acid digestion method with reverse aqua regia. It was found that reaching a temperature of 210°C for sample’s digestion is necessary to get EI recoveries of greater than 95% and pre-digestion grinding was found to be beneficial to minimise variation in data and get relative standard deviation (RSD)of less than 5%. The validation results showed good linearity (R2>0.995) over a wide range with low limits of detection (LoDs) and limits of quantification (LoQs). The calculated LoQs in ng/mL are As (5.86, 1.149), Cd (0.87, 0.037), Hg (2.23, 1.701) Pd (4.73, 0.041), Co (1.58, 0.299), Ni (1.74, 0.159) and V (7.64, 0.485) for ICP-OES and ICP-MS incorporated with collision reaction cell (CCT) respectively. Twenty-four commercially available pharmaceutical products including analgesic tablets, cough remedies, flu powders and antacids were analysed. Four products contained Cd in concentrations exceeding the permitted daily exposure limit (PDE) of 5μg/day when the maximum dose is taken, and nine products exceeded the 5μg/day PDE of Pb. This is especially concerning for the paediatric products because children are more susceptible to EI adverse effects as for example, they can absorb up to 40-70% of ingested Pb. The antacids were found to contain As and although the levels quantified were below the PDE (15μg/day), a speciation method using an ion-pair reversed phase high performance liquid chromatography (HPLC)-ICP-MS was optimised and validated according to ICH Q2B guidelines as no information regarding what species are present in such products is available in the literature. Four arsenic species were selected, arsenite (AsIII), arsenate (AsV), monomethyl arsonate (MMA) and dimethyl arsenate (DMA). Calibrations with R2>0.995 for all four species in the range of 1 to 50 ng/L and % recoveries>95% with RSD<5% were obtained. Arsenic was extracted from the samples using MW assissted extraction with 0.3M phosphoric acid at 55°C for 10 miutes, 75°C for 10 minutes and 95°C for 1 hour. The species were stable after being exposed to the extraction procedure (spiked recoveries >95%). This method was able to extract 95% or more of arsenic for all products. The ion-pair reversed phase chromatography was performed using a mobile phase: 10mmol/L tetrabutylammonium, 20mmol/L potassium dihydrogen orthophosphate and 2% methanol at pH 6 with a C18 column. The speciation analysis results for all the antacids showed that approximately 50% of the extracted As was present as the most toxic AsIII form. The work demonstrates some of the potential issues with the new regulations and the availability of suitable solid SRM and seeks to provide workable solutions for the analysis.

Epidermal Langerhans cells (LCs) and multiple subsets of dermal dendritic cells (dDCs) make skin a valuable route for vaccination, offering the potential for antigen-sparing immunisation. The interconnected immunological functions of dDC subsets and LCs are not fully understood however. This Thesis therefore aimed to explore the interactions of skin immune cells with viral pathogens and vaccines to inform the development of future therapeutics and intradermal vaccines. LCs and dDCs were isolated from ex vivo human skin tissue using a walkout protocol which allowed the enrichment of the migratory cells from the tissue. LCs and dDCs were infected with a lentiviral vector encoding GFP, allowing study of post-entry HIV viral restriction. The study uncovered the existence of a SAMHD-1-independent antiviral factor unique to LCs. LCs and dDCs from ex vivo skin were used to examine the cross-presentation of an inactivated influenza virus-derived matrix peptide to CD8+ T-cells. Two CD11c+ subsets of dDCs were found to potently cross present the antigen. Delivery of VLPs, which lack genetic material, markedly reduced cross-presentation, suggesting that viral genetic material is vital for dDCs to activate cross-presentation pathways. Future work is required to determine if this is true of other influenza peptides or pathogens. Vaccine delivery studies performed using murine and human models found that dDCs were responsible for the greatest uptake of ovalbumin peptide antigen and LCs did not migrate out of the epidermis in the first 4 hours after inactivated influenza virus vaccine delivery respectively. Collectively, this work highlighted the importance of dDCs in antigen uptake and cross-presentation to prime cytotoxic T-cell responses. Innovative delivery methods such as microneedles offer a means of accessing the dermal compartment in a pain-free manner, though further work is required to determine the optimal combination of vaccine formulation and delivery method to harness the immunostimulatory abilities of dDCs.

