abstracts presented at the6th international workshop on

Abstracts presented at the 6th International Workshop onAdverse Drug Reactions and Lipodystrophy in HIV

25–28 October 2004, Washington, DC, USA

ORGANIZING COMMITTEECo-chairs for 2004Andrew Carr St Vincent's Hospital, Sydney, AustraliaVeronica Miller The George Washington University, Wash., DC, USAKathleen Mulligan University of California at San Francisco, San Francisco, Calif., USA

Jacqueline Capeau Faculty of Medicine Saint Antoine, INSERM, Paris, FranceDavid Cooper University of New South Wales, Sydney, AustraliaStefan Mauss Center for HIV and Hepatogastroenterology, Düsseldorf, GermanyMorris Schambelan University of California at San Francisco, San Francisco, Calif., USAIan Weller Royal Free and University College Medical School, London, UK

SCIENTIFIC COMMITTEEBen Cheng Forum for Collaborative HIV Research, Wash., DC, USAJudith Currier University of California, Los Angeles, Calif., USAMichael Dubé Division of Infectious Diseases, University of Indianapolis, Ind., USAJulian Falutz Immune Deficiency Treatment Center, Montreal General Hospital, Montreal, CanadaSteven Grinspoon Massachusetts General Hospital, Boston, Mass., USACarl Grunfeld University of California at San Francisco, San Francisco, Calif., USAYasmin Halima International AIDS Society, Clinical Trials Partnership, London, UKTim Horn Physicians’ Research Network, New York, NY, USADonald Kotler Columbia University, New York, NY, USAJens Lundgren University of Copenhagen, Hvidovre, DenmarkSimon Mallal Royal Perth Hospital, Perth, AustraliaEsteban Martínez IDIPAPS - Hospital Clínic, University of Barcelona, Barcelona, SpainGraeme Moyle Chelsea and Westminster Hospital, London, UKRobert Munk New Mexico AIDS INFO NET, Arroyo Seco, NM, USAPeter Reiss University of Amsterdam, Amsterdam, The NetherlandsWilly Rozenbaum Hôpital Tenon, Université Pierre et Marie Curie, Paris, FranceJames Stein University of Wisconsin Medical School, Madison, Wis., USAStephen Sturley Columbia University, New York, NY, USAFrancesca Torriani University of California at San Diego, San Diego, Calif., USAKevin Yarasheski Washington University, St Louis, Mo., USA

Plenary SpeakersEnzo Bonora Ospedale Maggiore, Verona, ItalyCourtney Fletcher University of Colorado, Denver, Col., USAEric Fliers University of Amsterdam, Amsterdam, The NetherlandsRobert Hegele Blackburn Cardiovascular Genetics Laboratory, London, ON, CanadaJoep Lange Academic Medical Centre, University of Amsterdam, Amsterdam, The NetherlandsTimothy Osborne University of California, Irvine, Calif., USA

ORGANIZING SECRETARIATInternational Medical Press 2–4 Idol Lane

London EC3R 5DD, UKTel: +44 20 7398 0700Fax: +44 20 7398 0701www.intmedpress.comEmail: [email protected]

Antiviral Therapy 9:6

ABSTRACT CONTENTS

ORAL PRESENTATIONS

Page Title and presenting author Abstract

Plenary Session

L1 Neuroendocrine regulation of fat metabolism P1E Fliers

L1 Implications of antiretroviral toxicity in the developing world P2E Katabira

L1 Non-HIV lipodystrophy P3R Hegele

L2 Clinical pharmacology of NRTI-triphosphates P4C Fletcher

L2 The role of SREBPs in regulation of lipid metabolism P5T Osborne

L3 The Metabolic Syndrome and cardiovascular disease: lessons P6from the Bruneck StudyE Bonora

Session IL3 Effects of recombinant growth hormone on visceral fat 1

accumulation: pilot study in HIV-infected adolescentsA Viganò

L4 Effects of a growth hormone releasing factor (GRF) 2analogue in HIV patients with abdominal fat accumulation: a randomized placebo-controlled trialS Grinspoon

L4 The effect of recombinant human growth hormone treatment 3on circulating leptin and adiponectin concentrations in patients with HIV-associated fat accumulationJC Lo

L5 Plasma adiponectin and IL-6 predict insulin sensitivity in patients 4with HIV infection independently of body fat distributionDN Reeds

Session IIL5 A structural basis for the effects of HIV protease inhibitors on 5

GLUT4 activityP Hruz

©2004 International Medical Press

L6 Mechanisms of insulin resistance in HIV-seronegative 6individuals acutely treated with ritonavir boosted indinavir and atazanavir regimens DA Doran

L6 Ritonavir acutely induces insulin resistance in healthy 7normal volunteersGA Lee

L7 Abacavir hypersensitivity syndrome: results of clinical, 8immunological and genetic screeningE Phillips

Session IIIL7 HIV-lipodystrophy is characterized by insulin resistance for 9

leucine and glucose metabolism: dissimilar to type 2 diabetes KE Yarasheski

L8 Antiretroviral therapy and markers of insulin resistance in the 10Multicenter AIDS Cohort StudyTT Brown

L8 Objective evaluation of HIV lipodystrophy case definition in a 11randomized 96 week clinical trialA Carr

L9 Prospective, partially randomized, 24-week study to compare 12the efficacy and durability of different surgical techniques and interventions for the treatment of HIV-related facial lipoatrophyG Guaraldi

Session IVL9 Zidovudine inhibits thymidine phosphorylation: a novel site of 13

potential toxicity in non-mitotic cellsE McKee

L10 Uridine abrogates the adverse effects of stavudine and 14zalcitabine on adipose cell functions UA Walker

L11 In vivo, nucleoside reverse transcriptase inhibitors alter 15expression of both mitochondrial and lipid metabolism genes independent to HIV infection PWG Mallon

L11 Differential effects of nucleoside reverse transcriptase inhibitor 16(NRTI) regimens on adipocyte mitochondrial DNA depletion in HIV-infected patientsD Nolan

Session VL12 Steatohepatitis is associated with increased hepatic expression 17

of SREBP-1 in HIV-infected lipodystrophic patients J Capeau


L12 In vivo evidence for impaired peripheral fatty acid trapping in 18HIV-1 lipoatrophyJPH van Wijk

L13 Ritonavir causes hypertriglyceridaemia in APOE*3-Leiden 19transgenic mice by impairing triglyceride clearanceM den Boer

L13 The metabolic effects of intermittent antiretroviral therapy 20with and without IL-2 (ACTG A5102)P Tebas

Session VIL14 Intact insulin-mediated glucose uptake despite severe 21

endothelial dysfunction with 4 weeks of indinavir in healthy menSS Shankar

L14 Ritonavir and indinavir promote lipid-induced atherosclerosis 22but inhibit endothelial denudation-induced neointimal hyperplasia in miceNM Schildmeyer

L15 Pre-eclampsia in HIV-infected pregnant women receiving 23highly active antiretroviral therapy is associated with endothelial dysfunction and insulin resistanceM Larrousse

L15 Endothelial dysfunction in HIV-infected patients on CART 24does not improve even when lipid profiles improve on pravastatinPA Sklar

POSTER PRESENTATIONS

Mitochondrial DisordersL19 Non-invasive assessment of hepatic mitochondrial toxicity 25

in HIV-infected patients with normal serum lactate by 13C-methionine breath test M Banasch

L19 Addition of tenofovir to a didanosine-based HAART does not 26increase mitochondrial DNA depletion but decreases cytochrome c oxidase function and mitochondrial massG Garrabou

L20 Mitochondrial studies in adipose tissue of HIV-infected 27patients without fat redistribution S López

L20 Mitochondrial DNA depletion in asymptomatic HIV-infected 28patients receiving didanosine plus stavudine-based HAART regimen seems to be compensated by up-regulatory mechanisms S López


L21 HIV infection is associated with increased skeletal apoptosis 29assessed by TUNELS López

L21 Uridine pharmacokinetics of Mitocnol, a sugar cane extract 30UA Walker

L22 Zidovudine inhibits thymidine phosphorylation: a novel site 31of potential toxicity in non-mytotic cellsMD Lynx

L22 Thymidine and zidovudine methabolism in isolated rat heart: 32zidovudine inhibition of thymidine phopsphorylationEE McKee

L23 The impact of nucleoside reverse transcriptase inhibitor (NRTI) 33treatment duration and insulin resistance on fasting arterialized lactate levels in patients with HIV infectionJC Lo

L23 The developmental stage determines the effect of NRTIs on 34adipocyte mtDNA depletion and adiponectin productionG Behrens

L24 Mitochondrial dysfunction of HAART-related hyperlactataemia 35is demonstrable by non-invasive studies G Garrabou

L24 Platelet contamination affects mitochondrial DNA (mtDNA) 36levels significantly in peripheral blood mononuclear cells from HIV patientsM Gerschenson

L25 Do statins enhance HIV therapy-induced mitochondrial toxicity? 37MP de Baar

HepatotoxicityL25 Effects of antiretroviral therapy (ART) on liver enzymes of 38

Brazilian children with AIDSAC Montes-Gil

L26 Assessment of the liver function in HIV-infected patients 39treated with antiretroviral drugs in CameroonO Lowe

Adipocyte BiologyL26 Adipocyte viability and function but not inhibition of 40

preadipocyte differentiation is compromised by indinavirG Behrens

L27 Nevirapine did not alter cell differentiation, lipid metabolism, 41insulin response and survival in cultured adipocytesM Caron

L27 PIs and NRTIs with adverse effects on adipocyte lipid 42metabolism and survival alter the production of pro-inflammatory cytokines and adiponectin in adipocytesC Lagathu


L28 Comparative effects of atazanavir alone and in combination 43with low concentration of ritonavir on triglyceride and cholesterol synthesis in vitroMA Noor

L28 Effects of HAART on resistin and perilipin mRNA expression 44in mouse 3T3-L1 and primary human adipocytesPV Nerurkar

L29 Interactions between T lymphocytes and pre-adipocytes 45increase HIV production and apoptosis of lymphocytes and block adipocyte differentiationDE Lewis

Body CompositionL29 Body composition changes in treatment-experienced 46

HIV-infected patients initiating a tenofovir-containing antiretroviral regimenG Tsekes

L30 Reversibility of lipoatrophy associated with highly active 47antiretroviral therapy 1 year after switching from stavudine to tenofovir disoproxil fumarateG Tsekes

L31 Body composition changes in treatment-experienced 48HIV-infected patients: 10 years of follow-upG Tsekes

L31 Soft tissue augmentation with polymethymethacrylate (PMMA) 49for correction of lipodystrophy related body fat atrophyMS Serra

L32 The proportion of patients reporting body fat redistribution 50was unchanged between week 48 and week 120 in subjects receiving fosamprenavir/ritonavir in Study APV30005 S Walmsley

L32 Oxymetholone as therapy to maintain body composition in 51HIV-positive menA Urbina

L33 Intrathoracic fat in HIV-infected patients 52A León

L33 Estimated prevalence of HIV-associated adipose redistribution 53syndrome (HARS) — abnormal abdominal fat accumulation — in HIV-infected patientsN Muurahainen

L34 Measurement of fat mass in HIV patients: comparison between 54anthropometry and bioelectric impedanceC Miralles

L34 Abdominal subcutaneous adipose tissue (SAT) and visceral 55adipose tissue (VAT) measurements in HIV+ adults: influencesof measurement site KJ Ellis


L35 Evaluation of HIV-associated lipodystrophy using standardized 56photos: a reproducibility studyRB Cavalcanti

L35 Loss of weight in the era of HAART is associated with 57elevated PBMC proviral DNA levelsCM Shikuma

L36 Usefulness of anthropometry in detecting paediatric 58lipodystrophy A Viganò

L36 Safety and efficacy of intradermal poly-L-lactic acid 59(Sculptra™) injections in patients with HIV-associated facial lipoatrophyDR Mest

L37 Bio-Alcamid™, a high-volume injectable prosthesis for 60facial reconstruction in HIV-related lipoatrophy: report on 100 patientsLC Casavantes

L37 Fosamprenavir/ritonavir and nelfinavir have comparable 61effects on body fat changes in antiretroviral-naive patients: 48-week results from the SOLO studyS Walmsley

L38 Longitudinal changes in body shape and metabolic parameters 62in HIV-infected, treatment-naive patients initiating NNRTI regimens in South IndiaS Saghayam

Other ToxicitiesL38 Antiretroviral protease inhibitors prevent L6 muscle cell fusion 63

by reducing calpain activity SP Colby-Germinario

L39 The effect of different combination therapies on oxidative 64stress markers in HIV infected patients in CameroonJ Oben

L39 Use of emtricitabine (FTC) as part of HAART in an inner-city 65clinic setting results in no appearance of pigmentation changes in HIV-positive patients of colourW Jordan

L40 Impact of race on nervous system side-effects of subjects 66taking efavirenzDW Seekins

L40 Adverse reaction of nevirapine (NVP) in antiretroviral-naive 67HIV-seropositive subjectsSK Dey

L40 A practical preclinical model for assessing the potential for 68unconjugated hyperbilirubinaemia produced by HIV protease inhibitorsDJ Kempf


L41 Concurrent statin therapy blunts CD4+ cell gain in combination 69antiretroviral therapy (CART) treated HIV-infected patients UH Iloeje

L41 Tenofovir and how to assess renal function in patients on 70antiretroviral therapyS Mauss

L42 Functional impairment of NRTI-related mitochondrial 71DNA-depletion in primary human T lymphocytes B Setzer

L43 Incidence and risk factors for nucleoside analogue 72transcriptase inhibitors toxicity in treated HIV-HCV co-infected patientsA Milinkovic

L43 Withdrawn by author 73

Clinical ManagementL44 Why do patients with HIV stop antiretrovirals used as part of 74

an initial highly active antiretroviral regimen? JD Lundgren

L44 The SCOLTA Project: an Italian approach to monitoring the 75safety of new antiretroviral drugs T Quirino

L45 Increased frequency of prescriptions and associated costs of 76drugs for the treatment of antiretroviral (ARV)-induced metabolic disordersL Normén

L45 Effectiveness of a dedicated lipodystrophy clinic in reducing 77hyperlipidaemia in HIV-infected individualsV Carter

L46 Rosiglitazone for HIV lipoatrophy: 84 weeks follow-up 78A Carr

L46 Association of risk of toxicity with drug levels of saquinavir 79when boosted with ritonavir in the MaxCmin1&2 trialsJD Lundgren

L47 Impact of switching PI-containing antiretroviral therapy on 80lipodystrophy and metabolic complications, APROCO Cohort Study (ANRS EP11)R Lassalle

L47 Impact of structured therapy interruptions on plasma lipids 81and body fat in patients with primary HIV-1 infectionA Milinkovic

L48 Improvement in lipid profile in HIV-infected virologically 82controlled patients switched to a simple QD regimen: preliminary results of the COOL trial evaluating EFV/TDF vs EFV/TDF/3TCP Mercié


L48 Effectiveness of ezetimibe on hyperlipidaemia in 83HIV-1-infected patientsD Lee

L49 Reexposure to nucleoside analogues after lactic acidosis/ 84hyperlactataemia syndromeC Galera

L49 Persistent tuberculous adenitis due to an unsuspected drug 85interaction with efavirenz and rifabutinH Edelstein

L50 Are patients given adequate dietary information when 86starting on a lopinavir/ritonavir-containing HAART regimen?MN Phillpot

L50 Autologous fat transfer for the treatment of HIV-related 87face lipoatrophy: a long follow-up experienceG Guaraldi

L51 Relapse of HIV-related buffalo hump after liposuction 88G Orlando

L51 Psychometric evaluation of patients undergoing surgical 89treatment of HIV-related facial lipoatrophyG Orlando

L52 Long-term follow-up of graft hypertrophy after autologous 90fat transfer for HIV-related face lipoatrophy (hamster syndrome 1 year later)G Guaraldi

Insulin ResistanceL53 HOMA-IR in the assessment of insulin resistance in HIV patients 91

E Álvarez García

L53 Abdominal obesity predicts insulin resistance but not to fasting 92triglyceride concentration in males treated with PI-containing HAARTRB Cavalcanti

L54 Myo-inositol/D-chiro-inositol metabolism in HIV lipodystrophy 93syndromeJ Currier

L54 Resistin decreases after 3 months of rosiglitazone in 94HIV-infected patients with lipodystrophyDS Kamin

L55 No evidence for reduced insulin-mediated glucose uptake 95in skeletal muscle with 4 weeks of indinavir in healthy menSS Shankar

Lipid MetabolismL55 HAART modulates liver lipid metabolism through direct effect 96

on hepatocytes G Behrens


L56 Changes in nuclear gene expression resulting from NRTI-induced 97inhibition of mitochondrial transcription reveal links between mitochondrial dysfunction and lipid metabolismPWG Mallon

L56 Nucleoside reverse transcriptase inhibitors (NRTI) decrease 98adipocyte and monocyte mitochondrial (mt) messenger RNA transcription in the absence of changes in mtDNA or cell morphologyPWG Mallon

L57 Favourable increases in high-density lipoprotein cholesterol 99(HDL-C) in HIV-infected therapy naive subjects receiving fosamprenavir/ritonavir in the SOLO/APV30005 Study – 120 week analysisJ Flamm

L57 Long-term efficacy of nelfinavir, adverse events and 100lipodystrophyE Daniels

L58 Simplified Protease Inhibitor Trial (SPRINT): metabolic effects 101of once daily saquinavir SGC plus ritonavir (SQV/r) vs twice-daily indinavir plus ritonavir (IDV/r)M Harris

L58 Improvement in dyslipidaemias among patients receiving 102HAART after substitution of tenofovir DF (TDF) for stavudine (d4T) with or without use of a concomittant lipid lowering agent (LLA)J Gilmore

L59 Inhibition of reverse cholesterol transport in ritonavir-treated 103hamstersPV Nerurkar

L59 NRTI Sparing Trial: metabolic effects of nevirapine (NVP) + 104lopinavir/ritonavir (LPV/r) vs zidovudine/lamivudine (AZT/3TC) + NVP vs AZT/3TC + LPV/rM Harris

L60 Study of lipodystrophy in treated HIV patients and evaluation 105of their psychological problems F Jalali

L60 Association of lipid profiles in HIV-seropositive patients 106with antiretroviral therapy in a real world scenarioS Mauss

L61 Metabolic complication and fat changes in Thai HIV-infected 107patientsM Homsanit

L61 Dietary evaluation and metabolic alterations in HIV-related 108lipodystrophyG Guaraldi


L62 Increased CD36 expression on circulating monocytes during 109HIV infectionM Galli

L62 The reduction in serum adiponectin levels in HIV patients 110correlates with apolipoprotein-B clearanceM Shahmanesh

L63 Lipoprotein (a) as cardiovascular risk factor in HIV-infected 111patientsWO Richter

L63 Investigation of cytokine promoter polymorphisms in the 112tumour necrosis factor (TNF)-α, and interleukin (IL)-10 genes in HIV-infected patients with lipodystrophyH Fernandes

Cardiovascular DiseaseL64 Evaluation of blood lipid disorders and atherosclerosis 113

measured by the intima media thickness in a cohort of HIV-positive patientsF David-Ouaknine

L64 Impaired activity of pravastatin on carotid intima-media 114thickness in HIV-infected patientsF Boccara

L65 Coronary artery bypass graft in HIV-infected patients. 115A multicenter case control studyF Boccara

L65 Evaluation of coronary artery calcification by electron 116beam computed tomography (EBCT) in HIV-infected men receiving prolonged protease inhibitor (PI) therapyDM Parenti

L66 Endothelial dysfunction, adiponectin plasma levels and 117lipodystrophy in patients on antiretroviral therapyV Estrada

L66 Comparison of Framingham and PROCAM scores for risk 118assessment of cardiovascular disease in HIV-positive adults with metabolic complicationsL Normén

L67 Cardiovascular risk assessment with carotid ultrasound and 119coronary calcium score in a cohort of HIV-infected subjectsA Mangili

L67 HS-CRP is elevated in HIV-positive patients with a trend to 120increased levels in patients prior to coronary events I Woolley

L68 Percutaneous coronary intervention in HIV-infected patients: 121immediate results and long-term prognosisF Boccara

L68 Antiretroviral exposure and carotid intimal medial thickness in 122a multicentre Canadian HIV cohortM Smieja

L68 HIV viral protein R causes atrial cardiomyocyte mitosis, 123mesenchymal tumour, dysrhythmia, and heart failure W Lewis

L70 Blood pressure elevation and hypertension in paediatric 124patients with or without human immunodeficiency virus-associated lipodystrophyS Benavides


Antiviral Therapy 9:6 L1

ABSTRACT P1Antiviral Therapy 2004; 9:L1

Neuroendocrine regulation of adipose tissue

E Fliers

Academic Medical Center of the University of Amsterdam,Department of Endocrinology and Metabolism, The Netherlands

Neuroendocrine research has altered the traditional per-spective of white adipose tissue (WAT) as a passive storeof triglycerides. In addition to fatty acids, WAT producesmany hormones and can, therefore, be designated as a tra-ditional endocrine gland. It has an important role in theconversion of androgens to oestrogens and in glucocorti-coid metabolism. It also produces more recently identifiedhormones such as leptin, resistin and adiponectin. Manyof these hormones have both central and peripheral effectsupon intracerebroventricular administration. Leptin actsin the hypothalamic arcuate nucleus on POMC and NPYexpressing neurons that project to the paraventricularnucleus. These neuropeptidergic pathways are involved inthe neuroendocrine and behavioural response to starva-tion. Therefore, WAT actively participates in the integra-tive physiology of energy metabolism and eatingbehaviour. Conversely, WAT is controlled by humoral fac-tors, para- and intracrine factors and by neural regulation.Sympathetic nerve fibres innervate WAT and stimulatelipolysis leading to the release of glycerol and free fattyacids. Earlier pharmacological studies have shown effectson glycerol output in human WAT through nicotinic andmuscarinic receptors. Later studies in rats have clearlyshown a functional parasympathetic innervation of WAT.Selective parasympathectomy induces insulin resistancewith respect to glucose and fatty acid uptake in the den-ervated fat depot and has selective effects on local hor-mone synthesis. Thus, the CNS is involved in theregulation of WAT hormone production and hormonesensitivity. The parasympathetic brain stem nuclei displaya distinct somatotopy: separate sets of autonomic neuronsin the dorsal motor nucleus of the vagus innervate eitherthe visceral or the subcutaneous fat compartment. Recentexperiments show that this somatotopy extends to thehypothalamus. We therefore propose that the central ner-vous system (CNS) plays a major role in the hithertounexplained regulation of body fat distribution. Thedevelopments in this research area are likely to increaseour insights in the pathogenesis of metabolic disorderssuch as type 2 diabetes mellitus and lipodystrophy syn-dromes. Specifically, the adipose tissue redistribution seenin HIV lipodystrophy may be mediated via the central ner-vous system.


Implications of antiretroviral toxicity in thedeveloping world

E Katabira

Mulago Hospital, Kampala, Uganda.

Abstract not available.


Non-HIV lipodystrophy: focus on inheritedlipodystrophies

R Hegele

Blackburn Cardiovascular Genetics Laboratory, Robarts ResearchInstitute, London, ON, Canada

Lipodystophies can be either inherited or acquired, and thedifferent types can show substantial clinical differences.Because patients with inherited lipodystrophies haveinsulin resistance that progresses to type 2 diabetes, thesedisorders are considered to be monogenic model systemsfor the metabolic syndrome (MetS). Inherited lipodystro-phies can involve a complete loss of fat from childhood,such as that seen in autosomal recessive Berardinelli-Seipcongenital generalized lipodystrophy (CGL). CGL occursin two molecular forms: CGL1 (MIM 608594) resultsfrom mutations in AGPAT2 (MIM 603100) encoding1-acyl-sn-glycerol-3-phosphate acetyltransferase, andCGL2 (MIM 269700) results from homozygosity formutations in GNG3LG (MIM 606158) encoding seipin. Incontrast, familial partial lipodystrophy (FPLD) is charac-terized by fat loss from limbs and the gluteal region atpuberty, with preservation of central, facial and visceralfat. FPLD also occurs in two molecular forms: FPLD2(MIM 151660) results from heterozygosity for germlinemutations in LMNA (MIM 150330), encoding nuclearlamin A/C, while FPLD3 (MIM 604367) results frommutations in PPARG (MIM 601487), encoding peroxiso-mal proliferator-activated receptor (PPAR)γ. FPLD is asso-ciated with increased risk of several medical complications,including abnormal serum lipids, hypertension and dia-betes. FPLD is associated with increased risk of coronaryheart disease, particularly in women. Since FPLD has lateronset and is progressive, it may be an appropriate modelfor MetS and HIV-associated lipodystrophy. Careful sys-tematic phenotypic analysis of subjects with FPLD2 andFPLD3, an approach called ‘phenomics’, has defined dis-tinct stages in FPLD with particular clinical and biochemi-cal attributes. Fat loss appears to be the inciting event,

Abstracts

©2004 International Medical PressL2

followed by increases in serum insulin and free fatty acids,then dyslipidaemia, then hypertension, then diabetes andfinally vascular complications. Furthermore, fat loss ismore extensive in FPLD2 than in FPLD3, but the metabol-ic complications are more severe in FPLD3 than in FPLD2.Molecular genetic characterization of FPLD2 has revealedthat mechanisms related to the structure and function ofthe nucleoskeleton can lead to lipodystrophy. Moleculargenetic characterization of FPLD3 has confirmed thathuman PPARγ deficiency affects adipose tissue and leads toinsulin resistance, as expected from the mechanism ofaction of thiazolidinedione.


Clinical pharmacology of NRTI-triphosphates

CV Fletcher

Department of Clinical Pharmacy, University of Colorado HealthSciences Center, Denver, Col., USA

Nucleoside analogue reverse transcriptase inhibitors(NRTIs) are the current backbone of virtually all combi-nation antiretroviral therapies for the treatment of HIVinfection. NRTIs are actually prodrugs because the activemoiety is the triphosphate anabolite that is formed intra-cellularly. NRTI-triphosphates elicit their anti-HIV effectvia the inhibition of HIV reverse transcriptase, and pre-sumably toxicity via the inhibition of mitochondrial DNApolymerase gamma. Therefore, the most informed andrational use of these compounds fundamentally dependsupon a quantitative understanding of the clinical pharma-cology of intracellular NRTI-triphosphates.

NRTI combinations such as zidovudine and lamivu-dine are well-documented to act synergistically in termsof antiretroviral activity. The recently demonstrated pro-found rates of virological failure of certain triple NRTIregimens (such as abacavir, lamivudine and tenofovirDF), however, has demonstrated unanswered questionsabout the pharmacological basis for NRTI combinationsas well as intracellular drug–drug interactions. NRTIshave been extensively used clinically and are consideredsafe agents with well-described adverse effect profiles.However, serious toxicities including lactic acidaemia±microvesicular hepatic steatosis, pancreatitis, peripheralneuropathy, and lipoatrophy, which have incidence ratesup to 15% for lactic acidosis and up to 50% for lipoat-rophy, illustrate limited knowledge about risk factors,including patient characteristics associated with NRTIadverse drug reactions. These gaps in our clinical phar-macological knowledge of NRTIs and their pharmaco-logically active triphosphate moieties exist, to a largedegree, due to the challenges of intracellular NRTI-triphosphate quantitation.

Recent advances in analytical methodologies haveallowed the generation of new knowledge about clinicalpharmacological characteristics of NRTI-triphosphateswith findings of, for example, sex and disease state asso-ciations with triphosphate formation, and relationshipsbetween intracellular NRTI-triphosphate concentrationsand virological response. New insights into the clinicalpharmacology of NRTI-triphosphates hold promise as atool to advance the state of HIV therapeutics.


Transcriptional stimulation by the lipid sensingSREBPs

TF Osborne

Department of Molecular Biology and Biochemistry, University ofCalifornia, Irvine, Calif., USA

SREBPs are dimeric bHLHLZ proteins that activategenes in lipid metabolism. They are expressed as precur-sors and insert into ER and nuclear envelope membranes.To increase lipids, they are released from the membraneby regulated proteolysis and imported into the nucleus toactivate gene expression. The normal physiological signalthat triggers their proteolytic maturation is a change inmembrane sterol content. Recently, some HIV proteaseinhibitors have been shown to alter adipocyte and hepa-tic lipid metabolism possibly by altering SREBP matura-tion. Thus, a key to designing more specific proteaseinhibitors may require a better understanding of SREBPactivity. Three SREBP isoforms, 1a, 1c and 2, areexpressed at varying levels in different tissues. Homo-and heterodimers probably form and contribute to over-all SREBP activity, yet no studies have directly evaluatedformation or activation properties of individual SREBPhomo- and heterodimers. To assess activation by eachparticular SREBP dimer, we fused DNA encoding indi-vidual monomers together via a flexible polypeptide teth-er. The expressed tethered 1a and 2 homodimers, liketheir monomeric forms, activate more robustly than 1chomodimers. 1c as a heterodimer with either 1a or 2attenuates activity relative to 1a or 2 homodimers. Theseexperiments provide the first information showing thatboth activation domains in a dimeric transcription factorare required for maximal activity, and support a modelwhere changes in SREBP-1c protein that occur inresponse to insulin and LXR would increase or decreaseSREBP activity in cells where initial ratios of SREBP-1ato 1c are low or high, respectively. This model couldexplain why over-expression of SREBP-1a and SREBP-1c, specifically in liver, have similar phenotypes but theiroverexpression in adipose tissue has dramaticallyopposite effects. Here, 1c overexpression results in a

6th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV


phenotype resembling lipodystrophy including insulinresistance and diabetes. In contrast, similar over expres-sion of SREBP-1a resulted in massive adipocyte hyper-trophy. Because the ratio of 1c to 1a varies form a high inliver (10:1) to a low in spleen (1:10), regulated expressionof SREBP-1c by developmental, hormonal and nutrition-al signals would have pronounced different tissue specif-ic effects on SREBP dependent gene expression.


The Metabolic Syndrome and cardiovasculardisease: lessons from the Bruneck Study

E Bonora

University of Verona, Verona, Italy

Over the last two decades, many epidemiological datahave documented that type 2 diabetes, hypertension anddyslipidaemia cluster in the same individuals. Clinicaldata have indicated that central obesity and insulin resis-tance are common denominators in this cluster and prob-ably play a key pathogenetic role. The clinical phenotypeof the cluster was defined with different names butMetabolic Syndrome has been the most widely used termin recent years. Clinical criteria for the identification ofthe Metabolic Syndrome were established by the WHOand NCEP. NCEP criteria are simpler but WHO criteriaidentify a greater number of subjects.

We have recently evaluated prevalence, further biochem-ical abnormalities and cardiovascular risk of the MetabolicSyndrome in a large sample from the general population ofBruneck aged 40 to 80 years. We used both WHO andNCEP criteria and found that the Metabolic Syndrome isextremely common, with a prevalence of 34% with WHOcriteria and 18% with NCEP criteria. Irrespective of diag-nostic criteria, subjects affected showed biochemical abnor-malities suggesting a mild chronic inflammation, apro-thrombotic state, an increased oxidative stress and anendothelial dysfunction. In fact, we observed that subjectswith the Metabolic Syndrome had higher ESR and WBC,higher fibrinogen and oxidized LDL, and higher circulatingendothelial adhesion molecules (E-selectin, P-selectin,ICAM-1 and VCAM-1). As indicated by several longitudi-nal studies, these abnormalities represent further risk fac-tors for cardiovascular disease. Therefore, subjects with theMetabolic Syndrome have a complex cluster of several tra-ditional and non-traditional risk factors. Interestingly, mostof these non-traditional risk factors were associated toinsulin resistance in our study.

The 5-year follow-up of the Bruneck cohort document-ed that subjects with the Metabolic Syndrome had anincreased incidence of carotid atherosclerosis and coro-nary heart disease. The risk was two- to threefold higher

than in individuals without the syndrome. These resultsare consistent with those reported by other investigators.Therefore, a large proportion of individuals from the gen-eral population are at increased cardiovascular risk for thepresence of the Metabolic Syndrome. The identification ofthese subjects seems to be a major goal of preventive med-icine in the next future.

ABSTRACT 1Antiviral Therapy 2004; 9:L3

Effects of recombinant growth hormone onvisceral fat accumulation: pilot study in HIV-infected adolescents

A Viganò1, S Mora2, P Manzoni1, L Schneider1, S Beretta1, M Molinaro3 and P Brambilla1

1 Chair of Paediatrics, L Sacco Hospital, University of Milan,Milan, Italy; 2 Laboratory of Paediatric Endocrinology, IRCCS HSRaffaele, Milan, Italy; and 3 Department of Pharmacology, IRCCSS Matteo Hospital, Pavia, Italy

Background: Recent data showed that recombinanthuman growth hormone (rhGH) reduces abnormal viscer-al adipose tissue (VAT) accumulation in HIV-infectedadults with lipodystrophy. However, the administration ofrhGH may carry unwanted side effects. To date, no studieshave evaluated the effect of rhGH in HIV-infected lipody-strophic children with VAT accumulation. Methods: Prospective, open-label study of 24 weeks ofrhGH 0.028 mg/kg/daily. Nine HIV-infected adolescents(age range 10.6–18.7 years) with abnormal VAT accumu-lation (>41 cm2 at L4-MRI) were enrolled. The main out-come measure was change in VAT on L4-MRI. Changes inbody composition by DXA, glucose metabolism, lipidprofile and IGF-1 were also evaluated. Results: From baseline to week 24: BMI remained stable[21.3 (2.6) vs 21.2 (2.4)]; VAT decreased significantly[81.1 (35.6) vs 48.9 (25.8) cm2; P=0.01]; total fat per-centage decreased significantly [23.4 (7.2) vs 20.4 (7.6);P=0.03]; and total and regional fat mass decreased, butnot significantly. Lean mass increased significantly as atotal (38.6 vs 42.2 kg; P=0.0008), at arms (3.4 vs 3.9 kg;P=0.001), legs (12.9 vs 14.2 kg; P=0.0013) and trunk[19.3 vs 20.9 kg; P=.0035] level; bone mineral content(BMC) increased significantly (17.9 vs 19.5 g; P=0.03).IGF-1 level increased twofold within 1 month of rhGHand this increase was maintained throughout the 24weeks. Insulin and glucose areas under the curve fromOGTT did not change from baseline to 24 weeks; fastingand post-prandial glycaemia remained within normal lim-its at monthly controls. Fasting triglyceride levels did notchange significantly from baseline to week 24 (125 vs144 mg/dl); four children showed hypertrygliceridaemia(>150 mg/dl) at baseline and maintained it throughout the

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study. Fasting total (164 vs 158), HDL (47 vs 44) andLDL (92 vs 81) cholesterol (mg/dl) did not change; onlyone child showed hypercholesterolaemia (>200 mg/dl).No major side effects were detected; only one child com-plained of transient hand swelling. Conclusion: Our data suggest that 0.028 mg/kg/daily ofrhGH achieved a consistent VAT reduction, a significantincrease in BMC and lean mass and decrease in totaladiposity. Overall rhGH treatment was well tolerated andit was not associated with a worsening of glucose andlipid metabolism.


Effects of a growth hormone releasing factor(GRF) analogue in HIV patients with abdominalfat accumulation: a randomized placebo-controlledtrial

S Grinspoon1, D Kotler2, S Allas3, B Lussier3, A Chapdelaine3, JM Tellier3, MC Domec3,L Vachon3, T Abribat3 and J Falutz4

1 Massachussetts General Hospital, Boston, Mass., USA; 2 StLuke's Roosevelt Hospital Center, New York, NY, USA; and3 Theratechnologies, Inc., Montreal, Que., Canada and MontrealGeneral Hospital, Montreal, Que., Canada

Background: Patients treated with antiretroviral therapymay demonstrate accumulation of abdominal fat andreduced growth hormone concentrations. TH9507, a GRFanalogue with a longer half-life than the natural peptide,has been shown to increase IGF-1 levels within the physio-logical range and to safely improve body composition innon HIV-infected patients. Methods: We investigated the effects of TH9507 in patientswith HIV lipodystrophy and conducted a multicentre, ran-domized, double-blind, placebo-controlled study in 61 HIV-infected patients (54 men, 7 women) with increased waistcircumference (WC) (≥95 cm for men, ≥94 cm for women)and waist to hip ratio (WHR) (≥0.94 for men, ≥0.88 forwomen). Patients received placebo or TH9507 at 1 or 2 mgsc daily for 12 weeks. Visceral adipose tissue was assessed byCT scan at L4-5 and body composition by DXA. Results: Baseline mean age was 46 ±7 (SD), BMI 28 ±3 (SD)kg/m2, WC 102 cm ±8 (SD) and WHR 1.0 ±0.1 (SD). Studypopulation included patients with type 2 diabetes or glu-cose intolerance. At week 12, serum IGF-1 levels increasedover baseline by 59% and 80% at 1 mg and 2 mg, respec-tively (P<0.001 vs placebo). At 2 mg, lean body massincreased (+1.7 kg, P<0.01 vs placebo) and body fat waspreferentially lost at the trunk level (–1.1 kg, P<0.02 vsplacebo) with no significant change in limb fat. There wasa decrease in visceral fat (–15.7% for the 2 mg group,P<0.05 vs baseline; –5.4% for the placebo group, NS vs

baseline) whereas subcutaneous fat was preserved. Lipidprofile improved with reduction in cholesterol to HDLratio. Both doses were well tolerated including with regardto glycaemic control. Conclusion: These results demonstrate that a single dailyadministration of TH9507 for 12 weeks safely increasedlean mass, and decreased abdominal fat with a reduction invisceral fat and preservation of subcutaneous fat in patientswith HIV lipodystrophy. TH9507, a GRF analogue, may bea beneficial treatment strategy for this population.


The effect of recombinant human growth hormonetreatment on circulating leptin and adiponectinconcentrations in patients with HIV-associated fataccumulation

JC Lo1, K Mulligan1, PJ Havel2, MA Noor3, GA Lee1,J-M Schwarz1, C Grunfeld1 and M Schambelan1

1 University of California San Francisco, San Francisco, Calif.,USA; 2 University of California Davis, Davis, Calif., USA; and3 University of California San Francisco (current address: BristolMeyer Squibb Company, Princeton, NJ, USA)

Background: Adiponectin and leptin are proteins derivedfrom adipose tissue. While leptin is related to overall fatmass and recent energy balance, circulating adiponectinlevels are inversely correlated to visceral adipose tissuemass (VAT). Higher circulating adiponectin levels areassociated with increased insulin sensitivity and HDL-Clevels. We have previously reported that growth hormone(GH) treatment reduced fat mass and excess VAT inpatients with HIV-associated fat accumulation. In the pre-sent study, the effects of GH treatment on adiponectin andleptin levels and the relationship to adiposity as well aslipids, resting energy expenditure (REE) and insulin sensi-tivity were assessed in these patients. Methods: Ten men with HIV-associated fat accumulation(buffalo hump and/or abdominal obesity) received GHfor 6 months at 3 mg/day (n=5) or 1 mg/day (n=5). Aspreviously reported, serum lipids, total fat mass (DEXA),VAT (CT), REE (indirect calorimetry) and insulin sensi-tivity (euglycaemic hyperinsulinaemic clamp) were mea-sured at baseline and 6 months. In the current study, wereport serum leptin and adiponectin levels measured byradioimmunoassay at baseline and month 6 of GH treat-ment and the relationship (Spearman correlation coeffi-cient) between percent change in these adipocytokineswith percent change in total fat, VAT, HDL-C, REE andinsulin sensitivity. Results: Treatment with GH resulted in a significantincrease in adiponectin (4.8 ±2.3 to 5.8 ±2.4 mg/ml,P=0.02) and decrease in leptin levels (5.2 ±2.6 to 3.8


±2.2 ng/ml, P<0.05). Percent change in leptin (r=0.77,P=0.01) but not adiponectin (r=–0.09, P=0.80) correlatedwith percent change in total fat mass; neither was signifi-cantly correlated with change in VAT (leptin r=0.42,P=0.23; adiponectin r=0.35, P=0.33). Percent change inadiponectin was significantly correlated with change inHDL (r=0.65, P=0.04) and REE (r=0.65, P=0.04), but notwith insulin sensitivity (r=0.07, P=0.85). Conclusion: Treatment with GH is associated with anincrease in adiponectin and a reduction in circulating lep-tin levels. The reduction in leptin is probably related toGH-induced reduction in total fat mass. The mecha-nism(s) underlying the increase in adiponectin and its rela-tionship to increased HDL-C and REE are of potentialclinical interest and warrant further investigation.


Plasma adiponectin and IL-6 predict insulinsensitivity in patients with HIV infectionindependently of body fat distribution

DN Reeds, KE Yarasheski, WT Cade, BW Patterson,WG Powderly and S Klein

Washington University School of Medicine, St Louis, Mo., USA

Background: Metabolic abnormalities occur in 40% ofpatients who receive highly active antiretroviral therapy(HAART). It is unknown whether adipokines are related tometabolic abnormalities in these patients. Objective: To examine the relationships between plasmaIL-6 and adiponectin concentration, and insulin sensitivityin HIV+ patients with (HIV-LD) or without (HIV-NC)lipodystrophy (Carr et al., Lancet 2003). Methods: We studied 24 men matched for age, BMI andbody composition: 12 with HIV-LD receiving HAART (sixreceiving protease inhibitors) and 12 with HIV-NC (sixreceiving HAART, six naive to antiretroviral therapy).Intravenous infusions of 6,6–2H2 glucose and 2,2–2H2

palmitate were used to measure plasma glucose and palmi-tate rates of appearance (Ra) and glucose disposal rate(Rd), during euglycaemia and graded insulin infusions [20mU·m–2·min–1 (stage 1) and 50 mU·m–2·min–1 (stage 2)].Fat-free mass (FFM) was assessed by dual energy X-rayabsorptiometry. Results: Fasting HOMA was greater in HIV-LD than HIV-NC (3.0 ±0.4 vs 1.3 ±2.1, P<0.001). Glucose Ra (4.7±0.9 µmol·kgFFM–1·min–1 vs 2.6 ±0.4 µmol·kgFFM–1·min–1,P=0.008) and palmitate Ra (0.54 ±0.06 µmol·kgFFM–1·min–1

vs 0.27 ±0.02 µmol·kgFFM–1·min–1, P<0.001) were greater,and glucose Rd (39 ±3 µmol·kgFFM–1·min–1 vs 58±4µmol·kgFFM–1·min–1, P=0.001) was lower in HIV-LD thanHIV-NC, during stage 1 and stage 2, respectively. Basalplasma adiponectin concentration was ~40% lower in

patients with HIV-LD than HIV-NC (4.2 ±0.6 pg/ml vs7.4 ±1 pg/ml, P=0.02), and was ~40% lower in HIV-NCpatients receiving HAART than those naive to therapy(5.4 ±1.1 mg/dl vs 9.4 ±1.6 pg/ml, P=0.07). Adiponectinconcentration correlated with serum HDL (r=0.47,P=0.03) and triglyceride (r=–0.45, P=0.02) concentra-tions, stage 1 glucose Ra (r=–0.40, P=0.05) and stage 2glucose Rd (r=0.52, P=0.008). Plasma IL-6 concentrationwas not different between groups with HIV infection.Plasma IL-6 concentration correlated with glucose Rd (r=–0.51, P=0.01) during stage 2 and palmitate Ra(r=0.37, P=0.08) during stage 1. These correlations per-sisted after correction for age, limb fat mass and durationof HAART, HIV infection and PI exposure. Conclusion: Plasma adiponectin and IL-6 concentrationsindependently predict the sensitivity of stimulation of glu-cose disposal and suppression of lipolysis by insulin inpatients with HIV infection. Use of HAART is associatedwith a reduction in plasma adiponectin concentration.


A structural basis for the effects of HIV proteaseinhibitors on GLUT4 activity

P Hruz, J Hertel, H Struthers and C Baird Horj

Washington University, St Louis, Mo., USA

HIV protease inhibitors (PIs) act as potent reversible non-competitive inhibitors of GLUT4 and are known to con-tribute to alterations in glucose homeostasis duringtreatment of HIV infection. As aspartyl proteaseinhibitors, these compounds all possess a core pep-tidomimetic structure together with flanking hydrophobicmoieties. To determine the molecular basis for GLUT4inhibition, a family of related aromatic di- and tripeptidescontaining structural elements found in PIs were screenedfor their ability to inhibit 2-deoxyglucose transport in pri-mary rat adipocytes. This analysis identified the peptideoxybenzylcarbonyl-His-Phe-Phe-O-ethyl (zHFFe) as apotent inhibitor of zero-trans glucose flux with a Ki of25 µM, comparable with the binding affinities of PIs toGLUT4. In addition, like PIs, glucose transport inhibitionby this peptide was acute, noncompetitive and readilyreversible. Within a Xenopus oocyte expression system,200 µM zHFFe acutely and reversibly inhibited GLUT4-mediated glucose uptake by 69% compared with untreat-ed controls, whereas GLUT1 activity was unaffected at thesame peptide concentration. To characterize the direct pep-tide–protein interactions that mediate GLUT4 inhibition,intact adipocytes were treated with a photoactivatablederivative of zHFFe, zHFF-P-benzoylphenylalanine-[125I]-Tyr-O-ethyl. The results of this analysis showed that a pro-tein with molecular weight identical to GLUT4 (50 kDa)

L5

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was selectively labelled by the photoactivatable peptideand that 500 µM indinavir effectively protected againstphotolabelling. Furthermore, GLUT4 was found to selec-tively bind to a peptide affinity column containing thezHFF sequence. These data establish a structural basis forPI effects on GLUT4 activity and support direct binding ofPIs to the transport protein as the mechanism for acuteinhibition of insulin-stimulated glucose uptake. Moreover,the identification of isoform selective peptide inhibitors ofGLUT4 provides novel tools for studying both normal glu-cose transport and the alterations in glucose homeostasisseen in patients treated with HIV protease inhibitors.


Mechanisms of insulin resistance in HIV-seronegative individuals acutely treated withritonavir boosted indinavir and atazanavir regimens

DA Doran1, SP Jones1,2, C Lagathu3, L Evans4, IT Cambell4, DJ Stokes1, AP Yates5, M Caron3, M Pirmohamed2, DJ Back2, SH Khoo2 and DP Maclaren1

1 Research Institute for Sport and Exercise Sciences, LiverpoolJohn Moores University, Liverpool, UK; 2 Department ofPharmacology and Therapeutics, University of Liverpool,Liverpool, UK; 3 INSERM Unit, CHU St Antoine, Paris, France;4 University Hospital of South Manchester, Department ofAnaesthesia, Manchester, UK; and 5 Clinical ResearchDepartment, Manchester Royal Infirmary, Manchester, UK

Background: Protease inhibitor (PI) therapy has beenassociated with differential effects on lipid and glucosemetabolism. Single dose indinavir (IDV) induces insulinresistance in HIV-seronegative men presumably by block-ade of the insulin responsive glucose transporter GLUT4.By contrast, atazanavir has not been associated withaltered lipid or glucose metabolism nor with inhibition ofGLUT4 in vitro. In this three-arm study, we compared theeffects of a single dose of boosted atazanavir/ritonavir(ATV/RTV), indinavir/ritonavir (IDV/RTV) and placeboon insulin and exercise stimulated glucose disposal,GLUT4 mRNA and protein expression.Methods: Eighteen healthy male volunteers (25.6 ±5.1years) were randomly allocated to a single dose of place-bo (n=6), ATV/RTV (300/100 mg) (n=6), or IDV/RTV(800/100 mg) (n=6). One hour after drug administration,subjects performed 60 mins of cycling at 70% of VO2 max.They then underwent a 180 mins euglycaemic hyperin-sulinaemic clamp to assess the effects on insulin sensitivi-ty, glucose disposal and free fatty acids (FFA). At baseline,60 mins and post-exercise a percutaneous needle musclebiopsy was performed.

Results: There were no significant differences in plasmaglucose, insulin and FFA between the placebo, IDV/RTVor ATV/RTV arms at baseline or during the clamp. ATVand IDV plasma concentrations were within the expectedrange at 60 mins and remained so until the end of theclamp. Insulin-stimulated glucose disposal per unit ofinsulin (M/I) was significantly reduced in the IDV/RTVgroup (8.19 ±0.3) when compared with both the placebo(12.1 ±1.0 mg·kg·min–1per µU/ml, P<0.05) and ATV/RTV(14.58 ±1.0 mg·kg·min–1per µU/ml, P<0.05) arms. Thenon-oxidative component of total glucose disposal (stor-age) was lower in the IDV/RTV group (3.74±0.42 mg·kg·min–1), when compared with the placebo(5.62 ±0.60 mg·kg·min–1) and ATV/RTV arms (5.40±0.53) P<0.01. Total GLUT4 mRNA and protein did notdecrease in either IDV/RTV or ATV/RTV groups whencompared with placebo.Conclusions: IDV/RTV, but not ATV/RTV, acutelyinduces insulin resistance, an effect which is not mediatedby direct inhibition of GLUT4. The effect of these drugsin vivo on the intrinsic activity of GLUT4 warrants fur-ther investigation.


Ritonavir acutely induces insulin resistance inhealthy normal volunteers

GA Lee1, DD Mafong2, JC Lo1, JM Schwarz1, FT Aweeka1, K Mulligan1, M Schambelan1, and C Grunfeld1

1 University of California San Francisco, San Francisco, Calif.,USA; and 2 University Hospitals of Cleveland, Cleveland, OH,USA

Background: Some HIV protease inhibitors acutely causeinsulin resistance even in the absence of HIV infection,hyperlipidaemia or changes in body composition. Severalprotease inhibitors acutely inhibit the activity of theinsulin-responsive glucose transporter GLUT4 in vitro.Previously, a single dose of indinavir was shown todecrease insulin-mediated glucose disposal in healthy nor-mal volunteers. Here we report that a single dose of riton-avir also decreases insulin-mediated glucose disposal inhumans. Methods: In this randomized, double-blind, cross-overstudy, a single dose of ritonavir 800 mg or placebo wasgiven to six healthy HIV-negative men 2 h before assessmentof insulin sensitivity by euglycaemic hyperinsulinaemicclamp. Subjects reached therapeutic ritonavir levels at thestart of the clamp. Free fatty acid levels and substrate oxi-dation rates were measured during the clamp study. Results: There were no significant differences in bodyweight, fasting plasma glucose, serum insulin or lipid


levels before each study. During steady-state (120 to 180min), serum insulin reached levels of 606 ±40 and 577 ±52 pmol/l during placebo and ritonavir administra-tion, respectively. Glucose was maintained at approxi-mately 4.4 mmol/l under both conditions. The averageconcentration of ritonavir reached therapeutic levels dur-ing steady state (10.9 ±0.7 µM), and the 3-h area underthe curve (AUC) was 33.4 ±4.4 µM/h. Insulin-mediatedglucose disposal per unit insulin decreased by 21% (from10.1 ±0.9 to 8.0 ±0.8 mg/kg·min per µU/ml insulin,P=0.02). The non-oxidative component of total glucosedisposal decreased by 30% (from 6.1 ±0.5 to 4.3 ±0.5mg/kg·min, P=0.0004). Ritonavir 3-h AUC correlatedwith the percent change in non-oxidative glucose disposal(r=–0.90, P=0.01). Free fatty acid levels were not signifi-cantly different at baseline and were suppressed similarlywith insulin administration during both studies. Conclusions: A single dose of ritonavir in HIV-negative mendecreased total and non-oxidative insulin-mediated glucosedisposal as assessed by the euglycaemic hyperinsulinaemicclamp. The percentage decrease in non-oxidative glucosedisposal was correlated with ritonavir drug levels. Thesedata suggest that insulin resistance will worsen further withsupra-therapeutic drug levels; excess levels might be avoid-ed with therapeutic drug monitoring.


Abacavir hypersensitivity syndrome: results ofclinical, immunological and genetic screening

G Wong3, R Kaul2, K Shahabi2, D Nolan4, S Mallal4,S Knowles1, A Martin4, N Shear1 and E Phillips1,2

1 Sunnybrook & Women’s College Health Sciences Centre,University of Toronto, Toronto, Ont., Canada; 2 University HealthNetwork, University of Toronto, Toronto, Ont., Canada;3 University of Liverpool, Liverpool, UK; and 4 Centre for ClinicalImmunology and Biomedical Statistics, Royal Perth Hospital andMurdoch, Perth, Western Australia, Australia

Background: Cutaneous patch testing has been previouslydescribed as a potential diagnostic modality for patientshaving experienced abacavir (ABC) hypersensitivity reac-tion (HSR). However, the duration of positivity, correla-tion with in vitro lymphocyte responses to ABC andstrength of association of these tests with genetic markersis currently unknown.Objective: To explore associations between cutaneouspatch testing, lymphocyte proliferation, cytokine responseand genetic testing in previously patch test positivepatients clinically characterized as ABC HSR. Methods: Seven patients previously found to be positive onABC patch testing with histories consistent with ABC HSRbetween July 2000 and June 2003 were re-consented for

repeat 24-h patch testing, genetic testing (HLA-typing)and in vitro PBMC assays to measure drug-specific T-lymphocyte proliferation and cytokine [interferon-gamma(IFNγ) and tumour necrosis factor alpha (TNFα)] produc-tion in response to ABC 0.1 µg/ml and 1 µg/ml. HLA-typ-ing, proliferation and cytokine assays were also conductedon 11 abacavir tolerant controls. Immunological andgenetic tests were performed by laboratory staff blindedto clinical status. Results: Seven out of seven patients who had experiencedABC HSR a median of 25 months (range 10–45) previ-ously and who were patch test positive a median of 23months (range 7–42) previously were strongly positive at24 h on repeat testing. There were no significant differ-ences in CD4+ T cell cytokine production or proliferationbetween patch test positive HSR cases and ABC-tolerantcontrols. However, five out of seven (71%) cases showedsignificant CD8+ proliferation in response to ABC versusone out of 11 (9%) of the controls tolerating ABC(P=0.005), and CD8+ IFNγ production was seen in twoout of seven (29%) cases and no controls (P=0.04). HLA-B*5701 was present in seven out of seven patch test posi-tive patients versus one out of 11 controls who was takingand tolerating ABC (P<0.001). Conclusions: Patch testing appears to be consistently pos-itive even remote from the original ABC HSR and showedgood correlation with ABC-specific CD8+ T lymphocyteresponses and presence of HLA-B*5701. This stronglysuggests that ABC HSR is mediated by HLA-B*5701restricted CD8+ T lymphocytes, and suggests that clinical,genetic and immunological tests may have complementaryapplication in the prevention, diagnosis and managementof ABC HSR.


HIV-lipodystrophy is characterized by insulinresistance for leucine and glucose metabolism:dissimilar to type 2 diabetes

KE Yarasheski, D Reeds, S Mohammed, WT Cade, X Chen, WG Powderly and S Klein

Washington University School of Medicine, St Louis, Mo., USA

Background: Type 2 diabetes is characterized by impairedglucose tolerance and insulin resistance with respect toglucose metabolism, but not amino acid metabolism(Diabetes 2002; 51:2395). Objectives: To examine whether HIV-associated impairedglucose tolerance (IGT) and insulin resistance extends toamino acid metabolism. Methods: Glucose disposal rate and plasma leucine rate ofappearance (Leu Ra = whole-body proteolysis rate) weremeasured during the post-absorptive state and euglycaemic-


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hyperinsulinaemia using primed constant infusions of 6,6-2H2-glucose and 13C-leucine in three groups of subjects:eight seronegative control (three women, five men;BMI=28 ±1 kg/m2), 18 HIV+ non-lipodystrophic (twowomen, 16 men; BMI=26 ±1, HIV RNA=6361 ±3817copies/ml, CD4=521 cells/µl), and 19 HIV-lipodystrophic(Carr et al., Lancet 2003) subjects (four women, 15 men;BMI=26 ±1, HIV RNA=1704 ±951, CD4=554). Glucoseand Leu kinetics were examined at insulin infusion ratesof 20 and 50 mU/m2/min, which produced plasma insulinlevels of 240–280 and 570–670 pM. Fat-free mass (FFM)was measured using dual energy X-ray absorptiometry. Results: At the highest insulin level, glucose disposal ratewas lower (P=0.002; ANOVA) in HIV lipodystrophy (42±3; mean ±SEM) and HIV non-lipodystrophy (56 ±5),than in controls (69 ±7 µmol/kg FFM/min). Contrary totype 2 diabetes, whole body proteolysis rate was higher(P<0.001) at all insulin levels in HIV lipodystrophy(baseline 163 ±4; low insulin 147 ±4 and high insulin 139±4 µmol/kg FFM/hr) and HIV+ non-lipodystrophy (146±4, 132 ±4, 123 ±3) than in controls (138 ±3, 121 ±3,115 ±3). Plasma interleukin-6 (IL-6) levels tended to behigher (P=0.016) in HIV lipodystrophy (1.9 ±0.3) andHIV+ non-lipodystrophy (1.1 ±0.2) than in controls (0.9±0.2 pg/dl). Conclusion: These findings suggest that both whole-bodyglucose and amino acid (Leu) metabolism are insensitiveto the anabolic actions of hyperinsulinaemia in HIV, espe-cially in HIV lipodystrophy. Increased inflammatorycytokine (IL-6) levels may contribute to the failure ofhyperinsulinaemia to suppress whole-body proteolysis inHIV-infected people.Acknowledgement: Supported by DK59531, DK49393,DK54163, DK56341, AI25903.


Antiretroviral therapy and markers of insulinresistance in the Multicenter AIDS Cohort Study

TT Brown1, X Li2, SR Cole2, LA Kingsley3, FJ Palella4,SA Riddler3, JS Chmiel4, BR Visscher5, B Margolick2

and AS Dobs1

1 School of Medicine, Johns Hopkins University, Baltimore, Md.,USA; 2 Bloomberg School of Public Health, Johns HopkinsUniversity, Baltimore, Md., USA; 3 University of Pittsburgh,Pittsburgh, Pa., USA; 4 Northwestern University, Chicago, Ill.,USA; and 5 University of California Los Angeles, Los Angeles,Calif., USA

Background: Surrogate markers have not been used toestimate insulin resistance in a large cohort of HIV-infect-ed individuals with direct comparison with HIV-seroneg-ative controls.

Methods: 533 HIV-infected and 755 HIV-seronegativemen in the Multicenter AIDS Cohort Study were exa-mined at 6-month intervals between 1999 and 2003.Fasting serum measurements, antiretroviral therapy(ART) history, demographics and clinical variables werecollected at each visit. ART exposure was quantified intwo ways. Firstly, recent exposure was assessed by strati-fying men by ART in the preceding 6 months, that is, noART, mono-combo ART, highly active antiretroviral ther-apy (HAART) without a protease inhibitor (PI) andHAART with a PI. Secondly, cumulative exposure wasdetermined for the three major ART classes: nucleosideanalogues (NRTIs), non-nucleoside reverse transcriptaseinhibitors (NNRTIs) and PIs. Individual medicationswithin each class were also investigated. Two markers ofinsulin resistance were used as the primary outcome vari-ables and assessed with reference to the HIV-seronegativemen: QUICKI [1/(logGlucose + logInsulin)] and fastinghyperinsulinaemia (insulin >15 µU/ml). Analyses wereadjusted for age, body mass index, race, nadir CD4 cellcount, hepatitis C serostatus and family history of dia-betes mellitus.Results: Each of the HIV-infected groups had higher oddsof fasting hyperinsulinaemia and lower mean QUICKIcompared with the HIV-seronegative men. Cumulativeexposure to NRTIs had the most consistent effect, witheach additional year of exposure associated with anincreased odds of hyperinsulinaemia (OR=1.1, 95% CI:1.1, 1.2) and a lower QUICKI (–0.06, 95% CI: –0.09,–0.04). Cumulative exposure to PIs was associated withan increased odds of hyperinsulinaemia (OR per addition-al year of PI exposure=1.1, 95% CI: 1.0, 1.1), but a lessapparent difference in mean QUICKI (–0.01 per addition-al year of PI exposure, 95% CI: –0.05, 0.02). Of the indi-vidual medications examined, stavudine was associatedwith the highest risk of hyperinsulinaemia (OR=1.2, 95%CI: 1.2, 1.3). Conclusions: Fasting surrogate markers suggest increasedinsulin resistance in HIV-infected men which is indepen-dently related to both NRTI and PI exposure.


Objective evaluation of HIV lipodystrophy casedefinition in a randomized 96 week clinical trial

MG Law1, A Cheng2, DA Cooper1,3 and A Carr3

1 National Centre in HIV Epidemiology and Clinical Research,UNSW, Sydney, Australia; 2 Gilead Sciences, Foster City, Calif.,USA; and 3 St Vincent’s Hospital, Sydney, Australia

Introduction: An objective definition of HIV lipodystro-phy has been developed by the Lipodystrophy CaseDefinition Study Group, but the definition has not been



evaluated based on prospective data. Gilead 903 was arandomized, double-blind clinical trial comparing stavu-dine (d4T) with tenofovir (TDF), both in combinationwith lamivudine and efavirenz in 600 treatment-naiveadults. Lipodystrophy was reported as an unpromptedadverse event for 12% of patients randomized to d4Tcompared with 1% of TDF patients.Methods: DEXA scans were assessed and centrally readat 96 weeks in a subset of patients in Gilead 903. Anadapted version of the lipodystrophy case definitionbased on centrally read DEXA scans, not including CTscans and waist to hip ratio in the definition (variablesthat were both unavailable in Gilead 903) was used toobjectively define lipodystrophy. The accuracy of theadapted and original case definition models was 78%and 79%, respectively. Rates of lipodystrophy by themodified case definition were compared with rates diag-nosed through unprompted adverse event reports. Riskfactors for objectively defined lipodystrophy at week 96using the modified case definition were identified usinglogistic regression.Results: 255 (43%) patients in Gilead 903 had DEXAscan and other data available for the adapted case defin-ition. Of these patients, 17 (7%) reported lipodystrophyas an unprompted adverse event by week 96. The casedefinition identified a total of 48 (19%) patients withlipodystrophy, 40 (31%) receiving d4T and eight (6%%)receiving TDF. Of these 48 patients, only seven (15%)also reported lipodystrophy as an adverse event. In mul-tivariate analyses, objectively defined lipodystrophy wasassociated with d4T vs TDF [odds ratio (OR)=8.7,P<0.001], older age (OR=1.08 per year, P=0.001), femaleversus male (OR=3.7, P=0.001) and higher baselinetriglycerides (OR=1.32 per mmol/l, P=0.021). Conclusions: An adapted objective lipodystrophy casedefinition was more sensitive for diagnosing lipodystro-phy in Gilead 903 than unprompted adverse eventreports.


Prospective, partially randomized, 24-week studyto compare the efficacy and durability of differentsurgical techniques and interventions for thetreatment of HIV-related facial lipoatrophy

G Guaraldi1, G Orlando1, D De Fazio2, M Callegari2,G De Santis1, A Pedone1, A Spaggiari1, ABaccarani1, M Pinelli1, V Borghi1, G Nardini1, BBeghetto1 and R Esposito1

1 University of Modena and Reggio Emilia, Italy; 2 San RaffaeleHospital, Milan, Italy and Casa di cura S. Pio X, Milan, Italy

Background: Autologous fat transfer (AFT) surgery andinjection of reabsorbable or non-reabsorbable materialinto the lipoatrophic areas of the face are the only avail-able treatment for HIV-related facial lipoatrophy.Comparisons between treatment approaches have beenlimited.Methods: This is a prospective, multicentre, partially ran-domized study. Patients with facial lipoatrophy receivedAFT if residual truncal subcutaneous fat was present as asource of fat graft, otherwise they were randomized toreceive filler injections in the cheeks with readsorbablepolylactic acid (PLA) or non-reabsorbable polyacrylamide(PAC). Ultrasound evaluation was used to assess subcuta-neous thickness; Visual Analogue Scale (VAS) was utilizedto evaluate patient satisfaction. Subjects were followed for24 weeks following end of treatment.Results: 59 patients were evaluated: AFT (24), PLA (20)and PAC (15). The groups were well balanced for clinical,HIV-related variables, ultrasound measured thickness ofcheek fat and VAS measurements. PLA and PAC groupsreceived 5 ±1 and 6 ±2 injections, respectively. Six monthsafter end of treatment, VAS had increased in the AFTgroup from 24.7 ±16.4 to 70.2 ±21.9, P=0.000; in thePLA group from 20 ±20 to 82.8 ±16.7, P=0.001; and inthe PAC group from 25.3 ±21.7 to 82.8 ±17.1, P=0.000.ANOVA did not show any statistically significant differ-ences among the three groups either for VAS variable(F=1.287, P=0.288) or for delta thickness of dermal plussubcutaneous layer measured with ultrasound (F=0.59,P=0.55). Overall cheek thickness increased by a mean of6.4 ±7.6 mm. There was no statistically significant differ-ence in number of injections necessary to reach a satisfac-tory result (evaluated both by surgeon and patients)between the PLA and PAC groups, P=0.083. No seriousadverse events were observed in the sample. Conclusion: All three interventional techniques were high-ly effective in improving the aesthetic satisfaction of thepatients. There were no significant differences in subjec-tive objective variables among the three study arms.Longer follow-up is necessary to discriminate the mostsuitable treatment in terms of durability.