The aim of this research was to establish whether or not mouse interferon could reverse the phenotype of transformed cells so that they behaved in a more normal manner. For this study, clonal isolates of transformed cells from two continuous cell lines and fibroblasts extracted from mouse embryos were used. It was found that interferon could inhibit the growth of both normal and transformed cells. With several transformed clones interferon also reduced their saturation densities, which were normally several fold higher than those of the non-transformed parents. This suggested that interferon had induced a partial reversion to density-dependent growth control. Butyric acid also inhibited growth rate and acted additively with interferon when cells were treated with the two agents together. Interferon had a variable effect on the ability of dispersed cells to form colonies on plastic substrate in liquid media, but had a consistently greater effect on the ability of transformed cells to form foci on a monolayer of normal cells, and to grow suspended in agar, two growth conditions specific to the transformed state. It was concluded that interferon had inhibited focus formation and growth in agar by a combination of its growth inhibitory activity and an effect specific for the transformed phenotype. Interferon also affected the morphology of both normal and transformed cells. The cells became more spread-out, and in transformed cells there was a partial restoration of the microfilament bundle system. Despite these effects on the cytoskeleton, the extracellular matrix of fibronectin fibrils appeared to be little altered by interferon, except when added in conjunction with butyric acid. Under these circumstances, the fibronectin matrix became much more extensive. These data increase the likelihood that interferon's in vivo antitumour activity involved a direct effect on the tumour cells themselves, such that these cells behave more normally.

HIV associated lipodystrophy (HIVLD), an adverse effect of combination antiretroviral therapy (cART), further introduces complexity in the management of HIV infection. There is also increased of cardiovascular disease in HIV patients, even in the absence of HIVLD. cART impairs adipogenesis and dysregulates the secretion of adipokines, fat and glucose metabolism in the adipose tissue. This is seen with both protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors. The aim of this thesis was to examine the pathogenesis of cART-induced metabolic disturbances using in vitro models (adipocytes) and in vivo (gene association) studies. Plasma levels of IL18, an adipokine associated with insulin resistance, are elevated in HIVLD patients. The murine 3T3-F442A cell line was used as an in vitro model to assess the effects of PIs (lopinavir [LPV], ritonavir [RTV], atazanavir [ATV]) and NNRTIs (efavirenz [EFV]) in the presence or absence of telmisartan (TEL; 1-5μM). All antiretrovirals (ARV) resulted in a dose dependent increase in IL18 protein levels by ELISA (LPV, 330pg/ml+2.1 [p=0.008]; ATV, 267.3+5.1 [p=0.0001]; RTV, 265.7+2.2 [p=0.0005] and EFV, 98.6+2.4 [p=0.0004]) as compared to the vehicle control; this also correlated with IL18 gene expression determined by RT-PCR. Sequenom MALDI-TOF was used to genotype 14 single nucleotide polymorphisms (SNPs) in the IL18 gene in ARV-treated patients with (HIVLD+; n=115) and without LD (HIVLD-; n=51), but no association was identified. ARV treatment also resulted in the upregulation of NFATC4 gene expression (LPV, 1.9+0.05 [p=0.0015]; ATV, 2.1+0.1 [p=0.0007]; RTV, 1.4+0.05[ p=0.0002] and EFV, 1.8+0.85 [p=0.0001]) as compared to the vehicle. Co-incubation with TEL partially attenuated ARV-mediated upregulation of IL18 (1.6+0.2 [p=0.01) and NFATc4 (1.2+0.1 [p=0.0001]). siRNA knockdown of NFATc4 in adipocytes resulted in significant upregulation in the levels of adiponectin and PPARgamma and down-regulation in IL6 secretion levels following treatment with ARVs. The data suggest that ARV-induced upregulation of NFATc4 could be a mediator of ARV-induced adipocyte toxicity and potentially insulin sensitivity. This study also observed changes in the regulation of various miRNAs following ARV treatment. Validation experiments confirmed LPV to result insignificant downregulation of miR-103 (0.5+ 0.01[p=0.002]) and miR-107 (0.4+ 0.03[p=0.001]) along with their target gene, Cav-1 (0.6+ 0.01[p=0.01]) during adipogenesis; this was reversed by co-incubation with Tel. A systematic review of the genetic associations with HIVLD was also undertaken – this identified that polymorphisms in mitochondrial DNA and cytokines had been the primary ones studied using a candidate gene approach. The results showed that several SNP associations have been identified which are biologically plausible, but in almost all cases, replication of the findings in different cohorts has either been contradictory or limited. This may be due to several reasons including small sample sizes, different diagnostic criteria used for HIVLD, differences in genetic strategy, ethnic variation in genetic architecture and differences in drugs used across different cohorts. Although further studies in larger patient groups with HIVLD should be undertaken, this may not be possible given its rarity now in clinical practice, and perhaps the emphasis should switch to insulin resistance per se. In summary, PIs and NNRTIs upregulate proinflammatory cytokines with NFATc4 potentially playing a major role in their transcriptional upregulation. Differential regulation of specific miRNAs were also found to be important in ARV-mediated dysregulation ofadipogenesis; further validation work which evaluates their role in adipocyte toxicity in vitro and in vivo is now warranted. Whilst this study did not find any genetic association with HIVLD, future studies using larger sample sizes and better defined phenotypes are now required.