ABSTRACT 13 Antiviral Therapy 2004; 9:L9

Zidovudine inhibits thymidine phosphorylation: anovel site of potential toxicity in non-mitotic cells

EE McKee1,2, MD Lynx1,2, D Susan-Resiga2, ATBentley2, JP D'Haenens1,2, D Cullen2 and M Ferguson2

1 Indiana University School of Medicine South Bend Center forMedical Education, Notre Dame, Ind., USA; and 2 University ofNotre Dame, Notre Dame, Ind., USA

Abstracts


Zidovudine (3′-azido-3′deoxythymidine, AZT) is a thymi-dine analogue pro-drug that can be phosphorylated byhost cell enzymes to the triphosphate, which is a potentinhibitor of the viral reverse transcriptase. AZT has beena staple of highly active antiretroviral therapy (HAART)for many years. In the early days of AIDS therapy, AZTwas given alone at relatively high concentrations and itslong-term use was associated with various tissue toxici-ties, including hepatotoxicity and cardiomyopathy,associated with mitochondrial DNA depletion. In morerecent therapy, AZT in combination with other nucleosideanalogues is associated with a lipodystrophy that may beof mitochondrial origin. The triphosphate form of AZT(AZT-TP) is a known inhibitor of the mitochondrial poly-merase γ and has been targeted as the potential source ofthe mitochondrial DNA depletion. This laboratory’s pre-vious work with isolated rat heart mitochondria in short-term experiments suggested that AZT was notphosphorylated beyond the monophosphate (AZT-MP).AZT-MP accumulated in the matrix and AZT-DP andAZT-TP were not detected. Instead, we found that AZTwas a potent inhibitor of thymidine phosphorylation (7.0±1.0 µM) acting at the level of the mitochondrial thymi-dine kinase (TK2). In non-mitotic tissues such as car-diomyocytes, cytoplasmic thymidine kinase (TK1) is notexpressed and the cell’s supply of TTP is generated solelythrough TK2. We therefore suggest the novel hypothesisthat toxicity of AZT in non-mitotic tissue is caused by thedirect inhibition of TK2 limiting the cellular pool of TTP.The low level of TTP limits mitochondrial DNA replica-tion, ultimately leading to mitochondrial dysfunctionassociated with mitochondrial DNA depletion. Thishypothesis was tested in this work using two approaches.In one, an isolated perfused heart system was used toextend our investigation of AZT and thymidine phospho-rylation to the whole cell (Abstract 32, Susan-Resiga etal.). In the second, we sought to extend our observationsin isolated heart mitochondria to mitochondria isolatedfrom other tissues, beginning with liver mitochondria(Abstract 31, Lynx et al.). Thymidine was readily phos-phorylated to TTP in both of these systems, with TTP thepredominant product in the perfused heart and TMP thepredominant product in isolated liver mitochondria.However, the phosphorylation of AZT beyond AZT-MPwas not detected in either system. The production of TTPand AZT-MP in isolated liver mitochondria was an orderof magnitude higher than observed in heart mitochondria.Finally, AZT inhibited thymidine phosphorylation in bothsystems with IC50s of 8.9 ± 1.8 µM in liver mitochondriaand 24 ± 5 µM in the perfused heart. Acknowledgements: This work was supported by a grantfrom the National Institute of Health, HL 72710 to EEM.


Uridine abrogates the adverse effects of stavudineand zalcitabine on adipose cell functions

UA Walker1, M Auclair2, D Lebrecht1, M Kornprobst2, J Capeau2,3 and M Caron2

1 Medizinische Universitätsklinik, Department of Rheumatologyand Clinical Immunology, Freiburg, Germany; 2 INSERM U402,Faculty of Medicine Saint-Antoine, Paris, France; and 3 HôpitalTenon, Paris, France

Objectives: The adverse effects of nucleoside analoguereverse transcriptase inhibitors (NRTIs) have been attrib-uted to their mitochondrial toxicity. Beneficial effects ofthe pyrimidine precursor uridine on NRTI-induced mito-chondrial damage have recently been described in hepaticcells. In the present study, we assessed whether uridine canprevent the adverse effect of stavudine and zalcitabine onadipocyte functions in vitro.Methods: 3T3-F442A preadipocytes were exposed tostavudine (10 µM) or zalcitabine (0.2 µM) in the absenceor presence of uridine (200 µM) for 21 days before, and7 days after induction of differentiation. Lipid accumula-tion (oil red staining), apoptosis (flow cytometry), mito-chondrial DNA (mtDNA) levels and mitochondrialmembrane potential (JC-1) were evaluated at day 7 ofdifferentiation.Results: Prolonged treatment with stavudine or zalcitabinemarkedly altered adipose cell morphology. Adipocyteswere enlarged and contained lipid droplets of reduced sizeand number. Stavudine and zalcitabine significantlydecreased lipid accumulation (by 36 and 20%, respective-ly) and increased apoptosis (by 5.6- and 2.2-fold, respec-tively). Stavudine and zalcitabine markedly decreasedadipocyte mtDNA to residual amounts of 36% (P=0.006)and 45% (P=0.01), respectively. Both NRTIs also inducedmitochondrial depolarization, as shown by the 40–45%decrease of the JC-1 aggregate/monomer ratio.

Uridine had no intrinsic effect, but abrogated theadverse effects of both NRTIs on adipocyte morphologyand lipid staining. Uridine normalized apoptotic indices(1.1-fold for both NRTIs). Uridine prevented mtDNA-depletion by stavudine and zalcitabine (mtDNA-levels101% and 78% of control adipocytes, respectively) andnormalized mitochondrial respiration (JC-1 aggregate/monomer ratio). Conclusions: Uridine supplementation protects culturedadipocytes from the adverse effects of stavudine and zal-citabine on lipid accumulation, cell survival and mito-chondrial functions. The beneficial effect of uridine is seenwhatever the parameter tested, suggesting that the toxiceffects of these NRTIs could be linked to depletion of uri-dine or its metabolites inside the cells. Elevating the intra-



cellular uridine level could allow competition of uridinemetabolites with NRTIs at polymerase-gamma and thusprevent the decreased mtDNA synthesis. Uridine is aninteresting candidate in the prevention of the NRTI-induced lipoatrophy in vivo. Acknowledgement: This work was supported by grants ofINSERM and ANRS, and by the BMBF, KompetenznetzHIV/AIDS (Grant number: 01KI0211).


In vivo nucleoside reverse transcriptase inhibitorsalter expression of both mitochondrial and lipidmetabolism genes independent to HIV infection

PWG Mallon1–3, P Unemori1,3, R Sedwell1,3,A Morey4, M Rafferty5, K Williams5, D Chisholm6,K Samaras6, S Emery1, A Kelleher1–3, DA Cooper1–3

and A Carr2,3 for the SAMA investigators

1 National Centre in HIV Immunology and Clinical Research,University of New South Wales, Sydney, Australia; 2 HIV,Immunology and Infectious Diseases Clinical Services Unit, StVincent's Hospital, Sydney, Australia; 3 HIV ImmunovirologyResearch Laboratory, Centre for Immunology, St Vincent'sResearch Campus, Sydney, Australia; 4 Department ofAnatomical Pathology, St Vincent's Hospital, Sydney, Australia; 5Clinical Trials Centre, St Vincent's Hospital Sydney, Australia; and6 Garvan Institute of Medical Research, Sydney, Australia.

Mitochondria play an important role in lipid homeostasis,with mitochondrial dysfunction implicated in obesity andinsulin resistance. The mitochondrial dysfunction inducedby nucleoside analogue reverse transcriptase inhibitors(NRTIs) used to treat HIV infection, has been implicatedin the development of lipoatrophy. How this mitochondr-ial dysfunction affects lipid metabolism at a molecularlevel has not been prospectively described in vivo. Weexamined early changes (2 weeks) in mitochondrial andnuclear gene expression in adipose tissue from 20 HIV-negative volunteers exposed to 6 weeks of dual-NRTItherapy. We observed inhibition of mitochondrial RNA(mtRNA) transcription without significant mitochondrialDNA depletion. Decreases in mtRNA coincided withsimultaneous up-regulation of nuclear genes involved inthe regulation of mtRNA transcription (NRF1 andmtTFA) and fatty acid oxidation (PPARα and LPL), whilePPARγ, important for adipose differentiation and insulinresponses, was down-regulated. Many nuclear changescorrelated with changes in PPARγ co-activator-1 (PGC1)expression, suggesting a central role for this transcription-al co-activator in nuclear responses to mitochondrial dys-function. Peripheral blood monocyte COX1 expressionalso decreased, suggesting that monocytes may be surro-gates for drug-induced mitochondrial dysfunction. These

results characterize the central role of mitochondrial func-tion in adipose tissue metabolism in vivo, provide evi-dence to support the presence of feedback mechanismsbetween mitochondria and the nucleus in humans, andoffer an explanation for NRTI-induced lipoatrophy.


Differential effects of nucleoside reversetranscriptase inhibitor (NRTI) regimens onadipocyte mitochondrial DNA depletion in HIV-infected patients

E Hammond, D Nolan, E McKinnon, I James and S Mallal

Centre for Clinical Immunology and Biomedical Statistics,Murdoch University and Royal Perth Hospital, Perth, WesternAustralia

Background: Clinical trials data have provided strong evi-dence that NRTI therapy significantly influences risk oflipoatrophy. Here we examine associations betweenstavudine, zidovudine and non-thymidine NRTI regimens,as well as demographic and HIV disease-related variables,and adipocyte mitochondrial DNA (mtDNA) depletion.Methods: Adipocyte mtDNA depletion was assessed usingreal-time PCR in 130 subcutaneous fat samples from 87HIV+ individuals, including 34 ART-naive samples and 96NRTI-exposed samples. Current NRTI therapy: 35 stavu-dine, 42 zidovudine and 19 non-thymidine NRTI (aba-cavir or tenofovir). Linear regression models were utilizedwith adjustment for multiple measurements in individuals. Results: Among ART-naive (n=34), median mtDNA con-tent was 1427 copies/cell (range: 413–6570). No signifi-cant correlations were detected between mtDNA levelsand age (mean ±SD, 45 ±10), race (non-white: 15%), CD4T-cell count (357 ±403) or HIV RNA (4.59 ±1.1) (P>0.1). Among NRTI-treated individuals,zidovudine was associated with mtDNA depletion com-pared with ART-naive (median 761, range: 94–2846copies/cell, P=0.0001), while mtDNA depletion was evenmore marked with stavudine (median 250, range: 61–2287copies/cell) compared with ART-naive or zidovudinegroups (P<0.0001). A significant decline in mtDNA levelswas also observed within 2–12 months of commencingstavudine or zidovudine therapy (n=14, mean reductionfrom ART-naive baseline: 263 copies/cell/month,P=0.005).

In contrast, non-thymidine regimens were associatedwith similar mtDNA levels to ART-naive controls (medi-an 1675, range: 916–4180 copies/cell, P=0.8), and initiat-ing these regimens was not associated with significantchanges in mtDNA over time (mean reduction of 69copies/cell/month, P=0.6). Among NRTI-experienced

Abstracts


patients, switching from stavudine to zidovudine or aba-cavir was associated with a marked increase in adipocytemtDNA (3–11-fold increase, P=0.01) over 1–24 months(median 6 months). After adjustment for effects of choiceof NRTI therapy in a mixed effects analysis, no significanteffects of HIV protease inhibitor therapy (current PI: 38%,P=0.7) nor age (P=0.9) were detected. Conclusions: These data are concordant with evidencethat lipoatrophy risk is differentially and specifically asso-ciated with stavudine or zidovudine use, providing furtherevidence that NRTI-induced mitochondrial toxicity iscentral to lipoatrophy pathogenesis. Alternative NRTIregimens were not associated with adipocyte mtDNAdepletion in this study, nor were demographic/disease-related factors


Steatohepatitis is associated with increasedhepatic expression of SREBP-1 in HIV-infected lipodystrophic patients

M Lemoine1,2, V Barbu1,2, JP Bastard2,3, D Wendum1,M Maachi2,3, F Paye1,2, R Poupon1,2, PM Girard1, C Housset2,3, J Capeau2,3 and L Serfaty1,2

1 Saint-Antoine Hospital, Paris, France; 2 INSERM U402, Paris,France; and 3 Tenon Hospital, Paris, France

Objectives: ART-induced lipodystrophy could be associat-ed with hepatic steatosis. However, due to the presence ofinsulin resistance, these patients could be at risk of steato-hepatitis and liver injury. In rodents, the transcription fac-tors PPARs and SREBP-1, controlling hepatic glucose andlipid metabolism, are probably involved in steatosis.Ritonavir increased hepatic expression of SREBP-1 andsteatosis. We therefore examined liver pathologicalchanges and expression of PPARα, PPARγ1, γ2 andSREBP-1 in HIV-infected lipodystrophic patients. Methods: Three groups of patients were studied. Group 1:nine HIV lipodystrophic patients (including PI in eightpatients), without HBV or HCV infection, who had liverbiopsy because of elevated transaminases; Group 2: ninepatients with non-alcoholic fatty liver disease (NAFLD),age and BMI matched with Group 1; Group 3: 10 con-trols without steatosis. All patients had determination ofserum fasting glucose, insulin, triglycerides, leptin andadiponectin levels. Hepatic expression of PPARs andSREBP-1 was assessed by real-time RT-PCR. Results: HIV-LD and NAFLD patients compared withcontrols had higher insulin (median 6.9 and 15.7 vs2.8 mU/l, P<0.05) and lower adiponectin levels (median5.2 and 6.4 vs 12 µg/ml, P<0.05). Liver histology inHIV-LD patients showed steatohepatitis in four out ofnine cases, associated with portal fibrosis in all cases.

Hepatic expression of SREBP-1 was higher in HIV-LDpatients than in NAFLD or control patients (median 90 vs50 and 40 104 copies/µg RNA, respectively, P<0.05).HIV-LD patients with steatosis had significantly higherSREBP-1 expression than those without steatosis (median175 vs 85 104 copies/µg RNA, P<0.05). Hepatic expres-sion of PPARα, PPARγ1, and PPARγ2 was not signifi-cantly different between the three groups. Conclusion: In patients with ART-related lipodystrophy1) steatohepatitis is frequent, 2) hepatic SREBP-1 mRNAis overexpressed as compared with NAFLD patients orwith controls, 3) SREBP-1 overexpression is associatedwith steatosis and 4) expression of PPAR genes is not sig-nificantly modified. These results suggest that SREBP-1contributes to the pathogenesis of fatty liver in thesepatients.


In vivo evidence for impaired peripheral fatty acidtrapping in HIV-1 lipoatrophy

JPH van Wijk1, M Castro Cabezas1,2, EJP deKoning1,3, TJ Rabelink3 and IM Hoepelman1

1 University Medical Center Utrecht, Utrecht, The Netherlands;2 St Franciscus Gasthuis Rotterdam, Rotterdam, The Netherlands;and 3 University Medical Center Leiden, Leiden, The Netherlands

Background: The use of highly active antiretroviral thera-py in HIV-infected patients has been associated withchanges in fat distribution (lipodystrophy), insulin resis-tance and dyslipidaemia and increases the risk for athero-sclerosis. Lipodystrophy may result from impairedperipheral free fatty acid (FFA) trapping, which may causeenhanced hepatic FFA flux and consequently VLDL over-prodution and exaggerated postprandial lipaemia. Methods: We have investigated FFA, hydroxybutyric acid(HBA, a marker of hepatic FFA oxidation) and triglyc-eride (TG) changes after a single oral fat challenge (10 h,50 g/m2) in HAART-treated HIV-infected male patientswith (LIPO, n=26) and without lipodystrophy (NON-LIPO, n=12) and in healthy normolipidaemic controls(n=35). Immunological parameters and duration of HIVand HAART were similar between the LIPO and NON-LIPO group. Area under the curves (AUCs) for FFA andTG were significantly higher in the LIPO group (7.9 ±2.1and 45.0 ±12.9 mmol·h/l, respectively) compared with theNONLIPO group (6.1 ±1.1 and 22.7 ±10.7 mmol·h/l,respectively) and healthy controls (5.9 ±1.2 and 17.0 ±3.9mmol·h/l, respectively). The postprandial increase inketone bodies was two times higher in the LIPO group(1954 ±1123 µmol·h/l) compared with the NONLIPOgroup and healthy controls (1188 ±567 and 1067±501 µmol·h/l, respectively, P<0.05 for each). There was



no difference in HBA-AUC and FFA-AUC between theNONLIPO group and healthy controls. In HIV-infectedpatients, HBA-AUC was negatively associated with BMI(r= –0.50, P<0.05), total body fat mass (r= –0.40, P<0.05)and hip circumference (r= –0.38, P<0.05) and positivelyassociated with incremental FFA-AUC. Conclusions: The present data suggest elevated hepaticFFA flux in HIV lipoatrophy, most likely as a result ofinadequate incorporation of FFA into TG in adipocytes.Impaired peripheral postprandial FFA uptake may lead toincreased hepatic FFA flux, VLDL overproduction andincreased concentrations of atherogenic postprandial TG-rich lipoproteins in these patients.


Ritonavir causes hypertriglyceridaemia in APOE*3-Leiden transgenic mice by impairing triglycerideclearance

M den Boer1,2, JFP Berbée1,3, PCN Rensen1,3,JA Romijn2, P Reiss4, M van der Valk4, PJ Voshol1,2

and LM Havekes1,2,5

1 TNO Prevention and Health, Leiden, The Netherlands;2 Department of Endocrinology, LUMC, Leiden, The Netherlands;3 Department of General Internal Medicine, LUMC, Leiden, TheNetherlands; 4 Department of Infectious Diseases, AMC,Amsterdam, The Netherlands; and 5 Department of Cardiology,LUMC, Leiden, The Netherlands

Objectives: The protease inhibitor ritonavir (RTV)increases plasma triglycerides (TG) both in humans and inmice. Our aim was to determine the mechanism underly-ing the RTV-induced hypertriglyceridaemia in APOE*3-Leiden transgenic mice which have a human-likelipoprotein profile.Methods: Female APOE*3-Leiden transgenic mice werefed a western-type diet with or without RTV added(35 mg/kg body weight/day).Results: After administration of RTV for 3 weeks, plasmaTG increased from 2.7 ±0.6 to 5.3 ±1.9 mM (P<0.01),which was specific for VLDL. We first determined whetherincreased production of VLDL-TG could be the cause ofthe increased plasma TG. However, when compared withuntreated mice, hepatic VLDL-TG production was notchanged (138.5 ±41.2 vs 177.4 ±59.9 µmol TG/kg/h incontrols; P>0.05). Interestingly, compared with control ani-mals, RTV caused a 2.5-fold increased postprandial TGresponse upon an intragastric olive oil load, which suggestsimpaired TG clearance (5.0 ±2.5 vs 2.0 ±1.1 mM at 2 hafter gavage; P<0.05). Indeed, clearance of intravenouslyinjected [3H]TG-labelled VLDL-like emulsion particles wasdecreased in RTV-treated mice compared with untreatedcontrols (t1/2 19.3 ±10.5 vs 5.0 ±1.3 min; P<0.05).

Conclusion: From these data, we conclude that RTV caus-es hypertriglyceridaemia by impairing plasma TG clearancerather than by stimulating hepatic VLDL-TG production.


The metabolic effects of intermittent antiretroviraltherapy with and without IL-2 (ACTG A5102)

P Tebas1, K Henry2, D Cherng3, D Katzenstein4,J Schmitz5, H Valdez6, N Jahed7, M Vargas7,L Myers8 and W Powderly9

1 University of Pennsylvania, Pa., USA; 2 University ofMinnesota, Minn., USA; 3 Harvard University, Cambridge, Mass.,USA; 4 Stanford University, Stanford, Calif., USA; 5 University ofNorth Carolina, NC, USA; 6 Case Western University, Cleveland,OH, USA; 7 AACTG Operations Center, Bethesda, Md., USA;8 Frontier Science & Technology Research Foundation, Mass.,USA; and 9 Washington University School of Medicine in StLouis, Mo., USA

Background: CD4-driven antiretroviral therapy is beingevaluated in large trials. The metabolic effects of this strat-egy are largely unknown. ACTG 5102 evaluated the utilityof IL-2 and pulses of ART based on CD4 cell counts as astrategy to reduce the costs and toxicity of continuous ARV. Objective: To evaluate the effects on lipid and glucosemetabolism of IL-2 and prolonged TI. Design: Forty-seven HIV+ subjects on ART with CD4+ T-cell counts >500 cells/mm3 and HIV RNA levels<200 copies/ml were randomized to receive or not three 5-day cycles of IL-2, 4.5 million units subcutaneously twicedaily every 8 weeks (n=23 and 24, respectively) for 18weeks. Then they discontinued ARV until the CD4 cellcount dropped below 350. The median follow-up off ARVis greater than 1 year. Twenty-six of the subjects receivedNNRTI-based therapy. Fasting glucose, insulin and lipidparameters were evaluated every 8 weeks initially and atweeks 2, 4, 8 and every 8 weeks after TI. Results: Three cycles of IL-2 did not affect lipid or glucosemetabolism. After 48 weeks of TI there were significantdecreases of triglycerides (from 172 mg/dl, –20%,P<0.001), total cholesterol (from 213 mg/dl, –15%,P<0.001) and LDL cholesterol (126 mg/dl, –12%,P=0.008). There were no significant changes in glucose orinsulin levels or HOMA-IR. Lipid changes occurred rela-tively early after interruption (within the first 4 weeks). Conclusions: Three cycles of IL-2 do not have significantmetabolic effects on subjects receiving stable antiretroviraltherapy. Treatment interruption is associated with immediateand sustained decreases in cholesterol levels (both LDL andHDL) and TG. The effects on glucose and insulin metabo-lism were limited in this cohort. A strategy of intermittenttherapy can decrease the cardiovascular risk associated

Abstracts


with ARV therapy and provide insight into which of themetabolic abnormalities observed in treated patients areHIV- or ARV-related.


Marked impairment of endothelial functionwithout insulin resistance in healthly men treatedwith the HIV-1 protease inhibitor indinavir

SS Shankar1,3, MP Dubé1,2, RV Considine1,3,L Christner1,3 and HO Steinberg1,3

1 Indiana University School of Medicine, Indianapolis, Ind., USA;2 Division of Infectious Diseases, Indianapolis, Ind., USA; and3 Division of Endocrinology, Indianapolis, Ind., USA

Background: The HIV-1 protease inhibitor indinavir (IDV)impairs endothelial function and basal nitric oxide (NO)-dependent tone in healthy men. Endothelial dysfunction iscommonly observed in states of insulin resistance. Wehypothesized that IDV-induced endothelial dysfunctionoccurs as a result of IDV-induced insulin resistance. Methods: We assessed insulin sensitivity, endothelial func-tion and insulin-mediated vasodilation in 16 lean healthymale subjects before and after 4 weeks of IDV 800 mgtwice daily. Insulin sensitivity was measured using theeuglycaemic hyperinsulinaemic clamp for 240 mins. Weassessed endothelial function by measuring changes in legblood flow (LBF) in response to intra-arterial administra-tion of graded doses of the endothelium-dependentvasodilator, methacholine chloride. Results: Our subjects were 37 ±3 years old, with BMI of 25±1 kg/m2, body fat of 19.6 ±1.9%, total cholesterol: 171 ±8mg/dl; LDL-cholesterol: 98 ±7 mg/dl; HDL-cholesterol: 50±4 mg/dl; triglycerides: 140 ±39 mg/dl, and resting LBF of0.207 ±0.015 l/min. There was no significant change in anyof these parameters after IDV. Plasma adiponectin levelsincreased after IDV (16.4 ±2.2 µg/ml pre-IDV, 19.1±2.3 µg/ml post-IDV, P<0.05). Normal, robust endotheli-um-dependent and insulin-mediated vasodilatory responseswere present at baseline. After IDV, there was a markedblunting of endothelium-dependent vasodilation (258±43% pre-IDV vs 60 ±13% post, P<0.05) and insulin-mediated vasodilation (70 ±10% pre-IDV vs 16 ±6% post,P<0.05). In spite of these dramatic effects on vascular func-tion, there was no significant change in the steady-statewhole body glucose-disposal rate with IDV (8.0 ±0.6mg/kg/min pre-IDV vs 7.5 ±0.6 post, P=NS). Conclusions: Four weeks of IDV markedly impairsendothelial function and insulin-mediated vasodilation,without significant impairment of whole-body glucosedisposal. Thus, it appears unlikely that insulin resistanceplays a major role in the induction of the endothelial dys-function seen in this human model of IDV monotherapy.

Acknowledgements: This study was supported by thesegrants from the NIH: HL 72711, AI 052852, and GeneralClinical Research Center grant M01-RR00750.


Ritonavir and indinavir promote lipid-inducedatherosclerosis but inhibit endothelial denudation-induced neointimal hyperplasia in mice

NM Schildmeyer, ZWQ Moore, JT Nickel, CJ Fichtenbaum and DY Hui

University of Cincinnati College of Medicine, Cincinnati, OH, USA

The use of protease inhibitors (PIs) for treatment of HIVinfection is associated with increased risk of peripheraland coronary artery disease (CAD). The pathogenesis ofCAD is a complex process involving macrophage infiltra-tion and lipid accumulation in the vessel wall, endothelialdysfunction and the migration of medial smooth musclecells (SMC) to the intima, followed by their proliferationand matrix deposition to form an occlusive plaque.Although PI therapy has been shown to induce hyperlipi-daemia and insulin resistance, thereby increasing the riskof lipid-laden foam cell formation and deposition at thevessel wall, whether PI therapy also contributes to thepathogenesis of CAD by modulating vascular cell activa-tion has not been explored. This study used the mousemodel to evaluate the effects of two different PIs, ritonavirand indinavir, on early (lipid-laden foam cell deposition)and late (SMC hyperplasia after endothelial injury) eventsof CAD. Administration of ritonavir 1.25 µg/kg/day orindinavir 1.875 µg/kg/day exacerbated foam cell-enrichedatherosclerotic lesions in LDL receptor-deficient C57BL/6mice fed an atherogenic diet for 16 weeks. To evaluate theeffects of PI on SMC activation in response to endothelialinjury, fat-fed wild-type FVB/N mice were treated with orwithout PI for 16 weeks. Endothelial denudation in thecarotid arteries was accomplished with a resin-modifiedcatheter probe. The carotid arteries were dissected andexamined histologically after 14 days. Despite a similarincrease in plasma lipid levels in the PI-treated mice,neointimal hyperplasia – as characterized by SMC migra-tion and proliferation – in the injured carotid arteries wasdramatically reduced in PI-treated mice as compared withvehicle-treated controls. In vitro cell culture experimentsalso showed ritonavir and indinavir inhibition of SMCmigration and proliferation in response to PDGF. Theseresults indicated that PI therapy promotes CAD mainlythrough its effects on metabolic parameters that lead tolipid-laden foam cell formation instead of vascular SMCactivation, thus suggesting that lowering plasma lipid andglucose levels may alleviate the accelerated CAD eventsassociated with PI therapy in HIV-infected patients.



Acknowledgement: This work was supported by NIHGrant HL65915.


Pre-eclampsia in HIV-infected pregnant womenreceiving highly active antiretroviral therapy isassociated with endothelial dysfunction andinsulin resistance

M Larrousse, E Martínez, A Suy, M Lonca, E de Lazzari, A Milinkovic, L Zamora, A León, M Laguno, JL Blanco, J Mallolas, S Pisa, S Hernández, V Cararach, JA Vanrell, JM Gatelland O Coll

Hospital Clinic Barcelona, Barcelona, Spain

Background: We have recently reported an unexpectedhigh risk for pre-eclampsia in HIV-infected pregnantwomen receiving highly active antiretroviral therapy(HAART). Although this adverse effect was clinically asso-ciated with the duration of exposure to HAART prior topregnancy, the underlying pathogenesis remains unknown. Methods: Stored plasma samples immediately prior to theestimated date of conception (time point 1), during preg-nancy (time point 2), and in the puerperium (time point 3)for HIV-infected pregnant women who developed pre-eclampsia (cases) and at similar time points for randomlyselected HIV-infected pregnant women who did not devel-op pre-eclampsia (controls) were used for measuringinsulin (IRMA, Med-Genix Diagnostics, Fleunes, Belgium)and P- and E-selectin (R&D System, Minn., Minnesota).Insulin, and P- and E-selectin measurements were com-pared with the Kruskall–Wallis test.Results: There were nine cases and nine controls.P-selectin (ng/ml) showed a trend to increase over timeboth in cases and controls, but the increase in cases wassignificantly higher than in controls (time point 1: 26.9±13.2 vs 13.1 ±4.2, P=0.0087; time point 2: 45.5 ±26.3 vs21.1 ±11.4, P=0.0211; time point 3: 60.6 ±24.3 vs 36.1±10.7, P=0.0136, respectively). Although there was atrend towards higher E-selectin (ng/ml) values in casesthan in controls at the different time point studies, differ-ences were not statistically significant (time point 1: 39.1±15.1 vs 27.3 ±13.5, P=0.0998; time point 2: 34.9 ±13.4vs 33.5 ±15.6, P=0.8369; time point 3: 43.7 ±10.7 vs 31.8±13.1, P=0.0523, respectively). Because the volume ofstored samples prior to pregnancy and in the puerperiumwas scarce, insulin could be measured only during preg-nancy. Insulin (mU/l) during pregnancy was significantlyhigher in cases than in controls (time point 2: 36.0 ±33.2vs 9.4 ±6.5, P=0.0311, respectively).Conclusion: Endothelial dysfunction and insulin resis-tance may be potential underlying mechanisms involved

in the pathogenesis of pre-eclampsia in HIV-infected preg-nant women receiving HAART.


Endothelial dysfunction in HIV-infected patientson CART does not improve even when lipidprofiles improve on pravastatin

PA Sklar1,2, JR Grubb2, JD Voell3, G Zalos4, WCBlackwelder2, JA Metcalf 3, A Rupert5, MTGladwin2, J Witek1, RT Davey3, H Masur2 andRO Cannon4

1 Drexel University, Philadelphia, Pa., USA; 2 Clinical Center, NIH,Bethesda, Md., USA; 3 NIAID, NIH, Bethesda, Md., USA; 4 NHLBI,NIH, Bethesda, Md., USA; and 5 Science ApplicationInternational Corporation, Frederick, Md., USA

Background: Atherogenic dyslipidaemia is commonamong HIV+ individuals on CART and there is evidencethat this may translate into premature cardiovascular(CV) disease. An empiric approach has been to modifylipids and thus decrease CV risk through the use of HMGCo-A reductase inhibitors.Methods: 23 HIV+ patients on stable CART completed arandomized, placebo-controlled, crossover study to eval-uate the effect of pravastatin (40 mg) on endothelial func-tion. Endothelial function (EF) – central to vascularpathophysiology – was assessed by flow-mediated vasodi-lation (FMD) of the brachial artery.Results: At baseline, HIV+ individuals demonstratedabnormal EF compared with an otherwise healthy HIV–population (mean ±SEM, 7.0 ±0.5% HIV vs 10.1 ±0.9controls, P=0.002). As expected, active treatment withpravastatin significantly improved total-C (mean differ-ence ±SEM, –36 ±5 mg/dl, P<0.001), LDL-C (–30 ±4,P<0.001) and triglycerides (–69 ±25, P=0.01); but notHDL-C or measures of insulin resistance. Despite theoverall improvement in metabolic risk profiles, pravas-tatin yielded no consistent or significant improvement inEF (mean difference in FMD ±SEM between drug andplacebo, –0.3 ±0.8%, P=0.68; means 7.0% on drug,7.3% on placebo). Furthermore, pravastatin did not yielda significant decline in C-reactive protein (CRP) values(mean difference ±SEM, –0.4 ±1.9 mg/l, P=0.85; means6.3 on drug, 6.7 on placebo); there was no significantcorrelation between FMD and CRP at baseline (correla-tion coefficient = –0.16, P=0.45)Conclusions: While pravastatin improves the dyslipi-daemia of HIV+ individuals on CART this does not trans-late into improved EF. Persistently elevated CRP valuessuggest that there may be an ongoing stimulus towardsCV risk which has yet to be elucidated.

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POSTER PRESENTATIONS



Non-invasive assessment of hepatic mitochondrialtoxicity in HIV-infected patients with normalserum lactate by 13C-methionine breath test

M Banasch1, O Goetze2, I Hollborn1, B Hochdorfer3,N Brockmeyer3, WE Schmidt1 and F Schmitz1

1 Department of Internal Medicine 1, University of Bochum,St Josef Hospital, Bochum, Germany; 2 Department ofGastroenterology and Hepatology, University of Zurich, Zurich,Switzerland; and 3 Department of Dermatology, University ofBochum, Bochum, Germany

Objectives: Mitochondrial toxicity related to HIV-infectionand antiretroviral therapy (ART) is associated with abroad range of metabolic disorders such as hepatic steato-sis, lactic acidosis and lipodystrophy syndrome. Recentlythe non-invasive, non-radioactive 13C-methionine breathtest (MBT) has been proven to detect mitochondrialimpairment in HIV-infected patients with symptomatichyperlactacidaemia. Since elevated serum lactate is no pre-condition for mitochondrial toxicity we investigated thevalidity of the 13C-methionine breath test in variouspatient groups with normal serum lactate values. Methods: The MBT was performed in 14 healthy controlsand 46 patients with chronic HIV infection: 13 patientswith no treatment experience, 23 patients with asympto-matic disease who had received ART for longer than1 year and 10 patients with long-term ART (>5 years) andclinical signs of lipoatrophy. After an overnight fast, thevolunteers received 2 mg/kg body weight [methyl-13C]-labelled methionine. Hepatic mitochondrial decarboxyla-tion was measured by breath 13CO2 enrichment expressedas delta over baseline (DOB) every 10 min for 120 min byisotope ratio infrared spectroscopy. Allowing for the dif-ferent body weights, the kinetic variable cPDR (cumula-tive percentage 13C dose recovered over test period) wasobtained by mathematical analysis. Results: ANOVA analysis showed significantly decreased13C-exhalation in HIV-infected patients with evidence oflipoatrophy compared with healthy subjects (cPDR 2.42±0.9 vs 6.74 ±1.4, P<0.01). There was no statistical sig-nificance between healthy volunteers and patients withasymptomatic disease in our study (cPDR 6.74 ±1.4 vs7.02 ±1.1, P>0.5). Surprisingly, treatment-naive patientsalso exhibited significantly decreased hepatic mitochondr-ial decarboxylation function compared with healthy vol-unteers (cPDR 4.92 ±1.90 vs 6.74 ±1.4, P<0.05). Conclusions: The MBT is a simple, non-invasive methodof detecting hepatic mitochondrial impairment in HIV-infected patients. The altered 13C-metabolism in patientswith long-term treatment and signs of lipoatrophy wasexpected, reflecting the chronic mitochondrial toxicity of

ART. We are the first to show that therapy-naives alsoexhibit significant impairment of mitochondrial decar-boxylation function. We hypothesize that mitochondrialdysfunction might rapidly improve after initiating ART.Our results provide evidence that the MBT is an invalu-able diagnostic tool for both screening and follow-upmonitoring of hepatic mitochondrial function.


Addition of tenofovir to a didanosine-basedHAART does not increase mitochondrial DNAdepletion but decreases cytochrome c oxidasefunction and mitochondrial mass

G Garrabou1, S López1, AB Infante1, E Negredo2,J Puig2, L Ruiz2, E Sanjurjo1, J Casademont1,F Cardellach1, B Clotet2 and Ò Miro1

1 Mitochondrial Research Laboratory, Muscle Research Unit,Internal Medicine Department, Hospital Clinic of Barcelona,IDIBAPS, University of Barcelona, Barcelona, Catalonia, Spain;and 2 Lluita contra la SIDA and Irsicaixa Foundations, GermansTrias i Pujol Hospital, Universitat Autònoma de Barcelona,Barcelona, Catalonia, Spain

Purpose of the study: The nucleotide analogue tenofovirdisoproxil fumarate (TDF) has been reported to be free ofmitochondrial toxicity. In the present study, we evaluatethe effects of the introduction of TDF on a HAART sched-ule containing didanosine (ddI) on mitochondrial mass,mitochondrial DNA (mtDNA) content and cytochrome coxidase (COX) activity.Methods: Fifty HIV-infected patients receiving a ddI-based HAART schedule were recruited, and changed toddI plus TDF (300 mg/d) and nevirapine (400 mg/d)(n=25, cases) or maintained with the same HAARTscheme (n=25, controls). All patients were symptom-freewith undetectable viral load along the study. Peripheralblood mononuclear cells (PBMCs) were obtained at 0(baseline), 6 and 12 months. The quantity of mitochon-dria was assessed by the spectrophotometric measurementof the citrate synthase activity, the content of mtDNA byquantitative real-time PCR and the activity of COX(complex IV of the mitochondrial respiratory chain) byspectrophotometry. Results: Cases and controls maintained unchanged in allmitochondrial parameters at 6 months with respect tobaseline. Conversely, at 12 months we found that mtDNAcontent was reduced in both cases (24%, P<0.01) and con-trols (18%, P<0.05), while mitochondrial mass and COXactivity were found to be significantly decreased only incases (28%, P<0.05 and 47%, P<0.001; respectively). Conclusions: A decrease in mitochondrial mass, mtDNAcontent and COX activity is detected after 12 months of

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the addition of TDF to HAART schedules containing ddI.This diffuse deterioration of mitochondrial parameterscould be due to the effects of TDF itself, the increase ofddI concentrations caused by TDF or both. The relevanceof these biochemical findings in clinical practice remainsto be determined.Acknowledgements: Study supported by Fundación parala Investigación y la Prevención del SIDA en España(FIPSE 3102/00), Fundació la Marató de TV3 (02/0210),Redes de Investigación en Mitocondrias (V2003-REDC06E-0) y SIDA (Redg 173) and Suports a Grups deRecerca (2001/SGR/00379).


Mitochondrial studies in adipose tissue of HIV-infected patients without fat redistribution

S López1, G Garrabou1, E Martínez2, P Domingo3,J Fontdevila4, JM Gatell2, AB Infante1, X Gallart5,A Milinkovic2, F Cardellach1, J Casademont1 andÒ Miró1

1 Mitochondrial Research Laboratory, Muscle Research Unit,Department of Internal Medicine, Hospital Clinic, IDIBAPS,Barcelona, Catalonia, Spain; 2 Department of Infectious Diseases,Hospital Clinic, IDIBAPS, Barcelona, Catalonia, Spain; 3 Infectious Diseases Unit, Department of Internal Medicine,Hospital de la Santa Creu i Sant Pau, Universitat Autonoma deBarcelona, Catalonia, Spain; 4 Department of Plastic Surgery,Hospital Clinic, IDIBAPS, Barcelona, Catalonia, Spain; and5 Department of Orthopaedic Surgery and Traumatology(ICMEQ), Hospital Clinic, IDIBAPS, Barcelona, Catalonia, Spain

Objectives: Mitochondrial (mt) DNA depletion in adiposetissue (AT) has been reported in patients with highlyactive antiretroviral therapy (HAART)-related lipodystro-phy (LD). However, few studies have focused on the roleof HIV itself or HAART in the AT mtDNA decrease.Similarly, the effects of mtDNA depletion on mitochondr-ial respiratory chain (MRC) function has not been evalu-ated. The aim of the study was to assess the mitochondrialamount, function and mtDNA content in AT from HIVpatients prior to the development of LD.Methods: We studied AT mitochondria of 20 healthy con-trols (group A) and 17 HIV patients, the latter stratified intwo subgroups: one antiretroviral naive (n=8, group B)and the other one on HAART (n=9, group C). ATobtained from abdomen, thigh or arm was immediatelyfrozen in liquid nitrogen and stored at –80°C until analy-sis. We determined the mitochondrial amount in the AThomogenate by the spectrophotometric assay of citratesynthase (CS) activity, as well as the mitochondrial respi-ratory chain (MRC) function, by the specific activity ofcytochrome c oxidase (COX, complex IV of the MRC).

MtDNA was measured by quantitative real-time PCR andexpressed as the mtDNA to nuclear DNA ratio.Results: The mitochondrial amount was unaltered amonggroups. Conversely, groups B and C showed a markeddecrease in mtDNA content per cell when compared withuninfected individuals [54% and 60% of the control value(100%), respectively; P=0.001], as well as decreasedabsolute activity of COX (57% and 91% of the controlvalue, respectively; P=0.07). Similar results were observedwhen mtDNA and COX activity were normalized by themitochondrial amount (86% and 52% of the controlvalue, respectively; P=NS, for normalized mtDNA con-tent, and 72% and 66% of the control value, respective-ly; P=0.02, for normalized COX activity). We found apositive correlation between mtDNA content and COXactivity (R2=0.3, P<0.001).Conclusions: Decreased mtDNA content in AT of HIV-infected patients is closely associated with decreased COXactivity, irrespective of the presence of HAART, before LDappears. The exact role that HIV itself and HAART playin adipocyte changes remains to be determined.


Mitochondrial DNA depletion in asymptomaticHIV-infected patients receiving didanosine plusstavudine-based HAART regimen seems to becompensated by up-regulatory mechanisms

S López1, G Garrabou1, MR de la Concepción3, E Martínez2, E Pedrol4, AB Infeante1 M Giralt3, F Cardellach1, JM Gatell2, F Vilarroya3, J Casademont1 and Ò Miró1

1 Mitochondrial Research Laboratory, Muscle Research Unit,Department of Internal Medicine, Hospital Clinic, IDIBAPS,Barcelona; 2 Department of Infectious Diseases, Hospital Clinic,IDIBAPS, Barcelona; 3 Department of Biochemistry andMolecular Biology, University of Barcelona, Barcelona, Spain; and4 HIV-Unit, Department of Internal Medicine, Fundació Hospital-Asil de Granollers, Granollers, Catalonia, Spain

Objectives: The use of nucleoside analogues is uniformlyassociated with mitochondrial DNA (mtDNA) depletion,but diverse studies in asymptomatic patients have report-ed functional indemnity of mitochondria. We determinedwhether homeostatic mechanisms are able to compensatethis mtDNA depletion in patients receiving stavudine plusdidanosine (d4T+ddI), an antiretroviral association withgreat in vitro and in vivo capacity to decrease mtDNA.Methods: We included 28 symptom-free HIV-infectedindividuals: 17 on first-line antiretroviral regimen consist-ing of d4T+ddI for at least 6 months (case group) and 11naive subjects (control group). In peripheral bloodmononuclear cells we assessed: 1) the quantity of



mitochondria by citrate synthase activity, 2) the content ofmtDNA by quantitative real-time PCR, 3) COX-II expres-sion [subunit II of cytochrome c oxidase (COX), encodedby mtDNA] by Western blot, and 4) COX activity byspectrophotometry. Results: The quantity of mitochondria and the mtDNAcontent of cases (d4T+ddI) were decreased when comparedwith controls, whether calculated by cells or by mitochon-dria. The expression of COX-II and COX activity weresimilar in cases and controls. The expression of COX-IIwas found to be constant and independent of the mtDNAcontent, whereas it was closely related to COX activity.Conclusions: Decreased mitochondrial mass and mtDNAcontent are associated with ddI+d4T treatment, but theexpression of COX-II and COX activity remains unal-tered. These data suggest that, at least during the initialphases of treatment, up-regulatory transcriptional or post-transcriptional mechanisms compensate mtDNA deple-tion caused by ddI+d4T.Acknowledgements: Supported by Fundación para laInvestigación y la Prevención del Sida en España (FIPSE3102-00 and 3161/00A), Fundació La Marató de TV3(020210), Redes de Investigación en Mitocondrias(V2003-REDC06E-0) y Sida (173) and Suport a Grups deRecerca 2001/SGR/00379.


HIV infection is associated with increased skeletalapoptosis assessed by TUNEL

S López1, G Garrabou1, J Fernández-Solà1, EPedrol3, E Badia1, AB Infante1, E Martínez2, FCardellach1, JM Gatell2, J Casademont1 and Ò Miró1

1 Mitochondrial Research Laboratory, Muscle Research Unit,Department of Internal Medicine, Hospital Clinic, IDIBAPS,Barcelona; 2 Department of Infectious Diseases, Hospital Clinic,IDIBAPS, Barcelona; 3 HIV-Unit, Department of InternalMedicine, Fundació Hospital-Asil de Granollers, Granollers,Catalonia, Spain

Objectives: Increased apoptosis in CD4+ T lymphocytesplays an important role in the pathogenesis of HIV infec-tion, probably through several viral proteins that havebeen proposed to be directly or indirectly associated withthe dissipation of mitochondrial membrane potential,thereby inducing apoptotic cell death. The aim of thestudy was to assess whether this increase is also present inthe skeletal muscle of HIV-infected patients.Methods: We included 18 healthy individuals without HIVinfection as controls and five asymptomatic antiretroviral-naive HIV-infected patients. Immunohistochemical reac-tions using monoclonal antibodies for TUNEL(deoxyribonucleotidyl-transferase-mediated-dUTP-biotin

nick-end labelling) were performed on skeletal musclesamples from the deltoid muscle of the non-dominant armof each patient and control. The specimens were codedwith random numbers and read by three independent,blinded observers. The percentage of apoptotic cells wasdetermined by means of an apoptotic index that was cal-culated by dividing the total number of positive stainingmyocyte nuclei in the TUNEL assay by the total numberof the myocyte nuclei, and multiplying this value by 100.Results: The results were consigned in a semiquantitativescale (– if less than 1% of positive nuclei or cells were pre-sent, + if less than 1%, ++ if less than 5% and +++ if morethan 5%). The TUNEL assay data showed significantlyincreased apoptosis in skeletal muscle of 80% of HIVpatients when compared with uninfected individuals(P<0.01). None of the control individuals were positivefor TUNEL.Conclusions: Skeletal muscle of HIV-infected patientsexhibits increased apoptosis compared with healthy unin-fected individuals. Further studies are necessary to eluci-date the mechanisms by which HIV infection leads toincreased apoptosis.Acknowledgements: Supported by Fundación para laInvestigación y la Prevención del Sida en España (FIPSE3102-00 and 3161/00A), Fundació La Marató de TV3(020210), Redes de Investigación en Mitocondrias(V2003-REDC06E-0) y Sida (RG173) del Fondo deInvestigaciones Sanitarias and Suport a Grups de Recerca2001/SGR/00379.


Uridine pharmacokinetics of Mitocnol, a sugarcane extract

N Venhoff1, M Zilly2, D Lebrecht1, D Schirmer2, H Klinker2, J Thoden1, P Langmann2 and UA Walker1

1 Medizinische Universitätsklinik, Department of Rheumatologyand Clinical Immunology, Freiburg, Germany; and 2 MedizinischePoliklinik, University of Wuerzburg, Wuerzburg, Germany.

Background: In vitro data and limited in vivo data indi-cate that the supplementation of uridine may be beneficialin preventing and treating the mitochondrial toxicity ofpyrimidine nucleoside analogue reverse transcriptaseinhibitors (NRTIs) by abrogating mtDNA depletion.

NucleomaxX is a food supplement of potential use intreatment of mitochondrial toxicity as it contains Mitocnol,a sugar cane extract with a high percentage of nucleosides.However, the exact effects of Mitocnol consumption on theserum levels of uridine in humans are not known. Methods: Healthy, fasting, adult human probands (fourmale and four female) consumed 36 g of NucleomaxX by

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drinking 200 ml of orange juice in which the extract wasdissolved. Baseline serum levels of uridine were measuredby HPLC before drinking NucleomaxX and during thefollowing 24 h.Results: Mean (±SD) uridine serum levels at baseline were5.6 ±1.1 µM (males 5.8 µM, females 5.4 µM). AfterNucleomaxX consumption, uridine serum levels rosesharply and peaked after 1.3 h. The mean maximal uridineserum concentration (Cmax) was 152.0 µM (±29.2 µM).The Cmax range was 116.0–212.0 µM. The mean Cmax infemales was slightly but non-significantly higher (165.4±35.8 µM) compared with males (138.6 ±15.2 µM), pos-sibly due to the lower body weight and body surface areaof the former (mean body weight and body surface offemales: 62.3 kg and 1.71 m2) compared with the latter(males: 77.5 kg and 1.96 m2, respectively). Uridine waseliminated from the serum with an initial half-life of 2 hand a terminal half-life of 11.1 h. After 8 and 24 h, meanuridine serum levels were 19.3 µM (±4.7 µM) and 7.5 µM(±1.6 µM), respectively. The mean AUC calculated withthe linear trapezoidal rule between the time points in thetime data range was 736 µMh (±95 µMh) and was identi-cal between sexes. Adverse events were not observed.Conclusion: Mitocnol effectively increases uridine serumlevels in humans. Acknowledgement: This work was supported by theBMBF, Kompetenznetz HIV/AIDS (Grant number:01KI0211).


Zidovudine inhibits thymidine phosphorylation:a novel site of potential toxicity in non-mytoticcells

MD Lynx1,2, JP D’Haenens1,2, AT Bentley2, D Susan-Resiga2 and EE McKee1,2

1 Indiana University School of Medicine South Bend Center forMedical Education, Notre Dame, Ind., USA; and 2 University ofNotre Dame, Notre Dame, Ind., USA

Zidovudine (3′-azido-3′deoxythymidine, AZT) has been astaple of highly active antiretroviral therapy (HAART) formany years. Unfortunately, long-term use of AZT is alsoassociated with various tissue toxicities, includinghepatotoxicity and cardiomyopathy, associated with mito-chondrial DNA depletion. The triphosphate form of AZT(AZT-TP) is a known inhibitor of the mitochondrial poly-merase γ and has been targeted as the source of the mito-chondrial DNA depletion. Previous work in thislaboratory with isolated rat heart mitochondria in 2–3 hexperiments suggests that AZT is not phosphorylatedbeyond the monophosphate (AZT-MP), which accumu-lates with no detection of AZT-TP. Rather, AZT was

shown to be a much more potent inhibitor of thymidinephosphorylation (7.0 ±1.0 µM) than AZT-TP is of poly-merase γ (>100 µM), suggesting that depletion of mito-chondrial stores of TTP may limit replication. Thepurpose of this work was to investigate the hypothesisthat an identical mechanism might account for the hepa-totoxicity seen with long-term use of AZT. Isolated ratliver mitochondria were incubated with labelled thymi-dine or AZT, and the rate and extent of phosphorylationto nucleotides was determined by HPLC analysis of acidsoluble extracts of the incubated mitochondria. The resultsobtained showed that AZT is phosphorylated only toAZT-MP and that AZT inhibited the production of TTP,with an IC50 of 8.9 ±1.8 µM AZT. This is comparable withthe results seen in isolated rat heart mitochondria. Thekinetics of thymidine phosphorylation to TMP differsbetween heart and liver. The maximal velocity (Vmax) andaffinity constant (Km) are an order of magnitude higher inliver than in heart. Liver also displays either strong nega-tive cooperativity or the presence of two enzymes, one witha Vmax and Km similar to TK2 in rat heart mitochondriaand one with much higher Vmax and Km. This secondenzyme may represent several possibilities including conta-minating thymidine kinase 1, a different isoform of TK2,or another yet uncharacterized kinase. Acknowledgements: This work was supported by a grantfrom the National Institute of Health, HL 72710 to EEM.


Thymidine and zidovudine metabolism in isolatedrat heart: zidovudine inhibition of thymidinephopsphorylation

D Susan-Resiga1, AT Bentley1, J D'Haenens1,2, D Cullen2, MD Lynx1,2 and EE McKee1,2

1 University of Notre Dame, Notre Dame, Ind., USA; 2 IndianaUniversity School of Medicine South Bend Center for MedicalEducation, Notre Dame, Ind., USA

Zidovudine (3′-azido-3′-deoxythymidine, AZT) is a thymi-dine analogue pro-drug that is phosphorylated in the hostto AZT triphosphate (TP), which functions by inhibitingviral reverse transcriptase. AZT has been part of the highlyactive antiretroviral therapy (HAART) for many years;however long-term use of AZT was linked to various tissuetoxicities, including cardiomyopathy, and was associatedwith mitochondrial DNA depletion. So far, the prevailinghypothesis for AZT toxicity has been the inhibition of mito-chondrial DNA polymerase γ by AZT-TP.

Our previous work with isolated heart mitochondria(McKee EE et al., 2004), demonstrated that AZT wasphosphorylated only to the monophosphate (MP) underconditions in which thymidine (T) was readily



phosphorylated to the triphosphate. This suggested thatthe cardiac mitochondrial toxicity observed for this drugwas not likely to be caused by AZT-TP inhibition of themitochondrial replication polymerase. Rather, we demon-strated that AZT was a potent inhibitor of thymidinephosphorylation suggesting that mitochondrial DNAreplication was limited by the TTP pool.

The present work extends the above observations tothe intact isolated perfused heart and examines whole cellmetabolism of thymidine and AZT. Tritiated thymidine isreadily converted to labelled TTP in a time and concen-tration dependent manner in the perfused heart. Steady-state level of the TDP/TTP pool is reached in 60–90 min.The level of labelled TMP is quite low, suggesting thatthymidine kinase (TK) is the rate limiting step in phos-phorylation. The kinetics of TTP phosphorylation dis-plays negative cooperativity, similar to the observationswith cloned and purified mitochondrial TK2 (Wang andEricsson, 2000). Labeled AZT is converted in a time andconcentration dependent manner to AZT-MP, the onlyphosphorylated form detected even after 3 h of perfusion.AZT phosphorylation in perfused heart follows simpleMichaelis–Menten kinetics, as opposed to thymidine. Thepresence of AZT in the perfusate inhibits the conversionof labelled thymidine to TTP with an IC50 of 24 ±5 mM.These data taken together extend the observations madein isolated heart mitochondria and suggest that mitochon-drial toxicity in the heart may be caused by a limiting cell-ular pool of TTP lowering the rates of mitochondrialDNA replication.


The impact of nucleoside reverse transcriptaseinhibitor (NRTI) treatment duration and insulinresistance on fasting arterialized lactate levels inpatients with HIV infection

JC Lo, M Kazemi, PY Hsue, JN Martin, SG Deeks, M Schambelan and K Mulligan

University of California San Francisco, San Francisco, Calif., USA

Treatment with NRTIs is associated with hyperlactataemia,presumably mediated by NRTI inhibition of mitochondr-ial-DNA replication (mitochondrial toxicity). The cumu-lative effect of NRTI treatment could impair oxidativeglucose metabolism, favouring glycolysis and lactate pro-duction. We examined the association of NRTI treatmentduration and plasma lactate levels in HIV-positivepatients. Methods: Fasting arterialized venous samples (10-minhandwarming, 55°C) were obtained in HIV-infected out-patients in whom detailed antiretroviral histories wereavailable. Plasma lactate, glucose, pO2, serum insulin,

ALT and CD4 were measured. Insulin resistance was esti-mated by HOMA-IR. Percent body fat was measured byDEXA. We examined the independent association ofcumulative NRTI use and lactate levels using multivari-able linear regression. Results: 95 participants had adequate venous arterializa-tion (PO2 >55 mmHg). Mean age was 44.4 ±8.3 years;90% were men; 95% had NRTI exposure [median 5.6years, interquartile range (IQR) 4.3–9.6]; and 83% werecurrently on NRTIs. Arterialized lactate levels averaged1.24 ±0.46 mmol/l (range 0.53–2.72). Median HOMA-IRwas 3.37 (IQR 2.24–4.63). Only two had ALT >100 mg/dl.Longer duration of NRTI use was associated with a smallbut significant increase in lactate in univariate analyses(0.047 mmol/l increase per year of NRTI use, P<0.01),and remained so after adjusting for current NRTI use.Female sex, percent body fat, PI treatment duration andHOMA-IR were also associated with lactate level in uni-variate analyses, while fasting glucose, ALT and CD4were not. After adjustment for age, sex, diabetes, percentfat and PI duration, increasing duration of NRTI therapyremained significantly associated with lactate level(0.035 mmol/l increase/year NRTI, P=0.04). Alternatively,d4T duration or current use of d4T/ddI was not. Notably,when we added HOMA-IR to the above adjusted model,the relation between NRTI duration and lactate wasattenuated and no longer significant (0.024 mmol/lincrease/year NRTI, P=0.14), while HOMA-IR was sig-nificantly associated with lactate level (P<0.01). NRTIduration was also associated with HOMA-IR after adjust-ing for age, sex, percent fat and diabetes (P=0.03). Conclusion: Increasing duration of NRTI use is associat-ed with higher lactate levels that may be mediated, in part,through increasing insulin resistance. Acknowledgements: SCOPE study, NIH (AI052745,DK45833, DK54615, RR-00083), UCSF AcademicSenate, Doris Duke Foundation.


The developmental stage determines the effect ofNRTIs on adipocyte mtDNA depletion andadiponectin production

M Stankov, RE Schmidt and G Behrens

Clinical Immunology, Hannover Medical School, Hannover,Germany

Objective: To evaluate the in vitro contribution ofnucleoside-analogue reverse transcriptase inhibitor(NRTI) therapy to lipoatrophy as a result of mitochondr-ial DNA toxicity in adipose tissue. Methods: Relationships between adipocyte mitochondrialDNA (mtDNA) content, adiponectin production and the

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incubation with three different NRTI in 3T3 L1 cell linewere investigated. Longitudinal effects (21 days) of NRTIson different stages of adipocyte development and differ-entiation were studied. Adipocyte mitochondrial DNAdepletion was also assessed according to the particulardrug and developmental stage using real-time PCR. Fatcontent was measured with Oil Red O staining andadiponectin production by real-time PCR and ELISA. Results: Stavudine (d4T) incubation was associated withmore severe adipocyte mtDNA depletion, but heavilydepended on adipocyte proliferation. Stavudine alsodirectly leads to decreased adiponectin production.Zidovudine (AZT) incubation was associated withimpaired differentiation and decrease in adiponectin pro-duction. Effects of zalcitabine (ddC) on adipocytesmtDNA were stronger in preadipocytes with almost noeffect on differentiating cells and adiponectin production. Conclusions: Different NRTIs affect mtDNA content ofadipose tissue and its function through different mecha-nisms depending on the developmental stage. We provideevidence at the cellular level that d4T-induces mitochon-drial DNA depletion and that this depletion is associatedwith decrease in adiponectin production.


Mitochondrial dysfunction of HAART-relatedhyperlactataemia is demonstrable by non-invasivestudies

G Garrabou1, S López1, E Sanjurjo1, A Infante1,J Riba2, J Casademont1, F Cardellach1 and Ò Miró1

1 Mitochondrial Research Laboratory, Muscle Research Unit,Internal Medicine Department, IDIBAPS, University of Barcelona,Hospital Clinic of Barcelona, Barcelona, Catalonia, Spain; and 2Department of Orthopedic Surgery and Traumatology (ICMEQ),Hospital Clinic of Barcelona, Barcelona, Catalonia, Spain

Objectives: Hyperlactataemia is one of the most seriousadverse effects of highly active antiretroviral therapy(HAART). Mitochondrial DNA (mtDNA) depletion andmitochondrial respiratory chain (MRC) dysfunction isdemonstrable in liver or skeletal muscle, but they consti-tute invasive methods with limited clinical use. We describeherein a non-invasive technique to detect mitochondrialdysfunction in HAART-related hyperlactataemia.Methods: We have developed the forearm aerobic exercisetest (FAET) to measure oxygen saturation in forearmvenous blood before, during and after aerobic exercise.Lactate measurements before and after FAET assureabsence of anaerobic effort. We have performed FAET inasymptomatic HIV controls and in an HIV-infectedpatient undergoing HAART-related hyperlactataemia and6 months after resolution. Simultaneously we measured

mtDNA content by quantitative real-time PCR and enzy-matic MRC activities by spectrophotometry in biopsiedskeletal muscle homogenate. Results: The symptomatic hyperlactataemic patient has amtDNA content of 47% with respect to control values(100%), which increased to 110% 6 months after recov-ery. MRC activities of mtDNA encoded complexes I, III,IV and V were diminished during the acute episode [39%,61%, 64% and 45%, respectively, compared with con-trols (100%)] and returned to normal 6 months after-wards. The FAET performed on the hyperlactataemiccrisis showed a decrease of 46.44% in the mean venoushaemoglobin saturation during aerobic exercise withrespect to control values (100%), and this abnormal oxy-gen usage disappeared 6 months later.Conclusion: Mitochondrial functionality analysis and theFAET match clinical manifestations in an HIV patientundergoing symptomatic HAART-related hyperlac-tataemia and after clinical recovery. The FAET could be anon-invasive useful tool for the screening of mitochondri-al abnormal function and reduced oxygen consumption. Acknowledgements: Study supported by Fundación parala Investigación y la Prevención del SIDA en España(FIPSE 3102/00), Fundació la Marató de TV3 (02/0210),Redes de Investigación en Mitocondrias (V2003-REDC06E-0) y SIDA (Redg 173) and Suports a Grups deRecerca (2001/SGR/00379).


Platelet contamination affects mitochondrial DNA(mtDNA) levels significantly in peripheral bloodmononuclear cells from HIV patients

M Gerschenson, J Lloyd, DE LiButti, D Tran, B Shiramizu, L Day, M Marcelo and CM Shikuma

Hawaii AIDS Clinical Research Program, John A Burns School ofMedicine, University of Hawaii, Honolulu, HI, USA

Objectives: MtDNA copies/cell in peripheral bloodmononuclear cells (PBMCs) have been suggested as apotential marker for HIV lipoatrophy. Isolation ofPBMCs coincides with platelet contamination. Sinceplatelets contain mtDNA and no nuclear DNA, this couldskew results that would make inter- and intrastudy com-parisons difficult. In this study, mtDNA copies/cell werequantitated in PBMCs from HIV+ and HIV– patients withand without platelet contamination. Platelet removal wasverified by flow cytometry. Methods: PBMCs were isolated from blood of three HIVseronegative and three seropositive patients using a stan-dard Ficoll gradient protocol. Cells from each individualwere assayed fresh or viably frozen. Cells were labelledwith CD61 platelet-specific magnetic microbeads for



chromatographic removal of the platelets using Miltenyiauto MACS magnetic column separation. The elute(platelet and PBMCs) and flow through (PBMCs) frac-tions were evaluated by flow cytometry using CD61 PE(platelet-specific) and CD45 FITC (PBMC-specific) label-ing. MtDNA copies/cell were quantitated by real-timePCR pre- and post-chromatography on all cell fractions. Results: Platelet contamination varied significantly in Ficoll-isolated PBMCs. Seronegative and seropositive PBMCs hadan average 20% platelet contamination, ranging from3–33%. Chromatography reduced platelet contamination to5% in the seronegative and 10% in the seropositive PBMCs.MtDNA copies/cell were decreased in the chromatographi-cally isolated frozen HIV+ PBMCs (209 + 64) comparedwith the HIV– PBMCs (357 + 40), P=0.028. Conclusion: MtDNA quantification from PBMCs isolatedusing Ficoll separation may be affected by platelet conta-mination and this can be as much as 20%. Plateletremoval by magnetic chromatographic separation canminimize the variability caused by contamination.

This research was supported by National Institutes ofHealth, USA (RR16467 and MD000173).


Do statins enhance HIV therapy-induced mitochondrial toxicity?

MP de Baar1, KGM Smolders1, M Buitelaar1, JT Dekker1 and AH Klerkx2

1 Primagen, Amsterdam, The Netherlands; and 2 Department ofExperimental Vascular Medicine, Academic Medical Centre,Amsterdam, The Netherlands

Antiretroviral therapy causes mitochondrial toxicity-related adverse events, as well as dyslipidaemia with lowerHDL-cholesterol and higher LDL and total cholesterollevels as a result. This dyslipidaemia is often treated withstatins to reduce the total and LDL-cholesterol levels.Several studies have shown that statins also affect themitochondria both morphologically and physiologically,occasionally resulting in mitochondrial dysfunction. Weinvestigated the effects of statin treatment on mtDNA lev-els in PBMC, using the Retina Mitox assay that quantifiesthe mitochondrial and nuclear DNA ratio in one tube. Agroup of seven individuals with elevated cholesterol levelswere treated for 2 months with 40 mg simvastatin daily,whereas a control group of six individuals with elevatedtriglyceride levels were treated with 100 mg ciprofibratedaily. Samples were taken at day 0 and after 2 months andanalysed for mtDNA content in a blinded way. There wereno significant changes in mtDNA over time in the controlgroup on ciprofibrate (P=0.292), whereas the group onsimvastatin showed a significant increase from 361 to 429

mean copies mtDNA per cell (P=0.032). Statins inhibitHMG-CoA reductase, thereby reducing the synthesis ofcholesterol, as well as intermediates in the cholesterolbiosynthesis pathway like farnesyl pyrophosphate, whichalso acts as precursor for coenzyme Q10 (Co-Q10). Co-Q10 plays an important role in the electron transport ofthe oxidative phosphorylation in mitochondria and deple-tion might lead to disorders in the energy metabolism.Our first hypothesis is that depletion of Co-Q10 results inincreased levels of mtDNA to facilitate an increase ofmRNA encoding for proteins, such as the COX family ofproteins, involved in the oxidative phosphorylation.

Furthermore, cells were cultured in the presence ofnucleoside RT inhibitors (ddC, ddI, d4T), simvastatin,ciprofibrate and combinations thereof. We observed highertoxicity on cells that were exposed to nucleoside analoguesin combination with statins relative to any of the other cul-tures. Since treatment with nucleoside RT inhibitors canresult in mitochondrial toxicity-related adverse events, oursecond hypothesis is that this mitochondrial toxicity mightbe worsened by the treatment with statins due to a com-bined toxic effect on the mitochondria.


Effects of antiretroviral therapy (ART) on liverenzymes of Brazilian children with AIDS

AC Montes-Gil1, R Lorenzetti1, G Bergsten-Mendes1

and MMS Vilela2

1 Pharmacology Department; and 2 Pediatric Department, StateUniversity of Campinas Medical School, SP, Brazil

Objective: This study was aimed at determining the fre-quency of asymptomatic or symptomatic hepatic enzymeelevations in a cohort of HIV-positive children receivingART in an ambulatory setting and to associate it withpotential causative factors.Methods: Medical records of 152 children receiving ART,seen at Hospital das Clinicas between March 2003 andApril 2004 were prospectively analysed for a median dura-tion of 9.8 months (range 4–12), with a median number ofrepeat hepatic enzyme determinations of 2.6 (range 1–5)per patient. Of those children, 45.4% were girls and54.6% were boys. Their ages ranged between 0.6–18.2years (mean 7.48); 97.4% children acquired HIV infectionfrom vertical transmission and 2.6% from blood transfu-sion. Laboratory monitoring included aspartate amino-transferase (AST), alanine aminotransferase (ALT),alkaline phosphatase (AP) and gammaglutamyl transpepti-dase (GGT). Abnormal hepatic enzymes values were con-sidered when levels rose above the upper limits of normal(ULN). The scoring system was: grade 1 (1.1–4.9 × ULN)grade 2 (5.0–9.9 × ULN) and grade 3 (10.0–15.0 × ULN).

Abstracts


Results: 31 patients (20.4%) had augmented hepaticenzyme values on at least two determinations (AST:61.3%, ALT 45.2%, GGT 61.3%, PA: 48.4%). Of these,seven patients had hepatosplenomegaly with hepaticsteatosis. Only one patient had serological markers forhepatitis C virus. Hepatic disorders were related to: histo-plasmosis (one case), malnutrition recovery (one case),and co-administration of antituberculosis (ATB) drugs(six cases, 19.4%). A total of 24 patients (15.8%) were onregimens with two drugs, 115 (75.7%) were on three-drug regimens and 13 (8.6%) on four-drug regimens.Didanosine was the ARV related to most of the hepaticalterations in 83.7% of cases.Discussion: Our results suggest that ambulatory HIV posi-tive children receiving ART can display elevations of hepat-ic enzymes without associated evidence of the clinicaldiagnosis of hepatotoxicity. We observed mild to moderate,grade 1, usually no more than four times the upper limit ofnormal, asymptomatic elevations of hepatic enzymes in ourpatients. Our findings point to a relationship betweenhepatic enzyme elevations and stage C3 of HIV disease,and use of antiretroviral regimens containing four drugsand co-administration of antituberculosis drugs.


Assessment of the liver function in HIV-infectedpatients treated with antiretroviral drugs inCameroon

O Lowe and G Lando

University of Yaounde I, Yaounde, Cameroon

Objective: This work is aimed at assessing the effects ofantiretroviral drugs used in the management of the liverfunction of HIV-infected Cameroonian patients.Methods: An HIV-infected and non-infected cohort wasretrospectively examined. After consent was given, clinicaldata were recorded and blood samples were obtained forHIV antibody testing; HIV infection was confirmed byWestern blotting. CD4+ counts were performed in all HIVseropositive patients by flow cytometry. Treated patientswere stratified into groups according to their antiretrovi-ral therapy. Four hepatic markers [liver enzymes (GOT,GPT and ALP) and bilirubin] were assessed for all thepatients.Results: Of 110 subjects, 45 were HIV positive and treat-ed, 40 were HIV positive and not treated and 25 wereHIV negative (blood donors). We observed a significantincrease (P<0.05) of these liver markers in all HIV-infect-ed patients compared with the healthy control; all theparameters except ALP were significantly higher (P<0.05)in ARV-treated than in untreated patients. Regressionanalysis identified CD4 T cell counts <200, CDC stage C

and antiretroviral treatment as independent factors ofliver deterioration (GOT activity and bilirubin level weresignificantly higher (P<0.05) in ARV-treated than inuntreated patients). Age and sex can also be considered asrisk factors. Severe drug-related hepatotoxicity was seenin five (31.5%) patients treated with two nucleoside andone non-nucleoside analogues and two (10.53%) patientstreated with two nucleoside analogues and one proteaseinhibitor.Conclusion: HIV infection significantly increase livermarkers; this appear to be higher in treated patients. Oneof the parameters (GOT) appear to be a marker of sur-vival. The combinations used, in particular tritherapy, arehepatotoxic. Close monitoring of HIV-infectedCameroonian patients treated with antiretroviral drugs isrequired to improve their survival.


Adipocyte viability and function but not inhibitionof preadipocyte differentiation is compromised byindinavir



Objective: Atrophy of peripheral subcutaneous adiposetissue is a common side effect observed in many HIV-infected patients treated with combination antiretroviraltherapy. Several HIV protease inhibitors were found eitherto inhibit preadipocyte differentiation or to promoteadipocyte cell death. We aimed to investigate the effects ofHIV protease inhibitor indinavir (IDV) on adipogenesisand adipocyte survival using the 3T3-L1 preadipocyte cellline.Methods: Transcription profiles of lipoprotein lipase(LPL), the adipogenic transcription factors CCAAT/enhancer-binding protein α, CCAAT/enhancer-bindingprotein β and peroxisome proliferator-activated receptorγ as well as neuronal apoptosis inhibitor protein andadiponectin were investigated using real-time PCR.Adiponectin production was also determined by ELISA.Cytoplasmic triacylglycerol accumulation was measuredusing Oil Red O staining. Cell death apoptosis/necrosiswas asessed with Hoechst/propidium iodide staining andtrypan blue exclusion. Results: When induced to differentiate in the presence ofIDV (up to 100 µM), 3T3-L1 preadipocytes accumulatednormal levels of cytoplasmic triacylglycerol. In addition,they expressed normal levels of LPL, the adipogenic tran-scription factors CCAAT/enhancer-binding protein α,CCAAT/enhancer-binding protein β and peroxisomeproliferator-activated receptor γ. We were unable to find



any inhibitory effect of IDV on critical early events inpreadipocyte differentiation and completion of the mitoticclonal expansion phase. Adipocyte function, however, wasimpaired by IDV as judged by the expression ofadiponectin. IDV treatment of fully differentiated 3T3-L1adipocytes resulted in loss of cell viability and decreasedexpression of neuronal apoptosis inhibitor protein(NAIP). In contrast, cell proliferation and viability ofpreadipocytes were unaffected by IDV treatment. Conclusions: Molecular or cellular changes that occurduring acquisition of the adipocyte phenotype promotesusceptibility to IDV-induced cell death. We provide evi-dence for a direct inhibition of adiponectin production byIDV. These results suggest that IDV may promote adiposetissue atrophy by directly compromising adipocyte func-tion and not by inhibition of preadipocyte differentiation.


Nevirapine did not alter cell differentiation, lipidmetabolism, insulin response and survival incultured adipocytes

M Caron1, M Auclair1, M Kornprobst1, C Lagathu1

and J Capeau1,2

1 INSERM U402, Faculty of Medecine Saint-Antoine, Paris,France; and 2 Hôpital Tenon, Paris, France

Objectives/aim: Drug-associated adipocyte dysfunctionplays a role in the aetiology of the adverse symptoms thatoccur in HIV-infected patients treated with antiretrovirals.Protease inhibitors, nucleoside reverse transcriptaseinhibitors (NRTIs) and more recently the non-NRTIefavirenz were shown to affect adipocyte functions invitro. Thus nevirapine, the other NNRTI used with suc-cess in switching studies, was evaluated in vitro for itspotential to alter adipocyte functions.Methods: 3T3-F442A cells were treated with nevirapine(4–50 µM) all along the differentiation process.Differentiation was estimated at day 7 by the percentageof cells with lipid droplets and the protein expression ofadipocyte differentiation markers : SREBP-1, PPARγ andC/EBPα. Lipid metabolism was evaluated by lipid stain-ing, mRNA expression of fatty acid synthase (FAS) andadipocyte lipid binding protein 2 (aP2), and insulin acti-vation of lipogenesis. Insulin response was evaluated bythe insulin-induced tyrosine phosphorylation of theinsulin receptor β-subunit and IRS-1, and the activation ofERK 1/2 and Akt/PKB. Apoptosis was estimated by flowcytometry, and cytotoxicity by MTT lysis. Results: In the therapeutic range (4–25 µM), NVP did notalter adipose cell proliferation and differentiation. Indeedthe number of cells with lipid droplets (92–95% of totalcells), lipid staining, protein expression of SREBP-1,

PPARγ and C/EBPα, expression of FAS, PPARγ and aP2were unchanged. At higher concentrations (50–100 µM),NVP decreased lipid staining and expression of adi-pogenic markers. Cell response to insulin was not alteredby NVP up to 50 µM: insulin (100 nM) almost normallyincreased the insulin receptor β-subunit and IRS-1 tyro-sine phosphorylation and promoted ERK 1/2 andAkt/PKB activation. Insulin-induced lipogenesis was notaffected by NVP up to 10 µM. Up to 50 µM, NVP did notpromote cell toxicity and apoptosis (1–3% of cells in sub-G1). At higher concentrations (50–100 µM), NVPinduced toxicity and apoptosis.Conclusions/discussion: Thus, in the therapeutic range(4–25 µM), nevirapine had no effect on the main adiposecell functions measured . These data are of interest sinceNVP-containing regimens are often used as a switchingoption in patients treated with PI-containing regimens.Acknowledgments: This work was supported by grantsfrom INSERM and ANRS.


PIs and NRTIs with adverse effects on adipocytelipid metabolism and survival alter the productionof pro-inflammatory cytokines and adiponectin inadipocytes

C Lagathu1, JP Bastard1,2, M Auclair1, M Maachi1,2,M Kornprobst1, J Capeau1,2 and M Caron1

1 INSERM U.402 and IFR65 Saint-Antoine Faculty of Medicine,University Pierre and Marie Curie, Paris, France; and2 Biochemistry Department Tenon Hospital, Paris, France

Objectives: The lipodystrophic syndrome is a majoradverse effect of highly active antiretroviral therapy(HAART), associated with altered circulating levels andadipose tissue mRNA expression of pro-inflammatorycytokines (IL-6 and TNF-alpha) and adiponectin.Proinflammatory cytokines and adiponectin, which aresecreted by adipose tissue, regulate fat metabolism, insulinsensitivity and adipose cell apoptosis. We examined thedirect effects of individual antiretrovirals on lipid metab-olism and cytokine and adiponectin production by cul-tured adipocytes Methods: Differentiating 3T3-F442A cells and differenti-ated 3T3-L1 adipocytes were treated for 12 or 4 days,respectively, with protease inhibitors (PIs) indinavir, nelfi-navir, amprenavir, lopinavir and ritonavir, or nucleosidereverse transcriptase inhibitors (NRTIs) stavudine andzidovudine, at near-Cmax concentrations. Lipid metabo-lism was estimated by Oil Red O staining of intracellularlipids, mRNA expression of fatty acid synthase andadipocyte lipid binding protein 2, and insulin activation oflipogenesis. Apoptosis was estimated by flow cytometry.

Abstracts


The expression and secretion of pro-inflammatorycytokines (IL-6, TNF-alpha and IL-1-beta) and adiponectinwas evaluated by real-time RT-PCR and ELISA. Results: Chronic treatment of 3T3-F442A differentiatingadipocytes and differentiated 3T3-L1 adipocytes with PIsand NRTIs reduced lipid accumulation, mRNA expres-sion of lipid markers and insulin-induced lipogenesis. IL-6, TNF-apha, IL-1-beta and adiponectin expression andsecretion were markedly altered in differentiating 3T3-F442A adipocytes. PIs had either no effect on differentiat-ed 3T3-L1 adipocytes (TNF-alpha expression andsecretion) or their effect was less marked than in 3T3-F442A cells. Indinavir and amprenavir did not altercytokine or adiponectin secretion and expression bymature adipocytes. The effects of stavudine and zidovu-dine on differentiating and mature adipocytes were simi-lar, despite the difference of treatment procedure. Thedrugs with the strongest effect on TNF-alpha expressionalso increased adipocyte apoptosis, contrary to the drugsthat only moderately increased TNF-alpha expression. Conclusions: These results suggest that increased cytokineand decreased adiponectin secretion and expression inducedby some PIs and NRTIs may contribute to the adipose tissueloss (via apoptosis and lipid leakage) and the insulin resis-tance associated with the lipodystrophy syndrome.


Comparative effects of atazanavir alone and incombination with low concentration of ritonaviron triglyceride and cholesterol synthesis in vitro

MA Noor1,2, R Mulvey1, C Elosua1, F Wang1, RA Parker1 and OP Flint1

1 Pharmaceutical Research Institute, Princeton, NJ, USA; and2 Virology Medical Affairs, Plainsboro, NJ, USA

Objective: Treatment with some protease inhibitors (PIs)is associated with dyslipidaemia. Proposed mechanismsinclude effects on lipid and lipoprotein production inhepatocytes and adipocytes. Atazanavir is a potent once-daily PI that in combination with low-dose ritonavirshowed comparable clinical efficacy to lopinavir/ritonavirin treatment-experienced patients. Compared with riton-avir and lopinavir, atazanavir is associated with less or nodyslipidaemia clinically and less effect on lipogenesis invitro. We hypothesized whether combining atazanavirwith ritonavir at low concentration (≤2 µM) would negatethe favourable in vitro effect of atazanavir on lipogenesisreported previously.Methods: Lipogenesis was assayed as [14C]2-acetateincorporation into triglyceride and cholesterol in humanprimary adipocytes and HepG2 hepatoma cells.Triglyceride and cholesterol synthesis were quantified in

triplicates in the presence of atazanavir and lopinaviralone or in combination with ritonavir at various dosesapproximating therapeutic and boosted PI levels expectedin vivo. Comparisons are by t-test versus control.Results: As a single drug at boosted-equivalent concentra-tion (≤2 µM), ritonavir did not affect lipogenesis in eithercell line. At 3 µM, ritonavir modestly inhibited (15–30%)triglyceride and cholesterol synthesis in adipocytes (P<0.05for both). At therapeutic concentration (10 µM), ritonavirinhibited triglyceride synthesis in adipocytes and increasedtriglyceride synthesis in HepG2 cells by 50% (P<0.01).Atazanavir as a single drug up to 10 µM or in combinationwith ritonavir (≤2 µM) had little effect on triglyceride orcholesterol synthesis in HepG2 or adipocytes (<15%effect, P>0.2). In contrast, lopinavir as a single drug at 10µM increased (~50%) triglyceride synthesis in HepG2, andinhibited synthesis of triglycerides (~30%) and cholesterol(~50%) in adipocytes (P<0.01 for all). Combination oflopinavir and ritonavir (≤2 µM) had no additive effect.Conclusions: Atazanavir as a single drug at 10 µM or com-bined with ritonavir up to 2 µM has very little effect onlipogenesis, whereas lopinavir as a single drug at 10 µM or combined with ritonavir up to 2 µM affects lipo-genesis. The data are consistent with the current hypothe-sis that ritonavir-boosted atazanavir used clinically willmaintain the favourable lipid profile of unboostedatazanavir. Confirmatory clinical data are needed.


Effects of HAART on resistin and perilipin mRNAexpression in mouse 3T3-L1 and primary humanadipocytes

PV Nerurkar1, L Pearson1, JK Cope1, K Adeli2, J Frank1 and VR Nerurkar1

1 University of Hawaii at Manoa, Honolulu, HI, USA; and2 Hospital for Sick Children, University of Toronto, Toronto, Ont.,Canada

Efficacy of HAART is complicated due to various side effectsincluding decrease in subcutaneous fat (lipoatrophy),increased visceral fat and insulin resistance. Mechanisticstudies implicate decrease in adipose transcription factorssuch as peroxisome proliferator-activated receptor γ (PPARγ)and mitochondrial toxicity-associated apoptosis to beinvolved in lipoatrophy, while increased visceral fat couldarise from increased adipogenesis. PPARγ is involved in reg-ulating the expression of adipocytokine, resistin and lipiddroplet specific protein, perilipin, implicated in adipocytedifferentiation and obesity-associated insulin resistance. Theaim of our study was to test the combined effects of proteaseinhibitors (PIs) such as ritonavir (RTV), lopinavir (LPV/r) andnucleoside analogue reverse transcriptase inhibitors (NRTIs)



Abstracts

such as combivir (CBV, containing zidovudine + lamivudine)on the release of caspase-3 as a measure of apoptosis, pro-duction of reactive oxygen species (ROS) and mRNA expres-sion of PPARγ-regulated genes, perilipin and resistin in3T3-L1 mouse and primary human subcutaneous pre-adipocytes. Our data indicate that RTV, LPV and CBV, eithersingly or in combination, significantly increase adipocyte dif-ferentiation in mouse and primary human pre-adipocytes, asmeasured by Oil Red staining and cellular triglycerides levels(P<0.05), with a concomitant increase in both resistin andperilipin mRNA expression (P<0.05). Treatment with PIand/or NRTI had no effect on either ROS production orapoptosis in both cell types. Current strategies to manageHAART-associated metabolic complications include PPARγagonists such as rosiglitazone that increase insulin sensitivityand ameliorate lipoatrophy by increasing adipocyte differen-tiation. Based on our in vitro data, it would therefore appearthat PPARγ agonists may not be suitable for treating HIV-infected patients on HAART regimens that specificallyincrease adipocyte differentiation. Understanding the under-lying pathophysiological mechanisms can assist in identifyingnew therapeutic molecular targets to treat HAART-associat-ed metabolic disorders and/or newer drug design.

Supported by NCRR, NIH (G12RR003061 andP20RR011091) and the Hawaii Community Foundation(20012061) grants.


Interactions between T lymphocytes and pre-adipocytes increase HIV production and apoptosisof lymphocytes and block adipocyte differentiation

DE Lewis1, D Ng-Tang1, H Mersmann2 and A Balasubramanyam3

1 Department of Immunology, Baylor College of Medicine,Houston, Tex., USA; 2 Department of Medicine, Baylor College ofMedicine, Houston, Tex., USA; and 3 USDA-Children's NutritionResearch Center, Baylor College of Medicine, Houston, Tex., USA

Background: Adipocyte depots are associated with lymphnode aggregates, hence distinct functional interactionsmay occur between activated lymphocytes and adipocytes.We investigated the potential role of such interactions inthe pathogenesis of HIV lipodystrophy. Methods: Primary human pre-adipocytes (stromal vascu-lar cells) or 3T3-L1 mouse pre-adipocytes were incubatedwith uninfected or HIV-infected lymphocytes for varyingdurations, with or without direct contact, and HIV pro-duction, cell cycle characteristics, apoptosis and biochem-ical functions were measured. Results: Exposure of acutely HIV-infected T lymphocytesto human pre-adipocytes led to markedly increased HIV-1production in the lymphocytes. Chronically HIV-infected

lymphocytes exposed to pre-adipocytes underwent both ablock in G2/M of the cell cycle and an increase in apop-tosis. These effects were observed without direct contactbetween the pre-adipocytes and HIV-infected lympho-cytes, as well as after exposure to conditioned media, indi-cating that the effects were mediated by soluble, secretedfactors. There was no change in the viability of uninfect-ed lymphocytes exposed to pre-adipocytes.

After treatment with standard adipocyte differentiationfactors, pre-adipocytes experienced a reversible block indifferentiation when pre-exposed to lymphocytes, asdemonstrated by a reduction in Oil Red O staining andquantitative adipocyte gene expression. These effects wereobserved in both human and murine pre-adipocytes, indi-cating that they were not due to direct HIV-1 infection.The same results were observed with three differenthuman T lymphocyte lines, but not with two humanmacrophage lines.Conclusions: Significant interactions between pre-adipocytes and HIV-infected lymphocytes increase HIV-1production, G2/M arrest of the cell cycle, as well as apop-tosis in lymphocytes, and cause a marked differentiationblock in pre-adipocytes. The interactions are mediated bysoluble factors – potentially cytokines, adipokines or HIV-1Vpr. Identifying these factors and specifying mechanismsof action are important for understanding the pathophys-iology of HIV lipodystrophy.


Body composition changes in treatment-experienced HIV-infected patients initiating atenofovir-containing antiretroviral regimen

G Tsekes1, N Mangafas1, N Tsogas1, C Psathas1, I Vlachadami1, N Batakis2 and MC Lazanas1

1 3rd Department of Internal Medicine and Infectious DiseasesUnit, Red Cross Hospital, Athens, Greece; and 2 Department ofRadiology, Red Cross Hospital, Athens, Greece

Background: Lipodystrophy has emerged as a commonand disturbing problem among HIV-infected patients.Tenofovir disoproxil fumarate is a nucleotide analoguereverse transcriptase inhibitor recently introduced in thetreatment of HIV infection. The long-term effects of thisdrug on body composition have not been clarified. Objective: To detect body composition changes in a groupof treatment-experienced HIV-infected patients before andafter 48 weeks of treatment with a tenofovir-containingantiretroviral regimen. Methods: Seventeen treatment-experienced HIV-infectedpatients (11 males, six females; age 43.7 ±6.2 years) wereincluded in the study. Their antiretroviral regimen waschanged to tenofovir + nucleoside reverse transcriptase



inhibitor + either protease inhibitor (eight patients) ornon-nucleoside reverse transcriptase inhibitor (ninepatients), because of virological failure, lipodystrophy ormetabolic disturbances. No patient was placed on stavu-dine. Patients’ body composition was assessed at baselineand week 48 by dual energy X-ray absorptiometry(DEXA). Bone mineral content (BMC), fat (FAT) and leanbody mass (LEAN) were evaluated for whole body as wellas regionally (arms, legs and trunk). Statistical analysisinvolved Student’s t-test for paired samples for compari-son of means. Statistical significance was set at 0.05.Results are presented as means ±SD. Results: 1) No significant changes in body weight(BW1=70.8 ±16.5 kg, BW2=72.7 ±19.4 kg; P=0.072), leanbody mass (LEAN1=51.6 ±11.2 kg, LEAN2=51.3 ±11.3 kg;P=0.224) and bone mineral content (BMC1=2668 ±427 g,BMC2=2665 ±456 g; P=0.827) were observed between thetwo assessments. 2) Total fat mass significantly increased(FAT1=16.5 ±9.4 kg, FAT2=18.8 ±10.9 kg; P=0.026).3) Regional analysis showed a statistically significantincrease in both limb and trunk FAT (limb FAT1=5.3±3.9 kg, limb FAT2=6.2 ±4.2 kg; P=0.045 – trunkFAT1=10.7 ±5.9 kg, trunk FAT2=12.1±7.1 kg; P=0.025).4) Serum cholesterol significantly decreased (CHOL1=240±68 mg/dl, CHOL2=217 ±53 mg/dl; P=0.038) and high-density lipoprotein (HDL) cholesterol significantly increased(HDL1=44 ±13 mg/dl, HDL2=47 ±15 mg/dl; P=0.047),while serum triglycerides did not change significantly(TRIGL1=318 ±362 mg/dl, TRIGL2=235 ±229 mg/dl;P=0.098). 5) CD4+ T-cell count remained stable(CD4+

1=486 ±256 cells/µl, CD4+2=474 ±237 cells/µl;

P=0.667) and no patient demonstrated virological reboundduring follow-up. Conclusions: In a group of treatment-experienced HIV-infected individuals, switching to tenofovir-containingantiretroviral regimens resulted in some improvement inlipodystrophy and hypercholesterolaemia, without loss ofvirological control after 48 weeks of treatment.


Reversibility of lipoatrophy associated with highlyactive antiretroviral therapy 1 year afterswitching from stavudine to tenofovir disoproxilfumarate

G Tsekes1, N Tsogas1, N Mangafas1, D Koukios1, C Michalakeas1, A Feretis2 and MC Lazanas1

1 3rd Department of Internal Medicine and Infectious DiseasesUnit, Red Cross Hospital, Athens, Greece; and 2 Department ofRadiology, Red Cross Hospital, Athens, Greece

Background: Long-term use of stavudine in HIV-infectedpatients has been implicated in the development of lipoat-

rophy in all body regions. Switching to non-thymidine ana-logues has shown modest reversal of lipoatrophy, oftenwith unacceptable rates of HIV virological rebound. Theeffect of switching stavudine to tenofovir on lipoatrophyhas not yet been investigated. Objective: To determine if switching from stavudine totenofovir disoproxil fumarate could result in reversibilityof lipoatrophy in a group of HIV-infected patients withclinically evident lipodystrophy. Methods: Ten HIV-infected patients (seven males, threefemales; age 42.2 ±7.3 years) with lipodystrophy wereincluded in the study. All patients were receiving a stavu-dine-containing antiretroviral regimen for at least 1 year.Seven patients were also receiving a protease inhibitor andanother nucleoside analogue; three patients were alsoreceiving a non-nucleoside reverse transcriptase inhibitorand lamivudine. In all 10 patients, stavudine was replacedby tenofovir, without other medication changes. Thepatients were assessed at baseline and week 52 by dualenergy X-ray absorptiometry (DEXA). Bone mineral con-tent (BMC), fat (FAT) and lean body mass (LEAN) wereevaluated for whole body as well as regionally (arms, legsand trunk). Statistical analysis involved Student’s t-test forpaired samples for comparison of means. Statistical signifi-cance was set at 0.05. Results are presented as means ±SD.Results: Patients showed a significant increase in their bodyweight (BW1=68.2 ±9.9 kg, BW2=71.7 ±13.0 kg; P=0.011).Total FAT significantly increased (FAT1=12.4 ±6.5 kg,FAT2=16.1 ±8.0 kg; P=0.004 – %FAT1=17.79 ±9.12%,%FAT2=21.63 ±9.82%; P=0.004), while LEAN was notaffected (LEAN1=53.0 ±8.6 kg, LEAN2=52.9 ±9.0 kg;P=0.741). Fat gain was statistically significant in the armsand trunk, but not in the legs (arms FAT1=1.2 ±1.0 kg, armsFAT2=1.7 ±1.3 kg; P=0.027 – trunk FAT1=8.5 ±4.1 kg,trunk FAT2=11.0 ±5.3 kg; P=0.002 – legs FAT1=2.2 ±1.9kg, legs FAT2=2.9 ±1.7 kg; P=0.086). The whole bodyBMC remained stable between the two assessments(BMC1=2814 ±407 g, BMC2=2822 ±434 g; P=0.710).CD4+ T-cell count remained stable (CD4+

1=525 ±290cells/µl, CD4+

2=524 ±280 cells/µl; P=0.988) and no patientdemonstrated virological rebound during follow-up. Conclusions: In a small group of HIV-infected individualswith lipoatrophy, switching from stavudine to tenofovirresulted in significant improvement of lipoatrophy withoutloss of virological control after 52 weeks of treatment.


Abstracts


Body composition changes in treatment-experienced HIV-infected patients: 10 years of follow-up

G Tsekes1, N Mangafas1, N Tsogas1, I Salpigktis1, N Batakis2, V Georgiou3 and MC Lazanas1

1 3rd Department of Internal Medicine and Infectious DiseasesUnit, Red Cross Hospital, Athens, Greece; 2 Department ofRadiology, Red Cross Hospital, Athens, Greece; and 3Department of Medical Physics, Medical School, University ofAthens, Greece

Background: The extensive use of highly active antiretro-viral therapy (HAART) for the treatment of HIV-infectedpatients has been implicated in the development of lipody-strophy. However, there are insufficient data regarding thebody composition changes of HIV-infected individuals onlong-term HAART. Objective: To determine body composition changes in agroup of HIV-infected patients followed longitudinally (for10 years). Methods: Twenty-one treatment-experienced HIV-infectedpatients (18 males, three females; age at first assessment 41.0 ±6.3 years) were included in the study. Body compo-sition analysis was performed by dual energy X-rayabsorptiometry (DEXA) at baseline (1994) and 10 yearslater (2004). For each patient studied, fat (FAT) and leanbody mass (LEAN) were evaluated for whole body as wellas regionally (arms, legs and trunk). Statistical analysisinvolved Student’s t-test for paired samples for comparisonof means. Statistical significance was set at 0.05. Resultsare presented as means ±SD. Results: Patients exhibited a significant decrease in their bodyweight (BW1=74.0 ±9.1 kg, BW2=68.3 ±10.8 kg; P=0.001).Weight loss was exclusively due to fat loss (FAT1=19.6±7.3 kg, FAT2=14.1 ±6.4 kg; P=0.001), while lean was notaffected (LEAN1=51.8 ±6.4 kg, LEAN2=51.6 ±6.5 kg;P=0.783). Fat loss was statistically significant in both the arms(arms FAT1=2.7 ±1.1 kg, arms FAT2=1.3 ±0.9 kg; P<0.001)and the legs (legs FAT1=6.9 ±2.7 kg, legs FAT2=2.6 ±1.9 kg;P<0.001); trunk fat, however, did not change significantly(trunk FAT1=9.0 ±4.1 kg, trunk FAT2=9.3 ±3.8 kg; P=0.757).The trunk/limb fat ratio exhibited a significant increasebetween the two assessments (trunk/limb FAT1=0.94 ±0.39,trunk/limb FAT2=3.07 ±1.31, P<0.001). Significant increasein both serum cholesterol and triglycerides was also evident(CHOL1=175 ±38 mg/dl, CHOL2=218 ±61 mg/dl; P=0.020– TRIGL1=138 ±89 mg/dl, TRIGL2=274 ±231 mg/dl;P=0.032). Lipodystrophy was clinically evident in 71% ofpatients at follow-up.Conclusions: In a group of HIV-infected individuals receiv-ing HAART after the initial double NRTI regimen, a

considerable amount of limb fat was found to be lost in a10-year follow-up. Trunk fat and lean body mass did notchange significantly. The aforementioned morphologicchanges were accompanied by metabolic abnormalities(dyslipidaemia).


Soft tissue augmentation with polymethymetha-crylate (PMMA) for correction of lipodystrophyrelated body fat atrophy

MS Serra1and LK Oyafuso2

1 Clinica Marcio Serra, Rio de Janeiro, Brazil; and 2 Hospital deInfectologia Emilio Ribas, São Paulo, Brazil

Aim: We have been successfully treating facial atrophywith PMMA for the last 6 years, but in the last few yearspatients have started to complain about body shapechanges. Particularly in women, exacerbation of theappearance of superficial veins in arms and legs, and los-ing the shape of the hips have become problems, leadingto a need to change the way they dress. For both genders,the loss of fat in the buttocks can lead to pain if they areseated for a long time and can sometimes expose the anus,leading to problems with clothes again. Fat transplant isan excellent option for treating these areas, but not allpatients have a donor area. In these cases we have startedto perform PMMA implants for treatment of body fatloss-related lipodystrophy.Methods: Body atrophic areas were treated with a PMMAsolution containing methylcellulose and lidocaine.Treatment consisted of parallel and net-crossed retro-injections in the subcutaneous area of the PMMA solutionin three different concentrations: 30% for buttocks, upperlegs and around the knees; 30 or 20% for lower legs; and10% for arms and hands. A 5-day course of antibioticsand anti-inflammatory medications was prescribed foreach session, starting on day 1, right after the procedure.Patients were photographed and followed-up every 45days. Sessions were performed with a minimum intervalof 10–12 weeks for each treated area. Results: Twenty-two patients were included in this study,10 men and 12 women. Twenty-seven areas were treated:14 buttock areas, nine legs, three arms and one pubis andperineum area.

Buttocks were treated with 40 ml of PMMA solutionon each session, upper and lower legs with around20–30 ml, and arms and hands with 10 ml.

Patients needed, in median, two to three sessions toachieve good cosmetic results. Some patients reportedexcellent results in hiding the appearance of arm and legveins. Good improvement of the overall appearance ofarms and legs after filling the depressions between the



muscles was observed, and improvement of the expositionof the anus. All patients who had implantation on thebuttocks felt more comfortable when being seated for along time. Side effects were oedema and redness of thetreated area, and light to moderate pain that last for 2–3days, which improved with paracetamol. No infections orinflammatory granulloma were observed. Patients weresatisfied with the results of the treatment and reportedfeeling more self confident. Women were satisfied by thefact that they could wear trousers with shorter blousesagain and were able to wear shorter skirts, reportingimprovement in quality of life issues.Conclusion: Use of PMMA solution for treatment of bodycontours in lipodystrophy is shown to be safe and effectivewith good cosmetic results, helping patients to becomemore self confident and improving their quality of life.


The proportion of patients reporting body fatredistribution was unchanged between week 48and week 120 in subjects receivingfosamprenavir/ritonavir in Study APV30005

S Walmsley1, S Staszewski2, J Yeo3, D Thorpe3 andN Givens3

1 Toronto Hospital University Health Network, Toronto, Ont.,Canada; 2 University Clinic Frankfurt/M, Germany; and3 GlaxoSmithKline R&D US and Europe

Background: Few long-term (>48 wks) data of the inci-dence of fat redistribution have been presented fromprospective clinical trials. Methods: 120 wk data for 211 antiretroviral treatment-naive subjects who completed ≥48 wks on a fosampre-navir/ritonavir (FPV/r) 1400 mg/200 mg once daily regimenin SOLO (APV30002) and continued FPV/r once daily inAPV30005 are presented. Body composition changes (BCC)were assessed by physician and patient observation using astandardized questionnaire and anthropometry. Results: The majority of subjects were male (72%) and ofwhite (49%) ethnic origin, although a notable proportionof females (28%) and subjects of black ethnic origin (39%)were enrolled. History of CDC class C disease was 21%.Median baseline CD4 was 168 cells/µl, and median HIV-1RNA was 4.82 log10 copies/ml. A total of 186/211 (82%)retained abacavir/lamivudine as the NRTI backbone inAPV30005. A median increase in body weight of 3 kg atwk 48 (n=201), 4 kg by wk 96 (n=179) and 4 kg by wk 120(n=170) was observed. There was a median increase frombaseline (BL) in waist (hip) circumference of 2 cm (2 cm) atwk 48 (n=196), 3.3 cm (3 cm) at wk 96 (n=174) and3.5 cm (3 cm) at wk 120 (n=167). There was no medianchange in waist/hip ratio from BL over 120 wks (median

change 0.0). Of subjects with no BCC reported at BL,34/180 (19%) reported any fat redistribution at wk 48,32/160 (20%) at wk 96 and 29/156 (19%) at wk 120. Ofsubjects who did not report fat wasting at BL, only 8/188(4%) reported fat wasting at wk 48, 8/165 (5%) at wk 96and 8/161 (5%) at wk 120. Of subjects who did not reportfat accumulation at BL, 32/195 (16.4%) reported signs offat accumulation at wk 48, 32/173 (18.5%) at wk 96 and31/166 (18.7%) at wk 120. Conclusion: Over 120 wks, the majority of BCCs were fataccumulation combined with an increase in body weightbut no change in waist/hip ratio. Of note, the proportionof subjects reporting fat wasting, fat accumulation or anyfat redistribution change did not increase between wk 48and wk 120.


Oxymetholone as therapy to maintain bodycomposition in HIV-positive men

A Urbina, M Miller and I Hance

Saint Vincent Catholic Medical Center, New York, NY, USA

Background: Wasting in HIV patients, which is the loss oflean body mass (LBM), has been shown to predict deathin HIV. While protease inhibitors (PIs) are improvingchances of survival in HIV, studies show that people whoexperience success with PIs regain fat mass, rather thanLBM, when they regain weight. Androgenic/anabolicsteroids (AAS) have been shown to improve LBM in HIVand to reduce the fat redistribution syndrome associatedwith PI therapy.Methods: Open-label, 24-week pilot study of up to 20HIV-positive subjects who had a past or current Dx ofwasting (greater than 5% weight loss), and were pre-treated with either nandrolone decanoate or oxandrolonefor at least 12 weeks to stabilize weight. They were alsorequired to be on stable antiretroviral therapy for at least30 days and throughout the study. Subjects were switchedto oxymetholone (Oxy) 50 mg once daily, an oral 17-alpha AAS approved for the treatment of anaemia; thedose of Oxy could be increased to 50 mg twice daily.Subjects were evaluated for efficacy and safety at weeks 4,8, 12, 18 and 24.

Clinical effectiveness was defined as either no change orimprovement from BL in body cell mass (BCM) as mea-sured by BIA, body composition, body weight and QOL.Safety was monitored with AEs, labs and vital signs. Theprimary population for analysis was the ITT sample,defined as all subjects who had at least one dose of Oxy andone post-BL measure. Hypothesis testing used two-sidedtests performed at the 0.05 level of significance. Pairedt-tests and Wilcoxon Signed Rank tests were employed.


Abstracts

Results: A total of 16 HIV-positive men were successfullyswitched from prior AS therapy to Oxy. BCM was main-tained over the 24-week period, with a mean increase of2.2 lbs (P=0.091) at endpoint. There was an increase in fatfree mass (FFM) at all weeks, with a significant improve-ment in FFM at endpoint (P=0.027). Lipids decreasedoverall, especially HDL-cholesterol and triglycerides.Bilirubin and AST showed no change over time; alkalinephosphatase decreased and ALT showed a slight increase;overall effects on liver function were not clinically impor-tant. CD4 values increased over time. Testosterone valueswere increased at weeks 18 and 24 (mean increase of 214and 113 ng/dl, respectively), possibly indicating the addedvalue of AAS over anabolic steroids. Conclusions: Oxymetholone 50 mg can be consideredeffective in maintaining lean body mass in HIV-positivepatients, without an adverse impact on CD4 count, liverfunction or vital signs.


Intrathoracic fat in HIV-infected patients

A León, A Biglia, E Martínez, JL Blanco, M Sánchez,E de Lazzari, A Milinkovic, M Larrousse, M Laguno,M Lonca, J Mallolas and JM Gatell

Hospital Clinic, Barcelona, Spain

Background: The impact of HIV infection or antiretrovi-ral therapy on the intrathoracic fat compartment isunknown.Methods: Clinically stable HIV-infected adult patientsirrespective of exposure to antiretroviral therapy and non-HIV-infected healthy volunteers, both without clinical evi-dence of body fat changes consistent with lipodystrophyand adjusted for age and gender, were recruited as casesand controls, respectively. Thoracic and abdominal fatassessed by computed tomography was comparedbetween cases and controls. Results: There were nine (33%) women and 18 (67%) menin each group. Nineteen (70%) cases had been taking anti-retrovirals for a median of 8 months [interquartile range(IQR): 6–11]. There were no significant differences in age,CD4 cells, intrathoracic and subcutaneous thoracic fat,and intra-abdominal and subcutaneous abdominal fatbetween cases without (n=8) and with (n=19) antiretrovi-ral therapy, respectively. Median intrathoracic fat area was6.8 (IQR: 5.6–10.5) cm2 in cases and 5.6 (IQR: 3.9–6.7)cm2 in controls (P=0.025). Median subcutaneous thoracicfat area was 41.4 (IQR: 15.1–79.2) cm2 in cases and 102.5(IQR: 56.3–122.0) cm2 in controls (P=0.001, WilcoxonRank Sum test). Median intra-abdominal fat area was 41.1(IQR: 13.2–61.9) cm2 in cases and 55.8 (IQR: 41.9–92.3)cm2 in controls (P=0.056, Wilcoxon Rank Sum test).

Median subcutaneous abdominal fat area was 68.4 (IQR:56.4–117.2) cm2 in cases and 197.4 (IQR: 130.0–258.3)cm2 in controls (P<0.001). The median ratio of intratho-racic to subcutaneous thoracic fat was 0.200 (IQR:0.110–0.356) in cases and 0.056 (IQR: 0.038–0.088)(P<0.001) in controls. The median ratio of intra-abdomi-nal to subcutaneous abdominal was 0.539 (IQR:0.247–0.905) in cases and 0.344 (IQR: 0.257–0.495) incontrols (P=0.163). Intrathoracic fat was positively corre-lated with subcutaneous thoracic fat both in cases (rho 0.4,P=0.037) and controls (rho 0.7, P=0.004). Conclusion: In HIV-infected adults without clinical evi-dence of lipodystrophy, intrathoracic fat content washigher than in healthy persons and positively correlatedwith intra-abdominal fat.


Estimated prevalence of HIV-associated adiposeredistribution syndrome (HARS) — abnormalabdominal fat accumulation — in HIV-infectedpatients

K Lichtenstein1, C Wanke2, K Henry3, M Thompson4,N Muurahainen5 and DP Kotler6

1 University of Colorado Health Sciences Center, Denver, Col.,USA; 2 Tufts-NE University School of Medicine, Boston, Mass.,USA; 3 Hennepin County Medical Center, Minneapolis, Minn.,USA; 4 AIDS Research Consortium of Atlanta, Atlanta, Ga., USA;5 Serono Inc., Rockland, Mass., USA; and 6 Columbia Universityand St Lukes-Roosevelt Hospital Center, New York, NY, USA

Background: A simple classification of HIV lipodystrophyincludes: lipoatrophy, lipohypertrophy or mixed. HARS isproposed as a form of HIV fat maldistribution with abnor-mal accumulation of trunk fat, including VAT, which there-by involves lipodystrophy patients from lipohypertrophicand mixed groups. Its isolation as a clinical entity is basedon common metabolic consequences and projected out-comes. The specific aim of this exercise was to determineprevalence of HARS based on published surveys of HIVlipodystrophy patients.Methods: Using MEDLINE and other search mechanisms,we identified 15 surveys with >100 HIV patients in treat-ment that described the patients with subjectively reportedterms suggestive of HARS, such as increased abdominalgirth, abdominal lipohypertrophy, abdominal enlargement,central or abdominal fat accumulation, truncal obesity, cen-tral fat gain, increased abdominal wall thickness and/orpseudo-obesity. In this analysis, patients were identified ashaving HARS if they were described as having one or moreof these characteristics.Results: HARS was reported in 9% to 48% (mean 32%) ofHIV patients, see Table 1 below.



Conclusions: The prevalence of HARS, with or withoutperipheral lipoatrophy, averaged 32% of HIV-infectedpatients surveyed. This might be an overestimation becausemost surveys relied on patient self-report or physician diag-noses rather than objective quantification of body compo-sition including measurement of VAT. Studies usingobjective criteria to quantify VAT accumulation in HIVpatients with increased girth versus controls would providea more accurate estimate of the true prevalence of HARS.


Measurement of fat mass in HIV patients:comparison between anthropometry andbioelectric impedance

C Miralles1, E Álvarez-García2, M Las-Heras3, FBarreiro4 and MJ Márquez4

1 Internal Medicine, University Hospital Complex of Vigo (CHUVI),Vigo, Spain; 2 Clinical Laboratory, University Hospital Complex ofVigo (CHUVI), Vigo, Spain; 3 Emergencies Service, UniversityHospital Complex of Vigo (CHUVI), Vigo, Spain; and 4 UniversityHospital Complex of Vigo (CHUVI), Vigo, Spain

Background: There is no completely error-free method tomeasure body composition. The most accurate methods(densitometry, total body potassium, isotopic dilution anddual energy X-ray absorptiometry) are time consuming,require complex and expensive technology and well-trained staff. Bioelectric impedance and anthropometry arethe only two applicable options for daily use in clinicalpractice, although the former is less reliable than the moresophisticated methods for the latter and less reproducible.Objective: To compare the measurement of fat mass inHIV and AIDS patients using bioelectrical impedance andanthropometry.

Methods: The measurements were taken in 138 clinicallystable patients, 80 with AIDS and 58 infected with HIV(46 females and 92 males). The measurements of fat masswere made using both tetrapolar impedance (impedan-ciometro multifrecuencia Bioscan-97, BiologicalTecnologia Médica SA, Barcelona, Spain) and anthro-pometry (according to Durning and Womersly) and theresults compared using two statistical tests, the intraclasscoefficient and Pearson’s correlation coefficient.Results: Mean age was 35.69 ±7.87 years, weight 62.09±7.87 kg, and mean BMI 22.39 ±3.49. After using the twostatistical tests, we found a strong correlation betweenboth methods in the measurement of fat mass (r=0.911,P<0.001, cci=0.734)Conclusion: Both bioelectric impedance and anthropome-try are sensitive and simple methods to calculate fat massof HIV and AIDS patients. The strong correlationbetween the measurements indicates that both methodsare equally reliable.


Abdominal subcutaneous adipose tissue (SAT) andvisceral adipose tissue (VAT) measurements inHIV+ adults: influences of measurement site

KJ Ellis1, B Grund2, F Visnegarwala1, C Mullin2, CG Miller3, CE Chesson4, W El-Sadr5 and A Carr6

for the Terry Beirn Community Programs forClinical Research on AIDS (CPCRA) and the BodyComposition Substudy of Strategies forManagement of Anti-Retroviral Therapy (SMART-CPCRA 065C)

1 Baylor College of Medicine, Houston, Tex., USA; 2 University ofMinnesota, Minneapolis, Minn., USA; 3 Bio-ImagingTechnologies, Newtown, Pa., USA; 4 Social and Scientific

Table 1 (Abstract 53)

Study % HIV patients Number of Number of Total HIVCohort with HARS HARS patients Lipo patients patients

Bernasconi (2001) 27% 373 559 1359Bogner (2001) 27% 31 56 115Boufassa (2001 39% 269 403 685Chene (2001) 13% 16 37 120Dong (2001) 16% 19 21 116Heath (2001) 33% 346 518 1035Joly (2001) 44% 44 67 101Miller JE (2003) 35% 475 689 1350Lichtenstein (2001) 30% 323 529 1077Paparizos (2000) 9% 21 28 242Paton (2002) 41% 167 357 410Rakotoambina (2001) 20% 35 51 175Rozenbaum (1999) 48% 300 524 624Saint-Marc (2000) 31% 48 82 154Thiebaut (2000) 21% 120 221 581Overall 32% HARS 2587 HARS 4142 Lipo 8144 HIV


Abstracts

Systems, Inc., Silver Springs, Md., USA; 5 Columbia University,New York, NY, USA; and 6 St Vincent’s Hospital, Sydney, Australia

Background: Loss of SAT and an increase in VAT are com-mon with HIV lipodystrophy. Excess abdominal VAT is aknown risk factor for cardiovascular disease. Computerizedaxial tomography (CT) can be used to measure SAT andVAT areas in cross-sectional images of the abdomen. Theideal site(s) and number of abdominal images required tomeasure SAT and VAT is unknown. Methods: We obtained nine-slice abdominal CTs in 26HIV+ adults, one at the intervertebral L2–L3, L3–L4 andL4–L5 spaces, and two additional slices 7 mm superior andinferior to each space. We assessed the interchangeabilityamong the three intervertebral sites, and determined theeffects of imprecise positioning for each site. Results: Mean ±SD values (cm2) for SAT were 100 ±52 atL2–L3, 120 ±55 at L3–L4 and 150 ±69 at L4–L5, indicat-ing a 50% increase when moving lower on the abdomen. Incontrast, the corresponding mean VAT values were 178±129, 165 ±113 and 134 ±84 cm2, or a 25% decrease at thelowest position. Also, the mean VAT/SAT ratios decreasedacross the three intervertebral sites: 2.15 ±1.75 at L2–L3,1.68 ±1.46 at L3–L4 and 1.03 ±0.75 at L4–L5. At each site,analysis of variance (ANOVA) confirmed the significantdifferences between superior, central and inferior slices forSAT (P<0.0001) and VAT (P<0.01). Conclusions: The magnitude of the differences in abdomi-nal VAT and SAT values between the three intervertebralsites precludes their interchangeability for the assessment ofabdominal SAT and VAT. Using the VAT/SAT ratio does notovercome this limitation. Furthermore, the substantialdifferences in SAT and VAT values between the three slicesat an intervertebral site indicate that precise re-positioningof the scans is needed for longitudinal studies. In summary,our findings suggest that nine-slice abdominal CT at threeintervertebral levels provides a more complete assessmentof abdominal fat distribution than does single-slice CT.


Evaluation of HIV-associated lipodystrophy usingstandardized photos: a reproducibility study

RB Cavalcanti, M Christian, K Logue, R Kaul and S Walmsley

University of Toronto, Toronto, Ont., Canada

Objectives: One of the challenges in the study of HIV-associated lipodystrophy (HIV-LD) is the need to reliablyidentify affected individuals. Ratings by physicians andpatients using a questionnaire have been used in manystudies, but reproducibility has been suboptimal. By stan-dardizing lighting conditions and comparisons between

individuals, photography may allow more objective eval-uation of changes in body shape, and especially in facialfat. The present study aimed to evaluate whether physi-cian rating of standardized photos would result inimproved agreement between observers in rating presenceand severity of body shape changes in HIV-LD.Methods: Participants in a study of HIV-LD underwentphotographic sessions in standardized positions underuniform lighting conditions. Five physicians, experiencedin evaluating HIV-LD, independently rated photos using astandardized questionnaire. Features of HIV-LD beingevaluated included facial, arm, leg, dorsal fat pad andabdominal fat changes. These were rated for presence andseverity (mild, moderate or severe). Agreement overchance was measured using multiple-rater kappa for pres-ence or absence of HIV-LD. Weighted kappa (Kw) andKendall’s coefficient of concordance (W) were used toevaluate agreement between severity ratings.Results: Forty subjects (two females) were evaluated inthis study (mean age 46, mean CD4 138, mean durationof HIV 11.4 yr). All had at least one moderately severefeature of HIV-LD. Weighted kappa results were higherfor fat gain than fat loss (for example, facial fat loss(Kw=0.24), fat gain in the dorsal fat pad (Kw=0.60) andbuttock fat loss (Kw=0.10). In all evaluated areas, kappafell in the moderate (Kw=0.4–0.6) range or lower.Agreement over chance for presence versus absence ofHIV-LD was higher than agreement over chance for rat-ings of severity. However, even agreement on the presenceof HIV-LD fell in the fair to moderate range.Discussion: With the use of standardized photos in HIV-LD, reproducibility of ratings of body shape changes byphysicians is at best moderate. Inter-rater agreement wassimilar to ratings of HIV-LD after clinical examination.Standardized descriptors for levels of severity mayimprove reliability of questionnaires aimed at identifyingpresence and severity of HIV-LD changes.


Loss of weight in the era of HAART is associatedwith elevated PBMC proviral DNA levels

CM Shikuma, V Valcour, S Ratto-Kim, AE Williams,S Souza, M Gerschenson, JH Kim and B Shiramizu

Hawaii AIDS Clinical Research Program, John A Burns School ofMedicine, University of Hawaii - Manoa, Honolulu, HI, USA

Objective: Loss of weight continues to be seen in HIV+subjects in developed countries despite widespread avail-ability of potent antiretroviral therapy. Cataboliccytokines such as TNF-alpha are often elevated in thesesubjects. Residual HIV infection in cells of the monocyte/macrophage (M/MΦ) lineage have been demonstrated in



antiretroviral-treated HIV-infected individuals with unde-tectable plasma HIV-1 RNA by PCR. We hypothesize thatweight loss in the era of HAART may be secondary toresidual HIV infection in M/MΦ with resultant increasesin catabolic cytokine production and release. Methods: PBMC proviral DNA copies/cell (assessed byreal-time PCR), select plasma cytokine levels (assessed byELISA) and weight records over the first year of follow-upwere available and analysed in a subset of 67 HIV+ sub-jects participating in the Hawaii Aging with HIV longitu-dinal cohort study.Results: 17.0% had weight loss >10%, 14.9% had weightloss from >5% to 10%, 61.7% maintained stable weightwith less than 5% gain or loss, and 6.4% demonstratedweight gain. PBMC proviral DNA levels were higher inthose with weight loss >5% compared with those withstable or increasing weight (median 8.9 vs 0.9 copies/106

cells; P=0.006 by Mann–Whitney). Proviral DNA levelsremained higher in the 52% of subjects with plasma HIVRNA levels <50 copies/ml (median 8.9 vs 0.5 copies/106

cells, P=0.028 Mann–Whitney). Analysis in selected spec-imens to ascertain HIV DNA in subsets of PBMCs demon-strated that the majority of proviral DNA copies inPBMCs were in activated (CD14+/CD16+ by flow)macrophages. A correlation was demonstrated betweenproviral DNA levels and sTNFRII levels (r=0.72,P=0.043) (n=8). Conclusions: Weight loss in the era of HAART may bedriven by residual HIV infection in cells of the monocyte/macrophage lineage.Acknowledgement: Supported by NIH/NINDS U54 NS43049.


Usefulness of anthropometry in detectingpaediatric lipodystrophy

P Brambilla1, S Mora2, C Figini1, L Cafarelli1, V Giacomet1, P Manzoni1 and A Viganò1

1 Paediatrics, L Sacco Hospital, University of Milan, Italy; and 2Laboratory of Paediatric Endocrinology, IRCCS H S Raffaele,Milan, Italy

Background: Lipodystrophy is a frequent and relevantfinding in HAART-treated HIV-infected children. Itsdetection requires reference body composition techniqueswhile an anthropometry role has not been completelyestablished. The aim of our study was to evaluate the use-fulness of simple anthropometry in assessing both periph-eral lipoatrophy (LA) and truncal lipohypertrophy (LH)in comparison with MRI and DXA.Methods: During 4 years follow-up, a total of 102 com-bined measures (anthropometry and DXA-MRI) were

collected in 38 HIV patients (aged 2.9–19.9 years). Forthe assessment of LH, waist circumference (W) was com-pared with trunk fat mass by DXA and with intra-abdom-inal adipose tissue (IAT) by lumbar MRI. For theassessment of LA, arm circumference and triceps skinfoldwere compared with arm fat/lean ratio by DXA. Age andsex-specific published cut-offs were used for arm circum-ference, triceps, W and IAT; cut-offs for DXA wereobtained from our matched control population (166 sub-jects). Sensitivity (SN) and specificity (SP) of anthropo-metric variables were calculated.Results: Reduced arm fat/lean ratio, increased trunk fatand increased IAT were detected in 25, 18 and 40% ofmeasures, respectively. IAT and trunk fat were concordantin 71% of measures.

LA: arm circumference <5° c.le showed SP 96.0% andSN 24.0%. Triceps <3° c.le showed SP 86.7% and SN20.0% and triceps <10° c.le showed SP 74.7% and SN36.0%. Combining arm circumference and triceps, theresults did not improve: SP 98.6% and SN 18.2%.

LH: W (>98° c.le) in comparison with trunk fat had SP90.5% and SN 83.3%, while with IAT had SP 96.8% andSN 71.4%.Conclusion: The use of anthropometry may have animportant role in detecting LA and LH. W can be used toscreen subjects with increased trunk fat, even if a normalW does not exclude an increased IAT, especially whenmetabolic abnormalities are present. Arm anthropometrydoes not give satisfactory results for SN; the high SP ofarm circumference, however, allows us to identify patientswith LA. The better results obtained with arm circumfer-ence with respect to triceps could be due the better repro-ducibility of the first.


Safety and efficacy of intradermal poly-L-lacticacid (Sculptra™) injections in patients with HIV-associated facial lipoatrophy

DR Mest and G Humble

Blue Pacific Aesthetic Medical Group, Inc., Hermosa Beach, Calif.,USA

Objectives: Facial lipoatrophy, the loss of subcutaneousfat, projects itself as the most obvious marker of HIV.Initially associated directly with the disease, the undesir-able appearance of facial lipoatrophy is now a side effectof the very therapies that have made HIV a more man-ageable chronic condition. The stigma associated with alipoatrophic appearance can erode self-esteem, quality oflife and, for those with HIV, can affect treatment compli-ance. Poly-L-lactic acid (Sculptra™) has recently been rec-ommended for expedited approval by an FDA advisory


Abstracts

panel to treat lipoatrophy in this population. Poly-L-lacticacid is a non-animal, biodegradable, biocompatible andimmunologically inert polymer with a long history of safe-ty. This study evaluated the efficacy and safety of poly-L-lactic acid in improving HIV-associated lipoatrophy forup to 12 months.Methods: Patients in this single-site, open-label studyreceived 1–6 ml of poly-L-lactic acid injected intradermal-ly every 3 weeks for up to six sessions. Skin thickness inthe malar region was assessed with calipers prior to eachsession, after the last session and at 6 and 12 month fol-low-ups. Patient satisfaction was evaluated (1–5; 5=‘verysatisfied’) at each session and at 6 and 12 month follow-ups. Adverse events (AEs) were self reported throughoutthe trial.Results: Of 99 patients who completed treatment, 6 and 12month data are available for 68 and 35 patients, respective-ly. The mean percent change from baseline in skin thicknessat 6 and 12 months was 59.2% and 64.8%, respectively.Patient and investigator satisfaction scores were each 4.7and 4.8 at 6 and 12 months, respectively. No serious AEswere reported at 6 and 12 month follow-ups and there wereno clinically relevant laboratory findings. Injection-site AEswere nominal and transient. Small non-visible subcutaneouspapules were noted in 12.1% of patients.Conclusion: Poly-L-lactic acid demonstrated safety andefficacy in treatment of HIV-associated lipoatrophy up tothe last study time point of 12 months. Further studies arerequired to determine efficacy beyond 12 months. Thenovel mechanism of action of this compound as a volumeenhancer (rather than filler) is well suited to treat theeffects of all grades of lipoatrophy.


Bio-Alcamid™, a high-volume injectable prosthesisfor facial reconstruction in HIV-related lipoatrophy:report on 100 patients

LC Casavantes1and M Gottlieb2

1 Clinic’estetica, Tijuana, BC, Mexico; and 2 Polymekon USA, SanDiego, Calif., USA

Objective: To evaluate the safety and efficacy of a highvolume injectable prosthesis containing 96% apyrogenouswater and 4% biopolymer known as polyalkylimide (Bio-Alcamid™, Polymekon Italy).Methods: One hundred patients with HIV-related faciallipoatrophy were treated, determining the number of ses-sions, amount of product and time required for a com-plete correction. Basal facial lipoatrophy severity wasgraded according to Clinic’estetica classification (L Casavantes, Consideraciones clínicas de la lipoatrofiafacial asociada a infección por VIH, Dermatología CMQ,

accepted for publication). For safety evaluation, biopsieswere taken at different times after the procedures.Results: The volume of Bio-Alcamid™ needed to replacethe lost subcutaneous fat in these patients was relative tothe initial severity of lipoatrophy and the patient’s facialstructure. Treatment was very well tolerated and the facialdeficits were fully and permanently restored after two tofour sessions in 100% of the patients. In all cases, therecuperation time was minimal, ranging from no downtime to 3 days. Biopsies showed minor acute inflamma-tion with no signs of chronic inflammation or granuloma.Two minor adverse events that occurred were controlledwith no compromise to the desired final result. Conclusion: This study demonstrates that Bio-Alcamid™is a safe and effective treatment option for HIV-relatedlipoatrophy. The procedures were performed in an out-patient setting and, regardless of the severity level, excel-lent permanent restoration results were achieved to thesatisfaction of both patient and investigator.


Fosamprenavir/ritonavir and nelfinavir havecomparable effects on body fat changes inantiretroviral-naive patients: 48-week resultsfrom the SOLO study

S Walmsley1, A Horban2, R Jain3, C Garris3 and T Stark3

1 Toronto Hospital University Health Network, Toronto, Ont.,Canada; 2 Hospital of Infectious Disease, Warsaw, Poland; and3 GlaxoSmithKline R&D US and Europe

Background: To date, minimal prospective data fromcohorts or from randomized, comparative trials on the inci-dence of body composition changes (BCCs) have been pre-sented. Methods: SOLO was a randomized study in antiretroviraltherapy-naive, HIV-infected adults which compared the effi-cacy and safety of fosamprenavir/ritonavir once-daily(FPV/r, Lexiva®, Telzir®; n=322) with nelfinavir twice-daily(NFV; n=327), each administered with abacavir/lamivudine.BCCs were assessed by physician and patient observationusing a standardized questionnaire and anthropometry. Results: Demographic characteristics were well matched inthe two groups. Median baseline (BL) CD4: 170 cells/µl,median HIV-1 RNA: 4.81 log10 copies/ml. A medianincrease in body weight of 3 kg at week (wk) 48 wasobserved in both groups. There was a median increase fromBL in hip and waist circumference of 2 cm in the FPV/rgroup and 3 cm in the NFV group. Median waist/hip ratiodid not change. Of the subjects, 40/322 (12%) and 52/327(16%) reported at least one BCC at BL in the FPV/r andNFV groups, respectively, with the majority of these subjects



reporting fat wasting [25/322 (8%) and 37/327 (11%),respectively]. Of note, 14/30 (47%) and 16/35 (46%) ofsubjects with BCC at BL did not report any BCC at wk 48,in the FPV/r and NFV groups, respectively. Of subjects withno BCC reported at BL, 36/203 (18%) and 45/220 (20%)reported BCC at wk 48 in the FPV/r and NFV groups,respectively. Of subjects who did not report fat wasting atBL, only 4% (9/215 and 9/232) reported fat wasting at wk48 in both groups. Of subjects who did not report fat accu-mulation (FA) at BL, 15% (33/219) and 18% (44/238)reported signs of FA at wk 48 in the FPV/r and NFV groups. Conclusion: Over 48 wks, a comparable proportion of sub-jects reported BCC in the FPV/RTV and NFV groups com-bined with a similar weight gain but no change in waist/hipratio. The majority of changes were fat accumulation. Ofnote, in both groups a low rate of fat wasting was observedwhich may relate to abacavir/lamivudine.


Longitudinal changes in body shape and metabolicparameters in HIV-infected, treatment-naivepatients initiating NNRTI regimens in South India

S Saghayam1, N Kumarasamy1, S Solomon1, N Aliabadi2, AJ Cecelia1, P Balakrishnan1, G Shivaji3, T Flanigan4, K Mayer4 and C Wanke2

1 YRG Centre for AIDS Research and Education, Chennai, India;2 Tufts University School of Medicine, Boston, Mass., USA; 3 Women’s Christian College, Chennai, India; and 4 MiriamHospital, Brown University, Providence, RI, USA

Background: Generic NNRTIs are available in India. Weasked whether body shape and metabolic changes differedbetween the two NNRTI regimens as diabetes and CVDare common in the South Indian population.Methods: All were treatment naive; 37 subjects initiatedefavirenz (67% stavudine/lamivudine; 33% zidovudine/lamivudine), 12 initiated nevirapine (57% stavudine/lamivudine; 43% zidovudine/lamivudine) and 11 were nottreated. Anthropometry, BIA, fasting lipids, insulin andglucose tolerance were measured at baseline and 6months. Paired t-tests and Kruskal–Wallis were used todescribe changes within and between groups.Results: The mean ages in the treated and untreated groupswere 35 and 31 years; 67% of the entire cohort was male.All groups were comparable except for CD4 (efavirenz:175 cells/mm3; nevirapine: 143 cells/mm3; untreated:400 cells/mm3) and median viral load (efavirenz: 262 000copies/ml; nevirapine: 482 000 copies/ml; untreated:52 300 copies/ml). BMI (19.1 kg/m2) was low normal forSouth India.

Baseline: mean glucose, glucose tolerance, total choles-terol, triglycerides and LDL-c were within normal limits;

<10% had triglycerides >150g/dl. HDL was low in allgroups (<35g/dl; 65% efavirenz, 58% nevirapine and36% untreated).

Six months: data are given as mean increases. Bothtreatment groups had increases in CD4 (efavirenz: 154cells/mm3, P=0.000; nevirapine: 135 cells/mm3, P=0.003).100% efavirenz and 67% nevirapine achieved viral load<400 copies/ml. Increases (all P<0.05) were seen in BMI(efavirenz/nevirapine) (1.3/1.6 kg/m2), waist (3.8/3.0 cm),hip (2.7/4.0 cm) and mid arm circumference (1.5/1.8 cm).Waist:hip increased to 0.86 (P=0.041) in efavirenz. (Nochange in untreated group). On nevirapine, body fat wasunchanged and BCM increased (2.7 kg, P=0.013). Onefavirenz, body fat (2.0 kg, P=0.001) and BCM (0.8 kg,P=0.000) increased. Fasting glucose increased (efavirenz:4.2 mg/dl, P=0.02; nevirapine: 6.4 mg/dl, P=0.014);insulin and 2-h glucose did not. On nevirapine, TC, HDL,and LDL did not change; on efavirenz there were increas-es (P=0.000) in TC (34.1 mg/dl), HDL-c (19.9 mg/dl) andLDL-c (19.8 mg/dl) – still within normal limits. TG didnot change in any group. HDL <35mg/dl decreased to 8%on efavirenz.Conclusion: Six months of NNRTI–HAART improvedvirological and nutritional parameters in South India. Allbody shape changes were symmetrical on nevirapine;there was a trend to increased fat on efavirenz. Glucosetolerance did not change. Lipid parameters increased onefavirenz but not nevirapine. Most striking was theincrease in HDL on efavirenz.


Antiretroviral protease inhibitors prevent L6muscle cell fusion by reducing calpain activity

SP Colby-Germinario1, LE Chalifour1,3,A Antoneccchia2 and RJ Germinario1–4

1 Lady Davis Institute for Medical Research, Montreal, Que.,Canada; 2 Concordia University, Montreal, Que., Canada;3 McGill University Department of Medicine, Montreal, Que.,Canada; and 4 McGill University AIDS Centre, Montreal, Que.,Canada

Introduction: The antiretroviral protease inhibitors, indi-navir (IDV) and ritonavir (RTV) are used in highly activeantiretroactive therapy (HAART). Side effects from long-term HAART therapy include loss of muscle mass.Myoblasts, when cultured in media low in growth factorswithdraw from the cell cycle, express muscle-specific differ-entiation inducers and proteins and fuse to form myotubes.The neutral protease, calpain, is required for myotube for-mation and RTV decreased calpain activity in vitro.Results: We found lower calpain activity, but not calpainprotein in homogenates of RTV-treated L6 cells than in


Abstracts

control cultures. Importantly, L6 and C2C12 myoblastsdid not form myotubes when cultured with 10 or 20 µMIDV or RTV. Control and drug-treated L6 myoblastsshowed identical decreases in PCNA expression indicatingproliferation arrest. Similarly, the expression of the muscledifferentiation inducers MyoD and myogenin and theirdownstream target, myosin heavy chain, were expressed tosimilar levels in control and drug-treated cells. Conclusion: Thus, whereas muscle differentiation wasunaffected by protease inhibitors, calpain activity wasreduced and myotube formation prevented. We concludethat RTV and IDV reduced myotube formation by reduc-ing calpain activity. The data suggest that proteaseinhibitors included in HAART might be involved in mus-cle wasting by reducing muscle remodeling.


The effect of different combination therapies onoxidative stress markers in HIV infected patientsin Cameroon

JL Ngondi and J Oben

Nutrition, HIV and Health Research Unit, Department ofBiochemistry, Faculty of Science, University of Yaounde I,Cameroon

Objective: Oxidative stress in HIV-infected subjects maybe due to the use of antiretroviral therapy. We assessed theeffect of antiretroviral therapy (ART) on certain oxidativestress markers, malondialdehyde (as TBARS), albumin,protein carbonyl content and protein sulphhydryl groups. Methods: The study group comprised 85 HIV patients(34.8 ±9.27 years) on antiretroviral therapy, 65 HIVseropositive patients (32.16 ±10.85 years) on no treat-ment, and 90 HIV-seronegative patients (32.57 ±9.27years) as the control group. Plasma samples were analysedfor protein sulphhydryl, protein carbonyl, total protein,vitamin C, albumin and malondialdehyde. Results: Plasma TBARS (P<0.0001, 0.0001) as well ascarbonyl (P<0.0001, 0.0001) levels were significantlyhigher in HIV patients on ART compared with those notfollowing therapy or non-HIV-infected controls. On theother hand, the protein sulphhydryl group content wasnot different for patients on ART compared with thosewho were not on ART, but both were significantly lowerthan non-HIV-infected controls (P<0.0001, 0.001). Therewas a marked increase with the length of time on ART,the protein sulphyhdryls groups of patients with longer(16.1 ±3.44 months) duration of ART treatment washigher compared with those with shorter (3.31 ±1.62months) (P<0.0001) and medium duration (9.332.18months) (P<0.001). The combination treatment [(stavu-dine + lamivudine + nevirapine (d4T+3TC+NEV) with

zidovudine + lamivudine (AZT+3TC)] brought about areduction in the plasma concentration of protein sulphhy-drl groups as well as TBARS compared with stavudine +lamivudine + nevirapine (P<0.05) or the combination ofzidovudine + lamivudine with nevirapine or efavirenz(P<0.05). Vitamin C content was lower in the plasma ofpatients on the d4T+3TC+NEV treatment compared withthose on d4T+3TC+NEV in combination with zidovudine+ lamivudine (P<0.01) and those on zidovudine andlamivudine in combination with nevirapine or efavirenz(P<0.001). Conclusion: HIV infection therefore increases the oxida-tive stress process, while antiretroviral combination ther-apy increased protein oxidation.


Use of emtricitabine (FTC) as part of HAART in aninner-city clinic setting results in no appearanceof pigmentation changes in HIV-positive patientsof colour

W Jordan1, B Guyer2 and R Jefferson1

1 OAISIS Clinic, Los Angeles, Calif., USA; and 2 Gilead Sciences,Inc, Foster City, Calif., USA

Background: Increased skin pigmentation occurs with themajority of approved antiretroviral (ARV) drugs and alsononsteroidal anti-inflammatory agents, antimalarials, tetra-cyclines and psychotropics. Among ARVs, it was first notedwith zidovudine. Recent studies have shown a rareoccurrence of pigmentation changes in HIV-positive patientsof colour when treated with cytosine analogues. However,there are limited data evaluating the outcomes of pigmenta-tion changes with the use of FTC in a clinical cohort.Methods: This was a cross-sectional cohort analysis of 81HIV-positive, chronically infected patients of colour treat-ed at the OAISIS clinic (an inner-city clinic) with an FTCHAART regimen between August 2003 and July 2004 toevaluate any appearance of pigmentation changes. Thecohort will continue to be followed prospectively.Results: We retrospectively reviewed the charts of 81 HIV-positive patients treated with FTC as part of a HAARTregimen. This included 53 patients who were switchedfrom 3TC to FTC, as well as 28 patients who were naiveto antiviral therapy. Enrolled patients had the followingbaseline (BL) characteristics: mean age 25 years; 67/81(83%) were black, of whom 58 were male, six werefemale and three were transgender; and 14/81 (17%) wereLatino, of whom 11 were male and three were female.Mean time on an FTC-containing HAART regimen was30 weeks. In this patient population, we have observed noappearance of pigmentation changes in these patients afterthe initiation of FTC.



Conclusion: In our experience, the use of FTC in HIV-positive patients of colour has resulted in no appearanceof pigmentations changes, regardless of whether these areHAART-naive patients or HAART-experienced patients.


Impact of race on nervous system side-effects ofsubjects taking efavirenz

DW Seekins, LJ Lupo, JF Maa, LJ Bessen andSL Hodder

Bristol-Myers Squibb Virology, Princeton, NJ, USA

Background: AACTG NWCS214 identified a CYP2B6allelic variant that is more commonly observed in blacksubjects and is associated with higher efavirenz levels.ACTG 5097s demonstrated a 32% decreased efavirenz(EFV) clearance in African-Americans and Hispanics andan increased discontinuation rate among those withdecreased clearance. This study assesses possible associa-tions of race and incidence of nervous system symptoms(NSS) in a large prospective clinical trial. Methods: DMP266-006 was an open-label, multicentre,multinational, prospective study of 1266 subjects ran-domized to EFV + indinavir (IDV), EFV + zidovudine(ZDV) + lamivudine (3TC), or IDV+ZDV+3TC. A post-hoc analysis of 1228 subjects was performed to determinethe percentage of subjects with at least one NSS event dur-ing the first 96 weeks. Thirty-eight subjects other thanwhite, black or Hispanic subjects were excluded from thisanalysis. NSS included confusion, dizziness, stupor, agita-tion, amnesia, depersonalization, euphoria, hallucina-tions, insomnia, somnolence, abnormal thinking,impaired concentration and abnormal dreaming.Results: Percent of subjects with NSS event(s) through 96weeks:

White Black Hispanic Treatment group (n=763) (n=256) (n=209)

EFV+IDV 146/256 (57%) 37/86 (43%) 42/74 (57%)EFV+ZDV+3TC 151/251 (60%) 50/90 (56%) 39/70 (56%)IDV+ZDV+3TC 76/256 (30%) 20/80 (25%) 11/65 (17%)

Within each treatment group, white subjects had the high-est incidence of NSS, though observed differences werenot statistically significant (P>0.05).Conclusions: Despite concerns regarding different rates ofEFV clearance among some racial populations, this studyfound no evidence to suggest an increased risk of NSSamong blacks and Hispanics compared with whites.


Adverse reaction of nevirapine (NVP) in antiretroviral-naive HIV-seropositive subjects

SK Dey1 and NK Pal2

1 Department of Chest Medicine, Medical College, Calcutta,India; and 2 School of Tropical Medicine, Calcutta, India

Aims: With decreasing costs of highly active antiretroviraltherapy (HAART) since 2000 the affordability has goneup from a meagre 1% to about 10%. The most commonand cheapest regimen used is NVP+3TC+d4T (about60%) followed by NVP+3TC+AZT (about 30%). A studyof the most important adverse effect hypersensitivity inthe racial groups of this geographical area is essential asthere are no reports.Methods: 51 HIV-positive patients (males 80.4%) age21–49 years (mean 34.9 years), naive to HAART, mainlyfrom the West Bengal state in Eastern India, were studied.They received 3TC+AZT (standard dose) or d4T (as perbody weight) along with NVP 200 mg once daily for thefirst 14 days, increasing to 200 mg twice daily if no sideeffects were seen. CD4 count ranged from 16 to 238(mean 125.2). Viral load was not measured due toresource constraints. All patients contracted HIV by theheterosexual route.Results: Eight (seven males and one female) out of 51 sub-jects developed maculopapular rash all over the body.Seven developed the rash in the second week (medianeighth day), while in one it occurred immediately afterincreasing the dose to 200 mg twice daily on day 15.Conclusions: The high rate of hypersensitivity to NVP inAsian populations is a stumbling block towards increasingaccess for all to HAART, as other groups of antiretroviraldrugs are very costly. Newer approaches such as struc-tured interrupted therapy requiring fewer drugs need to bedevised in resource-constrained settings like India, hometo 5.1 million HIV-positive individuals. Alternatively,boosted protease inhibitors using indinavir 800 mg plusritonavir 100 mg twice daily instead of NVP may be used.


A practical preclinical model for assessing thepotential for unconjugated hyperbilirubinaemiaproduced by HIV protease inhibitors

DJ Kempf, K Marsh, JF Waring, DC Morfitt, PWerner, B Ebert, M Mitten, B Nguyen, JT Randolph,DA DeGoey & LL Klein

Abbott Laboratories, Abbott Park, Ill., USA


Abstracts

Objective: Unconjugated hyperbilirubinaemia produced bythe HIV protease inhibitors atazanavir and indinavir causesa significant incidence of jaundice and/or scleral icterus, andmay lead to therapy discontinuation. A previous report hasdescribed elevation of serum bilirubin in Gunn rats, whichare heterozygous for a polymorphism in the gene encodingUDP-glucuronosyltransferase (UGT), after treatment withindinavir; however, the elevations were not statistically sig-nificantly different from the vehicle. Our objective was theestablishment of a practical preclinical hyperbilirubinaemiamodel for screening new protease inhibitors. Methods: Gunn rats were administered a single100–250 mg/kg dose of various protease inhibitors boost-ed by a 50 mg/kg dose of ritonavir. After 4 h, blood sam-ples were drawn and split for serum bilirubin and drugconcentration analysis.Results: The boosting dose of ritonavir produced a small(mean 0.04 mg/dl) but statistically significant increase inbilirubin (P<0.001). Amprenavir/ritonavir, the negativecontrol, was not distinguishable from the ritonavir boosterdose (0.07 mg/dl, P=0.06). In contrast, indinavir/ritonavirand atazanavir/ritonavir produced significant elevations,even after a single dose (0.15 and 0.28 mg/dl, respective-ly, P<0.001 for each compared with ritonavir). The incre-ment in serum bilirubin produced by indinavir andatazanavir was dependent on the serum drug concentra-tion. An exploratory protease inhibitor under preclinicalinvestigation, A-681799, produced a significantly higherelevation than atazanavir (0.53 mg/dl, P<0.001), disqual-ifying it from further development.Conclusion: This practical preclinical model, which reca-pitulates the hyperbilirubinaemia produced by HIV pro-tease inhibitors in humans, may be useful for theidentification of new protease inhibitors that are devoid ofeffects on serum bilirubin.


Concurrent statin therapy blunts CD4+ cell gain incombination antiretroviral therapy (CART) treatedHIV-infected patients

UH Iloeje1, Y Yuan2, AC Moorman3, KC Wood4 andSD Holmberg3

1 Pharmaceutical Research Institute, Bristol-Myers Squibb,Wallingford, Conn., USA; 2 Bristol-Myers Squibb, Plainsboro, NJ,USA; 3 Centers for Disease Control & Prevention, Atlanta, Ga.,USA; and 4 Cerner Corporation, McLean, Va., USA

Background: The increasing prevalence of hyperlipi-daemia in HIV-infected patients has resulted in increasinguse of lipid-lowering therapy in these patients. We testedthe hypothesis that concurrent statin therapy may bluntCD4+ cell response in HIV-infected patients on CART.

Methods: Case control study with 1:1 matching, usingdata from the HIV-Insight database. Patients on statins(cases) were matched to non-statin-exposed patients (con-trols), on age, calendar year of observation, gender andbaseline CD4+ cell count. Patients were required to be oncontinuous CART >6 months. Outcome was change(increase) in CD4+ cell count 6 months from the indexdate [first use of CART (controls)/statin (cases) within thestudy period]. The study period was 1 January 1997 to 31December 2003. Nadir CD4+ cell count was the lowestvalue within 365 days prior to the index date; baselineCD4+ cell and viral load (VL) were the closest valueswithin 90 days prior to the index date; last CD4+ cellcount was that closest to the end date ±90 days. Statisticaltesting was by an ANCOVA model adjusting for baselineVL and duration of (known) HIV-test positivity Results: Groups: n=136 cases and 136 controls. In bothgroups, mean age was 44.5 years and 97.1% were males.At baseline for cases and controls: mean CD4+ cell countswere 440.5 and 433.6 cells/mm3 (NS); nadir CD4+ cellcounts were 339 and 334.1 cells/mm3 (NS); baseline viralload were 4.7×103 and 5.79×105 copies/ml (P<0.0001);duration of known HIV-test positivity 9.2 and 11.2 yrs(P=0.0002).

For cases versus controls: mean increase in CD4+ cellcount was 24 versus 68 cells/mm3 (P<0.0001), with amedian change of 16 versus 41 (P=0.07); mean percentageincrease in CD4+ cells was 12% versus 32% (P<0.0001),median of 3% versus 10% (P=0.08); and 36% versus47% (P=0.07) with CD4+ cell response ≥50 cells/mm3.Statin use was associated with diminished CD4+ cellresponse (ANCOVA model, P=0.04). Conclusion: These data suggest that statin therapy is asso-ciated with a blunting of CD4+ cell response in patientson CART. The clinical significance of this finding remainsto be determined.


Tenofovir and how to assess renal function inpatients on antiretroviral therapy

S Mauss, F Berger and G Schmutz

Center for HIV and Hepatogastroenterology, Düsseldorf, Germany

Objective: The increasing use of tenofovir in conjunctionwith anecdotal cases of nephrotoxicity has led to observa-tional studies on the influence of tenofovir on renal func-tion. Most studies use calculation of glomerular filtrationrate (GFR) based on serum creatinine. In this study weassessed three methods to estimate GFR in HIV-seroposi-tive patients on tenofovir or never-treated with tenofovir.Methods: Cross-sectional study comparing patients treat-ed with tenofovir (n=82) with patients on antiretroviral



therapy never treated with tenofovir (n=92). In serum,creatinine, cystatin C and electrolytes were measured andcreatinine, electrolytes and protein were measured in 24-hurine. In cases of proteinuria >130 mg/d a DISC-elec-trophoresis was performed. GFR was estimated by: 1) cal-culation of creatinine clearance using creatinine in serumand in 24-h urine; 2) calculation according to the modi-fied MDRD-study equation which is based on serum cre-atinine only; and 3) calculation by serum cystatin C, arecently established GFR marker. Results: Patients on tenofovir (n=82) showed a lower meanGFR estimated by creatinine clearance (97 ±49 ml/min)compared with 92 patients never treated with tenofovir(107 ±39 ml/min, P=0.03). The same result was obtainedwith cystatin C with 86 ±21 ml/min (tenofovir) comparedwith 97 ±20 ml/min (P=0.001). GFR estimated by serumcreatinine (MDRD formula) did not differ (tenofovir: 106±54 ml/min vs never treated with tenofovir 104 ±22ml/min, P=0.375).

Patients on tenofovir had a higher mean protein contentin urine (124 ±110 mg/d vs 94 ±55 mg/d, P=0.03). In total30 patients on tenofovir (36%) versus 15 control patients(16%) had proteinuria >130 mg/d (P=0.003). In the major-ity of patients the proteinuria was of tubular origin.Electrolyte excretion did not differ between the groups.Conclusion: Treatment with tenofovir is associated withmild renal dysfunction in a higher proportion of patients.GFR assessed by creatinine clearance or, alternatively, byserum cystatin C seem to be the appropriate methods.Calculation of GFR by MDRD-formula on the basis ofserum creatinine seems to be relatively insensitive. Forconvenience, determination of cystatin C in serum may bea useful screening marker for impaired renal function.Determination of 24-h proteinuria adds further informa-tion on tubular and glomerular dysfunction.


Functional impairment of NRTI-relatedmitochondrial DNA-depletion in primary humanT lymphocytes

B Setzer, M Schlesier and UA Walker

Medizinische Universitätsklinik, Department of Rheumatologyand Clinical Immunology, Freiburg, Germany

Background: The normal metabolism of primary humanT-cell mitochondria is unknown, as are the effects ofnucleoside analogue reverse transcriptase inhibitors(NRTIs) on these cells. Methods: Peripheral blood CD4 and CD8 T-lymphocyteswere purified from three male and three female healthyhumans, stimulated mitotically (anti-CD3/CD28), andexposed to NRTIs in different concentrations for 10 days.

Mitochondrial toxicity, lymphocyte number, function andviability were quantified daily. Results: Significance levels are indicated as follows: *P<0.05, **P<0.01 and ***P<0.001. In the absence ofNRTIs, the induction of mitosis led to an up-regulation(threefold**) of mitochondrial DNA (mtDNA) and of lac-tate production***. In CD4 lymphocytes, didanosine(11.8 µM, 35.4 µM, 59.0 µM and 118 µM) led to a super-imposed highly significant time- and dose-dependentmtDNA-depletion (at day 10: 73%*, 29%***, 24%***,and 23%*** of controls, respectively), reduced mtDNA-encoded COX subunits (at day 10: COXII/COXIV ratio:86%, 81%**, 55%** and 31%*** of controls, respec-tively) and increased lactate production (at day 10:139%*, 222%***, 276%*** and 312%*** of controls,respectively). Lymphocyte proliferation was reduced(93%, 75%*, 62%* and 55%***). As early as day 7,mitochondria were depolarized (JC-1 aggregate/monomerratio 90%, 69%**, 62%*** and 52%*** of controls).At day 10, but not at day 7, didanosine increased celldeath by apoptosis (122%, 197%**, 263%*** and278%*** of control values). Lymphocyte electronmicroscopy revealed enlarged mitochondria with unde-fined cristae, large vacuoles and inclusions. Similar toxic-ities were observed with 1.8 µM of zalcitabine (day 10:mtDNA 25%**, COXII/COXIV 55%**, lactate produc-tion 214%** and cell count 60%* of controls) and with36 µM of stavudine (mtDNA 40%**, COXII/COXIV70%*, lactate 175%** and cell count 41%** of con-trols). Zidovudine (71 µM) reduced cell counts andincreased lactate relatively early (34%** and 170%** ofcontrols), but mtDNA and COX II expression remainednormal. Practically identical findings were observed withrespect to all parameters in CD8 lymphocytes. Conclusions: MtDNA measurements in HIV patients arenot necessarily a marker for the mitochondrial toxicity ofantiretrovirals, although didanosine (and possibly alsoother NRTIs) do induce a time-dependent mtDNA-depletion in cultivated T lymphocytes, which is of poten-tial clinical relevance. In vitro, this mitochondrial toxicityleads to a functional impairment of the lymphocytes witha ‘lazy’ phenotype initially, followed by increased celldeath with prolonged exposure. Acknowledgements: We thank Dr K Müller, K Sutter andR Wirtz for their kind assistance with the electron micro-scope and the Deutsche Forschungsgemeinschaft (DFG)for its financial support (grant number Wa 1387/1-4).


Abstracts


Incidence and risk factors for nucleoside analoguetranscriptase inhibitors toxicity in treated HIV-HCV co-infected patients

M Laguno, A Milinkovic, E de Lazzari, J Murillas, E Martinez, JL Blanco, M Lonca, A Biglia, A Leon,M Larrousse, F Garcia, JM Miro, JM Gatell and J Mallolas

Infectious Diseases Unit, Hospital Clinic Barcelona, Spain

Background: Co-infection with HCV and HIV is notuncommon and therapies for both infections are current-ly available. A major drawback, however, could be apotential higher risk for toxicity due to the nucleosideanalogue reverse transcriptase inhibitors (NRTIs) con-tained in both therapies.Methods: Prospective study of consecutive HIV-HCV co-infected patients assigned to receive ribavirin (RBV) plusinterferon alpha (IFN). Clinical, laboratory and liverbiopsy histological variables were analysed.Results: Out of 113 patients included, 13 had no concurr-ent antiretroviral therapy due to preserved immunologi-cal status. Fourteen patients, all receiving concomitantantiretrovirals, showed laboratory data suggestingNRTI-associated toxicity consisting of increased levelsof amylase/lipase (n=3), hyperlactataemia (n=7) or both(n=4). None developed clinical pancreatitis. Four patientshad symptomatic hyperlactataemia but recovered in 2weeks after drug withdrawal. Median (IQR) time untildevelopment of NRTI toxicity was 3 (1–6) months.Therapy with ddI, ddI plus d4T, previous history of dia-betes and the baseline lactate level were variables associ-ated with NRTI toxicity in the univariate analysis.However, the only independent risk factor for NRTI tox-icity identified in the multivariate analysis was ddI use.NRTI toxicity was not associated with gender, age, alco-hol consumption, type of interferon, degree of steatosisand fibrosis in liver biopsy, presence of lipodistrophy,CD4+ cell count, HCV or HIV viral load, mitochondrialDNA and COX in liver tissue or antiretroviral therapycontaining d4T or protease inhibitors.Conclusion: Our study confirms that concomitant use ofribavirin plus didanosine should be avoided, and that rou-tine monitoring of lactate and pancreatic enzymes may becost effective.


Withdrawn by author.




Why do patients with HIV stop antiretroviralsused as part of an initial highly activeantiretroviral regimen?

A Mocroft1, AN Phillips1, V Soriano2, J Rockstroh3,A Blaxhult4, C Katlama5, A Boron-Kaczmarska6,L Viksna7, O Kirk8, JD Lundgren8 for the EuroSIDAstudy group

1 Royal Free Centre for HIV Medicine and Department of PrimaryCare and Population Sciences, Royal Free and University CollegeMedical School, London, UK; 2 Hospital Carlos III, Madrid, Spain;3 Universitäts Klinik, Bonn, Germany; 4 Karolinska Hospital,Stockholm, Sweden; 5 Hôpital de la Pitié-Salpêtière, Paris,France; 6 Medical University, Szczecin, Poland; 7 InfectologyCentre of Latvia, Riga, Latvia; and 8 Copenhagen HIV Program,Hvidovre Hospital, Copenhagen, Denmark

Background: Low adherence and toxicities among HIV-positive patients starting highly active antiretroviral ther-apy (HAART) can lead to discontinuation of therapy andtreatment failure. Little is known about hepatitis C (HCV)status and discontinuation of HAART. Methods: Poisson regression was used to determine fac-tors related to discontinuation of any part of an initialHAART regimen due to treatment failure (TF) or toxici-ties and patient/physician choice (TOX), and to investi-gate the relationship between HCV and discontinuationof a HAART regimen in 1052 patients staring HAARTafter 1999 from the EuroSIDA study.Results: At 1 year after starting HAART, 65% of patientsremained on their original regimen, 28% had changedand 7% were off all treatment. The most frequent reasonfor discontinuation was toxicities (31%). The incidence ofany discontinuation has decreased over time by 18% peryear (95% CI 11–24%, P<0.0001). The main decline wasamong patients who discontinued due to TOX. Patientswith HCV had a higher incidence of discontinuation dueto TOX during the first 6 months of HAART (incidencerate ratio (IRR) 2.14, 95% CI 1.05–5.92, P=0.035) orafter 6 months on therapy (IRR 2.09; 95% CI 1.02–4.28,P=0.044) compared with patients without HCV. Conclusions: Patients with HCV were more likely to dis-continue all or part of their HAART regimens due to tox-icity or patient/physician choice. Managing adverse eventsmust remain a key intervention in maintaining HAART.There is a need for further studies to describe the rela-tionship between HCV, specific antiretrovirals and differ-ent treatment strategies.


The SCOLTA Project: an Italian approach to monitoring the safety of new antiretroviral drugs

P Bonfanti, E Ricci, G Penco, D Migliorini, GMadeddu, S Melzi, C Magnani, D Zeme andT Quirino for the CISAI Group

CISAI Group, I Department of Infectious Diseases, 'L Sacco'Hospital, Milan, Italy

Aim: The SCOLTA Project (Surveillance Cohort Long-term Toxicity Antiretrovirals) is a system for on-line sur-veying of adverse reactions to recently commercializedantiretroviral drugs; a website was created(www.cisai.info). The aim is to assess the incidence of seri-ous and rare adverse events with new antiretroviral drugs. Methods: A prospective, observational, multicentre study;25 Italian departments of infectious diseases participatedin the project. All patients who were consecutively startedon lopinavir (LPN), tenofovir (TFV), peginterferon (IFN)and atazanavir (ATZ) were enrolled. All grade III or IVadverse events and all unexpected events are reported onthe website. Results: Between 1 October 2002 and 30 March 2004,1184 patients were enrolled: 837 males (70.7%), meanage 40.5 years (SD ±7.77) and mean CD4 lymphocytecount 314 (SD ±249); 1255 (87.4%) were experiencedand 181 naive. Mean follow-up is 10.7 months (SD±6.22).

The LPN/r cohort comprises 703 patients, the TFVcohort 585 patients, IFN 35 patients, ATZ 95 patients,T20 10 patients and TPN eight patients. A total of 100adverse events have been reported, 73 in the LPN/r groupand 15, seven, one and four with TFV, IFN, ATZ andT20, respectively. A total of 41 patients (3.4%) havestopped treatment because of toxicity.

The LPN/r cohort was big enough and followed up forlong enough (12.4 months) to permit an initial interimanalysis. Of the 703 enrolled, 493 (70.1%) are still receiv-ing treatment, 166 (23.6%) have stopped, and 44 (6.3%)are lost to follow-up. Treatment was stopped in 30 cases(4.3%) because of toxicity and in 28 (4%) because of fail-ure; 17 patients (2.4%) stopped treatment because ofgrade I or II events. The rate of adverse events per 100person-years was 14.2 on the basis of all patients treated,9.8 (95% CI 9.1–10.5) for naive patients and 15.0 (95%CI 14.6–15.4) for experienced patients. Conclusion: The online recording system for adverseevents helps to optimize the postmarketing pharmacosur-veillance and this data collection method gives timely‘real-life’ information to assess the impact of short- andlong-term toxicity of new antiretroviral drugs. In the


Abstracts

LPN/r cohort, the incidence of adverse events is lower innaive patients compared with experienced patients


Increased frequency of prescriptions and associated costs of drugs for the treatment ofantiretroviral (ARV)-induced metabolic disorders

L Normén1, B Yip2, JSG Montaner1–3, M Harris1,2, J Frohlich3, G Bondy3 and RS Hogg2,3

1 Canadian HIV Trials Network, Vancouver, BC, Canada; 2 BCCentre for Excellence of HIV/AIDS, Vancouver, BC, Canada; and3 University of British Columbia, Vancouver, BC, Canada

Background: Protease inhibitors (PI) may induce insulinresistance and dyslipidaemia in HIV+ adults. To preventcardiovascular disease, medication prescriptions to treatthese disorders have increased sharply in recent years.Objectives: To compare the increases in prescriptions andcosts of statins, fibrates and insulin sensitizing medicationsin HIV+ adults receiving ARV therapy with or without PIsin a specialized clinic for metabolic complications.Methods: HIV+ patients seen in a tertiary care clinic formetabolic complications (HIV Metabolic Clinic, HMC)between 1999–2003 were studied retrospectively. Onlypatients with complete ARV therapy records were included.Use of medications during the first 6 months after the base-line HMC visit was assessed using patient charts from theHMC and the pharmacy. Both charts have detailed formsrecording the names and dosages of medications dispensed.PI-users were categorized as such if PIs were part of theirlatest ARV regimen prior to the HMC baseline visit. Drugcosts in US$ were calculated based on the costs in the BCPharmacare Masterfile with a low-cost generic and a brandalternative, respectively. Pearson’s chi-square and t-testswere used to compare statistical differences. Results: In the analysis, 224 men and 13 women wereincluded. PI users constituted 86% of the participants. Forthe whole group, statin treatment increased from 19% atbaseline to 39% in 6 months; fibrates prescriptionsincreased from 17% to 46%; and insulin sensitizing agentsfrom 11% to 27%. The average monthly costs per patientof generic and brand-name statins were $12.95(SD=$18.32) and $13.63 (SD=$19.22), respectively. Forfibrates, the costs were $10.22 (SD=$12.11) and $14.91(SD=$17.78), respectively. For insulin sensitizing agents, thecosts were $8.32 (SD=$19.52) and $9.15 (SD=$20.43),respectively. There were no significant differences betweenPI users and non-PI users in rates of prescriptions of con-comitant drugs or their associated costs (P >0.05).Conclusion: In this specialized clinic of mostly PI-treatedpatients, the number of prescriptions of concomitant drugsto treat metabolic disorders increased over time, but associ-

ated costs did not differ between PI-treated versus non-PItreated HIV+ patients. Acknowledgements: This study was funded by theCanadian HIV Trials Network and Bristol-Myers SquibbCompany.


Effectiveness of a dedicated lipodystrophy clinic inreducing hyperlipidaemia in HIV-infectedindividuals

V Carter3, I Woolley1, K Costello1, A Dart2, K Lim3 and A Mijch1

1 Department of Infectious Diseases, Alfred Hospital, Prahran,Vic, Australia; 2 Department of Cardiology, Alfred Hospital,Prahran, Vic, Australia; and 3 Department of Nutrition, AlfredHospital, Prahran, Vic, Australia

Since March 1999, the Alfred Hospital has had a dedicat-ed, multidisciplinary clinic to address the problems of HIV-associated lipodystrophy with a focus on control ofhyperlipidaemia and management of cardiovascular risk.Included in the therapeutic team are infectious diseasesphysicians, a cardiologist, dietitians, occupational thera-pists, physiotherapists and nurses. A variety of strategiesare used, including dietary manipulation, lipid-loweringagents and change of antiviral therapy. Clinic guidelinesfor management were developed and promulgated,recommending referral of individuals with multiple risk fac-tors for cardiovascular complications. We have seen 52patients with average maximum total cholesterol of324.3 mg/dl [range 142.9–945.9 standard deviation(SD)=131] and an average maximum triglycerides of436.3 mg/dl (range 50.2–490.3, SD=440). The most recentlevels of all patients seen are equal or reduced for all patientsbut one, who has a small rise in triglycerides. Mean declinesare 106.2 for cholesterol (range of reduction 0–706.6) and266.4 for triglycerides (range of reduction –92.7 to 2316.7)to give current mean levels of 216.2 for cholesterol(SD=61.8) and 169.9 for triglycerides (SD=162.2), P valuesfor significance by t-test <0.001 for both cholesterol andtriglycerides. A dedicated multidisciplinary clinic is effectiveat reducing lipid levels significantly.



ABSTRACT 78

Antiviral Therapy 2004; 9:L46

Rosiglitazone for HIV lipoatrophy: 84 weeksfollow-up

D Carey1, C Workman2, G Rogers3, D Baker4, A Martin1, H Wand1, S Emery1, DA Cooper1,5 and A Carr5, ROSEY study group

1 UNSW, Sydney, Australia; 2 AIDS Research Initiative, Sydney,Australia; 3 University of Adelaide, Adelaide, Australia; 4 407Doctors, Sydney, Australia; and 5 St Vincent's Hospital, Sydney,Australia

Objectives: A 48-week, randomized, placebo-controlled,double-blinded study found rosiglitazone 4 mg twice dailydid not improve lipoatrophy in HIV-infected adults receiv-ing antiretroviral therapy, despite significantly improvinginsulin sensitivity and plasma adiponectin levels. Weassessed whether lipoatrophy might improve over longerfollow-up.Methods: 108 participants (55 previous placebo recipi-ents) were offered open-label rosiglitazone 4 mg twicedaily for weeks 48 to 96. Twelve participants (five fromthe rosiglitazone group) did not consent. During the open-label phase, one participant died (previous placebogroup), and 10 ceased rosiglitazone (six previous rosigli-tazone group), but continued follow-up. The study wasceased early as the blinded phase showed no benefit onlipoatrophy and adverse lipid effects. Participants com-pleted a final visit at this time. Data from weeks 84 and96 were combined to one time point (week 84).Results: Limb fat increased by 0.40 (SD=0.69) kg in therosiglitazone group and by 0.38 (SD=0.90) kg in the place-bo group at week 84 [mean difference 0.02 (95%CI,–0.46, 0.51) kg; P=0.93]. Independent baseline predictorsof greater increases in limb fat at week 84 were higher totalcholesterol (ρ=0.28, P=0.003) and greater subcutaneousthigh fat (ρ=0.04, P=0.01). There was no significancebetween group difference in subcutaneous mid-thigh fat(P=0.16), subcutaneous abdominal fat (P=0.52) or visceralfat (P=0.60) assessed by computed tomography; total bodyfat mass (P=0.80), total trunk fat (P=0.86), lean body mass(P=0.55) assessed by DEXA or the lipodystrophy case def-inition score (P=0.29); total cholesterol (P=0.72), triglyc-erides (P=0.25), glucose (P=0.51) or insulin (P=0.46). Themain adverse effects were asymptomatic hypertriglyceri-daemia (grade 3 or 4 in 58% and 49% of the rosiglitazoneand placebo groups, respectively, to week 84), hypercho-lesterolaemia (grade 3 or 4 in 26% and 18% of the rosigli-tazone and placebo groups, respectively, to week 84), andasymptomatic elevated creatinine kinase (grade 3 or 4 in42% and 20% of the rosiglitazone and placebo groups,respectively, to week 84).

Conclusion: Rosiglitazone 4 mg twice daily for 84 weeksdid not improve lipoatrophy in HIV-infected adults receiv-ing antiretroviral therapy.


Association of risk of toxicity with drug levels ofsaquinavir when boosted with ritonavir in theMaxCmin1&2 trials

JD Lundgren1, Z Fox2, US Justesen3, S Warmsley4,M Youle5, P Vernazza6, J Gerstoft7, M Losso8 andUB Dragsted1 on behalf of the MaxCmin trial groups

1 Copenhagen HIV Programme, Hvidovre University Hospital,Copenhagen, Denmark; 2 Royal Free & University CollegeMedical School, London, UK; 3 Odense University Hospital,Odense, Denmark; 4 Toronto Hospital, University Health Network,Toronto, Ont., Canada; 5 Royal Free Hospital, London, UK; 6 Kantonspital, St Gallen, Switzerland; 7 Rigshospitalet,Copenhagen, Denmark; and 8 Hospital JM Ramos Mejia, BuenosAires, Argentina

Background: Risk of drug-related toxicity is frequentlylinked to drug levels. Saquinavir drug levels are pharma-cologically boosted with ritonavir to optimize viral effica-cy. Few data exist on whether boosting results in increasedadverse events (AEs). Methods: Unplanned sub-analysis of the correlationbetween week 4 (W4) trough drug levels from the patientsin the saquinavir/ritonavir arms of the MaxCmin1&2 tri-als and adverse event outcome. Design and evaluation ofAEs and W4 drug levels were identical in both trials.Differences occurred in the choice of comparator. For thisanalysis patients were stratified based on quartiles ofdrug levels. Results: Of 309 patients initiating saquinavir/ritonavir,drug levels were available from 130 and 132 subjects atW4 and W48, respectively. Saquinavir and ritonavir druglevels were positively correlated with each other at bothtime points. Both the saquinavir [median: 1.04 µg/ml(IQR: 0.53–1.75)] and ritonavir [0.42 (0.29–0.65)] at W4were correlated with their corresponding levels at W48.No association was found between W4 saquinavir con-centrations and risk of treatment-limiting AEs, seriousAEs or premature treatment discontinuation. The propor-tion of patients with grade 3/4 AEs increased with increas-ing saquinavir concentrations (<25 percentile: 12%, >75percentile: 34%; P<0.05); only gastrointestinal (GI) toxi-city was significantly associated with saquinavir concen-trations at W4 (P<0.05). Absolute increases in cholesterollevels at W4 were independently associated with W4saquinavir quartiles (P=0.06). This association was notlinear; the significance was driven by patients in the uppersaquinavir quartile (0.5 mmol/l higher increase than other


Abstracts

quartiles). Ritonavir drugs levels were not associated withchanges in cholesterol. Conclusion: An apparent association between highersaquinavir concentrations at W4 with grade 3/4 GI toxic-ity and increases in cholesterol was identified in thisanalysis. Most patients had saquinavir drug levels that farexceed those required to inhibit wild-type HIV. This posthoc analysis did not have sufficient power to exclude asso-ciations with other toxicities. Conversely, the data suggestthat, as saquinavir troughs above 2 µg/ml far exceed theconcentration required to suppress wild-type HIV replica-tion, doses could be reduced without loss of efficacy aslong as the subjects have not acquired HIV with dimin-ished susceptibility to saquinavir.


Impact of switching PI-containing antiretroviraltherapy on lipodystrophy and metaboliccomplications, APROCO Cohort Study (ANRS EP11)

G Chene1, R Lassalle2, F Raffi3, C Michelet4, WRozembaum5, C Perronne6, C Roussillon7,F Alkaïed8, J-M Bard9, J Capeau10 and C Leport andthe Aproco-Copilote (ANRS EP11) Study Group11

1 INSERM U593, Bordeaux, France; 2 INSERM U593, Bordeaux,France; 3 Hôtel-Dieu Hospital, Nantes, France; 4 PontchaillouHospital, Rennes, France; 5 Tenon Hospital, Paris, France;6 Raymond Poincaré Hospital, Garches, France; 7 INSERM U593,Bordeaux, France; 8 Bichat-Claude Bernard Hospital, Paris,France; 9 Faculty of Pharmacy, Nantes, France; 10 INSERM U402,Paris, France; and 11 Bichat-Claude Bernard Hospital, Paris,France

Objectives: To study the evolution of clinical lipodystro-phy (LD) and metabolic abnormalities in patients contin-uing on a PI-containing regimen versus patients switchedto PI-sparing regimens.Methods: Observational cohort of patients started on PI-containing regimens in 43 French HIV clinics in1997–1998. LD was assessed using a standardized ques-tionnaire. Laboratory evaluation was performed underfasted conditions at month 12 or 20 (first measurement,M1) and 24 or 36 (second measurement, M2). A logisticregression was used to study determinants of abnormali-ties at M2.Results: A total of 76 patients switched to a PI-sparingregimen (equally nevirapine and efavirenz) at M1, and278 continued to receive the PI regimen until M2.Stavudine was associated in 59% (switch group) and 65%(maintenance group) patients at M1 (P=0.39) and in 45%and 59% at M2 (P=0.10), respectively. At M1, the switchgroup had higher mean CD4+ (496) than the maintenancegroup (419) (P=0.05). LD prevalence was 78% (24%

atrophy, 26% hypertrophy and 28% mixed syndrome) inthe switch group and 60% (21%, 15% and 24%) in themaintenance one (P=0.02). At M2, the prevalence was71% (29%, 19% and 23%) and 66% (23%, 14% and30%), respectively (P=0.29). Waist-to-hip ratio, biceps,triceps, supra-iliac skinfolds and mid-arm circumferenceremained stable at the two measurements in the twogroups. Mean HDL cholesterol levels (g/l) were higher inthe switch group than the maintenance one: 0.54 and 0.48at M1 (P=0.03); 0.50 and 0.47 at M2 (P=0.03). The evo-lution of mean cholesterol and triglyceride levels, gly-caemia and HOMA did not differ between the twogroups. In the multivariate analyses, prevalence of lipoat-rophy at M2 was significantly lower in patients who neverstarted stavudine (OR=0.34, P=0.003) than those whostopped it after M1 (OR=1.20, P=0.75) versus thosemaintaining stavudine (reference class).Conclusion: In an observational setting, patients whoswitched were those who succeeded in therapy or had LDsymptoms frequently. Switching to a PI-sparing regimenwas not associated with clear improvements in metabolicabnormalities after 1 year. Lower prevalence of peripher-al lipoatrophy at M2 may be explained by the use of othernucleosides than stavudine. Additional strategies to limitmetabolic complications are warranted in this context.


Impact of structured therapy interruptions onplasma lipids and body fat in patients withprimary HIV-1 infection

A Milinkovic, E Martínez, S Vidal, E de Lazzari, X Claramonte, O Sued, JL Blanco, JB Pérez-Cuevas,C Tortajada, J Mallolas, JM Gatell and JM Miro

Hospital Clinic, Barcelona, Spain

Background: The effect of discontinuing antiretroviraltherapy on the evolution of plasma lipids and body fat ispoorly known.Methods: After 1 year of successful antiretroviral therapywith either indinavir (n=10) or nelfinavir (n=3) plus stavu-dine and lamivudine, patients with primary HIV infectionhad four cycles of 2 months ‘on’ and 1 month ‘off’ andthen antiretroviral therapy was definitively withdrawn. Atthe end of each ‘on’ and ‘off’ period and every 3 monthsuntil 1 year after definitive antiretroviral therapy with-drawal, fasting plasma triglycerides and cholesterol, CD4cells and HIV-1 RNA were measured and body composi-tion was assessed (DEXA). Results were analysed as per-cent change in every two consecutive time points.Results: There were 11 men and two women. HIV-1 RNAafter each interruption was lower than at the diagnosis ofHIV-1 infection. CD4 cells and plasma lipids, which had



increased with antiretroviral therapy prior to the study,decreased after interruptions and increased towards base-line values after reintroductions. After definitive anti-retroviral therapy withdrawal, CD4 cells and plasmalipids showed progressive decreases, but were alwayshigher than at the introduction of antiretroviral therapy.The evolution of CD4 cells, triglycerides and cholesterolwas highly correlated. Extremity fat increased between4% and 9% in the ‘off’ periods, and it also increased evenfurther (16% each) in the first two ‘on’ periods, but finallydecreased (–0.4% and –7%) in the last two. After defini-tive antiretroviral therapy withdrawal, there was a pro-gressive fat gain in extremities (1%, –2%, 2% and 4%after 3, 6, 9 and 12 months, respectively). Fat changes inthe trunk were of lower magnitude than in extremities andnot consistent with interruptions; however, there was aprogressive fat loss in trunk after definitive antiretroviraltherapy withdrawal (-2%, –1%, 2% and –4% after 3, 6,9 and 12 months, respectively). Fat free mass remainedstable (range of changes: 0–2%).Conclusion: Structured therapy interruptions showed ashort-term positive impact on plasma lipids and body fatthat persisted 1 year after definitive discontinuation ofantiretroviral therapy.


Improvement in lipid profile in HIV-infectedvirologically controlled patients switched to asimple QD regimen: preliminary results of theCOOL trial evaluating EFV/TDF vs EFV/TDF/3TC

P Mercié1, A Trylesinski2, A Cabié3, C Michelet4, R Verdon5, C Katlama6, L Weiss7, JL Pellegrin8, D Sereni9, M Bentata10, J Durant11, A Simon6, L Morand-Joubert12, G Chêne13 and PM Girard12

1 Hôpital Saint-André, Bordeaux, France; 2 Gilead, Paris, France;3 Hôpital PZ Quitman, Fort de France, France; 4 HôpitalPontchaillou, Rennes, France; 5 CHU de Caen, France; 6 HôpitalPitié-Salpêtrière, Paris, France; 7 Hôpital Européen GeorgesPompidou, Paris, France; 8 Hôpital Haut-Levêque, Pessac, France;9 Hôpital Saint-Louis, Paris, France; 10 Hôpital Avicennes,Bobigny, France; 11 CHU, Nice, France; 12 Hôpital Saint-Antoine,Paris, France; and 13 INSERM U 593, Bordeaux, France

Objectives: Antiretroviral therapy induces metabolic abnor-malities and fat tissue redistribution. We evaluated serumlipid levels and morphological changes in virologically con-trolled patients taking HAART who switched to two sim-plified new regimens. Methods: 48 weeks, randomized, multicentre trial to assessthe benefits of switching patients under HAART for>6 months with viral load (VL) <50 copies/ml toefavirenz+tenofovir vs efavirenz+tenofovir+lamivudine. CD4

cell count, VL, fasting glycaemia and lipids were measured ateach visit. Changes in peripheral and central fat DEXA val-ues on 30 patients and CT scan in all patients [total abdom-inal tissue (TAT), subcutaneous abdominal tissue (SAT),visceral abdominal tissue (VAT), VAT/SAT, VAT/TAT andSAT/TAT ratios] were assessed at baseline (D0) and week 48.CT scan data for 78 patients at D0 and preliminary biologi-cal data on 60 patients at week 36 are presented. Results: 143 patients were enrolled, treated before ran-domization with two NRTIs + one NNRTI (43%) or PI(45%) (zidovudine+lamivudine 72% with NNRTI and PIrespectively) with mean age 42.1 ±10.1 years, 72% men,34% CDC stage C and mean HAART duration 3.71 ±1.9years. At D0, mean BMI was 23.44 ±3.41 kg/m2, medianVAT, SAT, VAT/TAT and SAT/TAT, were 82.5 cm2 (12;425), 137 cm2 (23; 477), 0.38 (0.09; 0.88) and 0.63 (0.12;0.96), respectively. At baseline, median total and LDL cho-lesterol were 5.3 mmol/l (2.8; 8.5) and 3.4 mmol/l (1.7;6.1). At week 36, median difference was –0.4 (–2.2; 1.4)(P=0.23) and –0.3 (–1.6; 1.5) (P=0.20), respectively; HDLdid not change. Median triglycerides levels was 1.4 mmol/l(0.3; 8.2) at baseline; decrease at week 36 was –0.4(–3.3;1.4) (P<0.004). At week 36, two out of four SAEpatients discontinued the trial (suicide attempt, pneumonia,dizziness and hepatic cytolysis), only one patient had VL>50 copies/ml (69 copies/ml at week 48) and ∆ CD4 atweek 36 was +23 (median CD4 of 475/mm3 at D0). TheDSMB recommended study continuation.Conclusions: Despite low lipid levels at baseline, switchingto a tenofovir based combination can mildly decrease totaland LDL-cholesterol and significantly reduce triglycerides.In patients with virologically controlled HIV infection,switching to a simplified maintenance regimen results in animproved lipid profile. The 48-weeks evaluation will assesslipid evolution and correlation between lipid disturbancesand morphological changes.


Effectiveness of ezetimibe on hyperlipidaemia inHIV-1-infected patients

FC von Lintig1, D Lee1, P Patel1, J Epp1, RE Barber1,2 and WC Mathews1

1 University of California San Diego, San Diego, Calif., USA; and2 Center for AIDS Research, San Diego, Calif., USA

Background: Ezetimibe is the first of a new class of lipid-lowering agents that do not inhibit hepatic P-450enzymes. It has not yet been studied in HIV-infectedpatients in the treatment of hyperlipidaemia.Objectives: The aim of this study was to estimate theeffect of ezetimibe on non-HDL cholesterol (non-HDLCh)


Abstracts

when added to other lipid lowering drugs (LLD) for thetreatment of hyperlipidaemia in HIV-infected patients.Methods: Retrospective analysis of medical records of 14HIV-1-infected male patients initiating ezetimibe therapyat 10 mg daily through a university-based HIV clinic(Owen Clinic). Data collected for each patient includedemographic information (age, gender and race), bodymass index (BMI), HAART regimen, duration of ezetim-ibe therapy, and pre-ezetimibe measures of CD4+ lym-phocyte counts and HIV viral loads. Because not allpatients had fasting lipid levels available, non-HDLChlevels were used and analysed at three different timepoints: prior to starting LLD (time point 1), on LLD butprior to adding ezetimibe (time point 2), and after addingezetimibe (time point 3). Differences in mean ±standarderror were estimated using repeated measures ANOVA.Pairwise comparisons were conducted using Bonferronicorrections for multiple comparisons.Results: Non-HDLCh levels were 257.4 mg/dl ±14.8 (timepoint 1), 224.3 mg/dl ±11.2 (time point 2), and 167.4±13.2 (time point 3). There was a 90.0 mg/dl or 35.0%reduction in non-HDLCh when comparing time point 1with time point 3 and a 56.9 mg/dl or 25.4% reduction innon-HDLCh when comparing time point 2 with timepoint 3. Repeated measures ANOVA showed a significantdifference among the three time points (F-ratio 17.87,P<0.00001). Bonferroni pairwise comparisons revealedthat the mean of time point 3 was significantly differentfrom the means of both time point 1 and time point 2.Conclusions: Ezetimibe appears to be useful in the treat-ment of hyperlipidaemia in HIV-1-infected patientsalready on LLD. Further prospective studies are needed toevaluate the safety, efficacy and potential drug interac-tions of ezetimibe in this patient population.


Reexposure to nucleoside analogues after lacticacidosis/hyperlactataemia syndrome

C Galera, C Redondo, G Poza, M Bermejo, P Arocaand A Sánchez

AIDS Unit, Internal Medicine Department, HUV Arrixaca Murcia,Spain

Background: Symptomatic hyperlactataemia syndrome(SHS) and lactic acidosis (LA) are serious adverse effectsof nucleoside analogue reverse transcriptase inhibitors(NRTIs), but NRTIs are essential components of theHAART regimen. The objective is to analyse the reintro-duction of NRTIs in patients who have recovered fromNRTI–SHS/LA.Methods: Observational prospective study. SHS wasdefinedasaHIVadult receivingaNRTI-containing regimen

with non-specific gastrointestinal symptoms and lactatelevels >5 mmol/l. If HCO3 levels were <20 mmol/l, LASwas diagnosed.Results: From 1 December 1999 to 1 July 2004, sixpatients have been diagnosed with SHS, five of them withlactic acidosis. All were males, 41–66 years old; HIV stageC3 (4/6) and A2 (2/6), with viral load <200 copies/ml inall but one at diagnosis. The NRTI regimens associatedwith SHS/LA were: lamivudine and stavudine (2), didano-sine and stavudine (3) and didanosine and stavudine withlamivudine (1). A patient died because of an SHS/LAepisode, and treatment was stopped between 1 to 6months in the five patients who survived. Clinical pro-gression or low CD4 count obliged the re-introduction ofHAART; in all the cases at least one and later two NRTIswere restarted. The new NRTI regimens introduced were:zidovudine/lamivudine (1), zidovudine/abacavir (2),lamivudine/abacavir (1), lamivudine/abacavir/didanosine(1), three of them with efavirenz, one with nevirapine, andthe last one only with the three-NRTI regimen. The re-exposure to each NRTI was: zidovudine 121 months(mth); lamivudine 121 months; abacavir 126 months anddidanosine 36 mths. During the first 18 months of a newregimen with NRTIs, lactate levels were measuredbimonthly, with peak levels always <3.2 mmol/l. Threeyears after NRTI re-exposure, all the patients have viralload below 200 copies/ml but one, and CD4 count >200cells/mm3, without new episodes of SHS/LA.Conclusion: Lactic acidosis syndrome is a potentially fatalcomplication of NRTIs. Re-exposure to other NRTIs,with a theoretically lower mitochondrial toxicity, is likelyto be safe and efficient. Symptoms and lactate levelsshould be closely monitored.


Persistent tuberculous adenitis due to anunsuspected drug interaction with efavirenz andrifabutin

H Edelstein and Y Cuadros

Highland General Hospital, Oakland, Calif., USA

Objective: We report two AIDS patients with persistenttuberculous adenitis who had sub-therapeutic rifabutinlevels despite megadoses of the drug due to an interactionwith efavirenz.Results: A 39-year-old Kenyan male with a CD4 count of120 cells/mm3 and a viral load of 117 000 copies/ml wastreated with daily efavirenz, tenofivir and lamivudine.After 2 weeks, a new left-sided cervical node appeared;aspirated material revealed pan-sensitive Mycobacteriumtuberculosis (TB). RIPE was initiated at standard dosesexcept for rifabutin (450 mg/d).



Over the next 6 months, the node persisted and a newright-sided node appeared. Drug levels of rifabutin, drawn2 h after ingestion, were undetectable (normal0.35–0.9 µg/ml). By month 11 the patient still had persis-tent adenopathy and sub-therapeutic drug levels(0.1 µg/ml) despite rifabutin 1350 mg/day.

Nevarapine was then substituted for efavirenz. Fifteendays later, the patient developed iritis, with toxic levels ofrifabutin (0.6 µg/ml). Dosages of rifabutin were adjustedand within 2 additional months the adenopathy had com-pletely resolved.

A 39 year-old AA male with pan-sensitive pulmonaryand nodal TB had a CD4 count of 24/mm3 and a viralload of >500000 copies/ml. RIPE was started at standarddoses (rifabutin 300 mg/day) under direct observationaltherapy. The patient clinically improved, and 6 weekslater he was started on efavirenz, tenofivir and lamivu-dine. New cervical adenopathy, fevers and anorexiaappeared 1 month after initiation of HAART. Aspirate ofthe nodes revealed +AFB stain and prednisone was initiat-ed with abatement of symptoms.

At month 5, a radiograph revealed increased pulmonaryadenopathy; expectorated sputum and a node aspirateshowed positive AFB stains with negative cultures. Isoniazidand riftabutin levels were undetectable. Nine months later,cervical adenopathy persisted and rifabutin levels were stilllow (0.3 µg/ml) despite doses of 900 mg/day. The patientwas then incarcerated. On his return, he stated that hislymph nodes had regressed and TB treatment was suspend-ed (2.5 years after his initial TB diagnosis). Clinical exami-nation and chest radiograph were normal.Conclusion: Rifabutin levels may be markedly decreasedwhen used with efavirenz, and recommended dose increas-es of rifabutin may not overcome this drug interaction.


Are patients given adequate dietary informationwhen starting on a lopinavir/ritonavir-containingHAART regimen?

MN Phillpot, E Kabaroff and TL Visser

Kobler Clinic, St Stephens Centre, Chelsea and WestminsterHospital NHS Trust, London, UK

Objectives: Kaletra (lopinavir/ritonavir), an Abbott man-ufactured protease inhibitor, is commonly used as part ofsecond-line HIV therapy.

Pharmacokinetic tests conducted by Abbott revealed amoderate fat meal containing 500–682 kcals was associ-ated with a mean increase of 48 and 23% in lopinavirAUC and Cmax respectively, relative to fasting. Abbottstate that the bioavailability of Kaletra increases withfood consumption and should be taken with food. The

HIV directorate at Chelsea and Westminster Hospitaladopted these guidelines but nothing specific was beingissued on what to eat with doses. Anecdotal evidencerevealed inconsistencies in the way patients were takingthe drug. We were concerned that patients may not beresponding to treatment effectively so investigated whatinformation was given to patients at the time of Kaletraprescription and how the drug was taken.Method: A list of patients taking a Kaletra-containingregimen was obtained (n=483). We aimed to consult 25%(n=126) in 1 month by telephone or during clinic visits.Every fifth patient on the list was chosen for interview toreduce bias. A questionnaire was designed and patientswere asked the following:

Were you given guidelines on how to take Kaletra attime of prescription?If yes, who by?Do you eat with your Kaletra doses?What do you eat with Kaletra?

A dietary recall was undertaken to obtain quantitativedata of food consumption.Results: 63 patients were interviewed (12.5%). 38 (60%)said they were told to eat with Kaletra, 20 (32%) saidthey were not given information and five (8%) could notremember. The main route of information supplied wasby the doctor and pharmacist. Regardless of informationissued, out of 101 doses analysed, 44 (43%) were takenwith adequate food and 28 (27%) were taken on anempty stomach.Conclusion: For those patients who said they were givenguidance on how to take Kaletra, a greater percentagetook it with the correct amount of food. However,patient error must be considered when carrying out ret-rospective questionnaires and dietary recall.

Further studies are required into the relationshipbetween dietary advice and drug levels, and considerationgiven to developing more specific dietary guidelines.


Autologous fat transfer for the treatment of HIV-related face lipoatrophy: a long follow-up experience

G Guaraldi1, G Orlando1, D De Fazio2, M Vigo2,I De Lorenzi2, A Rottino2, A Grisotti2, V Borghi1,G Nardini1 and R Esposito1

1 University of Modena and Reggio Emilia, Modena and ReggioEmilia, Italy; and 2 San Raffaele Hospital, Milan, Italy and Casadi cura S Pio X, Milan, Italy

Objectives: The aim of this open, prospective study was toassess subjective and objective efficacy and durability of autol-ogous fat transfer (AFT) in HIV+ people with facial atrophy.


Abstracts

Methods: Among 109 HIV-infected patients undergoingAFT for the treatment of facial lipoatrophy, 57 hadreached at least 6 months follow-up and have beenanalysed. The population was divided into three groupsaccording to post-surgery follow up (FU): group 1 (6–12months): 31 (54.4%) patients; group 2 (13–24 months):19 (33.3%) patients; and group 3 (25–36 months) seven(12.3%) patients. Subjective aesthetic results and satisfac-tion were assessed with the Visual Analogue Scale (VAS),objective efficacy and durability with ultrasound. Results: Patients’ baseline characteristics were: 32.1%female, mean age 43 ±6 years, 21% CDC group C, meanCD4 nadir 191 ±151 cells/µl, CD4 at surgery 582±248 cells/µl, median HIV-VL at surgery 9621 ±24 867copies/ml, mean HAART exposures 65 ±17 months andmean D4T exposures 44 ±19 months. Fat graft was har-vested mainly from subcutaneous abdominal fat (72%) orfrom dorsocervical buffalo hump (15%).

VAS improved in all the groups (VAS group 1 from 21±16 to 66 ±25, P=0.000; VAS group 2 from 36 ±19 to 62±24, P=0.013; and VAS group 3 from 36 ±21 to 72 ±11,P=0.013). ANOVA did not show any difference in face VASimprovement among the three follow-up groups, P=0.164.

Ultrasound evaluation showed an increase in cheek sub-cutaneous thickness (ST) in the three groups: group 1 ∆right cheek ST 3.3 ±3.05 mm, P=0.000, ∆ left cheek ST3.89 ±3.62 mm, P=0.000; group 2 ∆ right cheek ST 5.6±2.6 mm, P=0.000, ∆ left cheek ST 5.5 ±2.8 mm, P=0.000;and group 3 ∆ right cheek ST 5.89 ±4.67 mm, P=0.027, ∆left cheek ST 5.7 ±4.1 mm, P=0.020. ANOVA showed nodifference in subcutaneous thickness between groups. Conclusion: Our results showed that autologous fat trans-plant is effective and durable over time for correction oflipoatrophy in HIV-infected people.


Relapse of HIV-related buffalo hump after liposuction

G Orlando1, G Guaraldi1, D De Fazio2, A Grisotti2,V Borghi1, G Nardini1, B Beghetto1 and R Esposito1

1 University of Modena and Reggio Emilia, Modena and ReggioEmilia, Italy; and 2 San Raffaele Hospital, Milan, Italy

Objectives: Traditional and ultrasound assisted liposuc-tion and lipectomy are potential surgical treatments forbuffalo hump. The primary objective of this retrospectivestudy was to analyse the durability of the surgical result 1year after treatment and the mean time to reinterventiondue to buffalo hump relapse. The secondary aim was toverify subjective satisfaction for the aesthetic result. Methods: Patients treated with liposuction for buffalohump were analysed 1 year after surgery The objective

variable was ultrasound measurement of the subcuta-neous and dermal dorsocervical fat accumulation. Visualanalogue scale (VAS), MOS-HIV and assessment of bodychange and distress (ABCD) questionnaires were used totest patients’ satisfaction with the aesthetic result. Results: In a cohort of 53 consecutive patients treatedwith liposuction for buffalo hump, 20 HIV patients hadbeen analysed 1 year after surgery. 92.4% patients hadundergone mechanically assisted liposuction while 7.6%had ultrasound assisted liposuction. Patients’ baselinecharacteristics were: 30% female, mean age 43 ±7 years,mean BMI 26 ±3, HAART mean exposure 67 ±16months, mean CD4 cells nadir 150 ±38 cells/µl, mean glu-cose 98 ±12 mg/dl, mean cholesterol 214 ±45 mg/dl andmean triglycerides 191 ±120 mg/dl. Two patients (10%)asked for re-intervention due to buffalo hump relapse at amean time of 10.5 months. Fat removal in re-interventionwas poorer due to the increase of fibrosis.

Ultrasound did not show any statistically significant dif-ference in dorsocervical fat thickness: ∆ dermal thickness0.044 ±2.7, P=0.962, ∆ subcutaneous thickness 9.1 ±12.3,P=0.056. VAS improved from 25.6 ±20 to 59.6 ±24,P=0.000. MOS-HIV resulted in a significant improvementof the mental health score (∆ 8.7 ±14, P=0.025) and in aworsening of pain domain score (∆ 17 ±26, P=0.018).ABCD revealed an improvement in general body satisfac-tion (1 ±1.08 P=0.006), but not in the general psychosocialdistress due to buffalo hump (∆ 2.18 ±17, P=0.680).Conclusion: Objective evaluation with ultrasound didnot reveal a statistically significant variation of subcuta-neous and dermal fat thickness 1 year after surgery. 10%of patients needed a re-intervention. Patients expressedimproved satisfaction with body image after surgery,nevertheless surgery did not relieve the psychosocial dis-tress linked to buffalo hump as shown by the ABCDquestionnaire.


Psychometric evaluation of patients undergoingsurgical treatment of HIV-related facial lipoatrophy

G Orlando1, G Guaraldi1, M Vandelli2, M DePalma2, D Comelli2, G De Santis1, A Pedoni1, ASpaggiari1, A Baccarani1, M Pinelli1, D De Fazio3,V Borghi1, G Nardini1, B Beghetto1 and R Esposito1

1 University of Modena and Reggio Emilia, Modena and ReggioEmilia, Italy; 2 Psychosocial Service, Azienda Ospedaliera Policlinico,Modena, Italy; 3 San Raffaele Hospital, Milan, Italy and Casa di CuraS Pio X, Milan, Italy

Objectives: Surgery seems to be the only effective treat-ment for HIV-related face lipoatrophy. The aim of this



study was to analyse the role of available psychometricquestionnaires to valuate quality of life (QoL), depressionand body image perception in a cohort of patients under-going surgical treatment for face lipoatrophy.Methods: MOS-HIV, ABCD and Beck questionnaireswere used to evaluate the impact of surgery respectivelyon QoL, depression and body image perception 6 monthsafter surgery. A VAS was used to assess satisfaction withaesthetic results. Results: 59 HIV patients reporting facial lipoatrophy wereenrolled. 24 underwent an autologous fat transfer (AFT),20 received injections of polylactic acid (PLA) and 15 ofpolacrylammide (PAC).

VAS increased in AFT group from 24.7 ±16.4 to 70.2±21.9, P=0.000, in PLA from 20 ±20 to 82.8 ±16.7,P=0.001, in PAC from 25.3 ±21.7 to 82.8 ±17.1,P=0.000. QoL improved in the AFT group in the areasof vitality/energy, mental health and social functioningbut with P=NS. In the PLA group, cognitive functioningimproved (∆=13.5 ±24.6, P=0.038) and in the PAC groupmental health summary score (∆=4.6 ±6.9, P=0.020), cog-nitive functioning (∆=9.06 ±15.4, P=0.033), health dis-tress (∆=18.4 ±24.5, P=0.009), mental health (∆=13.2±17.1, P=0.007), social functioning (∆=20 ±30, P=0.018),role functioning (∆=21.8 ±32.7, P=0.017) improved.ANOVA showed statistically significant differences indomains of physical health score (P=0.015), cognitivefunctioning (P=0.005), health distress (P=0.020) and rolefunctioning (P=0.000) between the three groups.

Depression score improved in the AFT group withP=NS, in the PLA group (∆ score 3.4 ±6.09, P=0.022) andin the PAC group (∆ score 6.25 ±7.8, P=0.006).

Body image satisfaction (BIS) improved in all threegroups (AFT: DBIS 0.31 ±1, P=0.050; PLA: DBIS 1.1 ±1.3,P=0.002; PAC DBIS 1.07 ±1.3, P=0.010). Psychologicaland social distress score (PSD) improved in all groups:AFT DPSD 2.2 ±16, P=NS; PLA DPSD 18.2 ±20,P=0.011; PAC DPSD 13.6 ±16, P=0.011. Comparinggroups, PLA patients expressed a greater satisfaction withbody image than AFT patientsConclusion: AFT, PLA and PAC treatment resulted in animprovement of body image satisfaction. MOS-HIV ques-tionnaires showed improvement in QoL domains just inthe PLA and PAC groups. Beck scale revealed an amelio-ration of the depression score in PLA and PAC groups.


Long-term follow-up of graft hypertrophy afterautologous fat transfer for HIV-related facelipoatrophy (hamster syndrome 1 year later)

G Guaraldi1, G Orlando1, D De Fazio2, A Grisotti2,V Borghi1, G Nardini1, B Beghetto1 and R Esposito1

1 University of Modena and Reggio Emilia, Modena and ReggioEmilia, Italy; and 2 San Raffaele Hospital, Milan, Italy and Casadi Cura S Pio X, Milan, Italy

Objective: At the 5th International Workshop on AdverseDrug Reactions and Lipodystrophy in HIV we presentedfour patients with facial lipoatrophy who underwentautologous fat transplantation and had a disfiguring fatgraft hypertrophy of the face occurring at the same timeas recurrent fat accumulation in the tissue harvest site(buffalo hump three cases, abdomen one case). Patientsdescribed themselves as ‘hamsters’. This report focuses onthe clinical and surgical follow up of these cases.Methods: Patient 1 did not receive any surgical correctiondue to the occurrence of a prostatic lymphoma. Patient 2was treated with facial liposuction with very limited successdue to the development of a highly fibrotic cheek subcuta-neous tissue. Patient 3 was treated with facial liposuctionwith collection of 100 ml of fat from cheek subcutaneoustissue. Patient 4 was treated with cervico-facial face lifting.Ultrasound evaluation was performed to show the progres-sive change in cheek subcutaneous fat thickness.Results: Patients baseline characteristics were: mean age44 ±11 kg, mean BMI 27 ±4, mean HIV infection dura-tion 8.5 ±2.3 years, mean HAART exposure 75 ±6months, mean D4T exposure 60 ±11 months, mean PIexposure 54 ±15 months, mean CD4 nadir 124 ±121cells/µl, mean CD4 at surgery 436 ±234 cells/µl, mean glu-cose 107 ±25 mg/dl, mean triglycerides 366 ±44 mg/dland mean total cholesterol 209 ±32 mg/dl.

A progressive increase of subcutaneous fat (SF) thick-ness in cheeks for all the patients after first surgery wasnoted: at 6 months after surgery ∆SF right cheek 8.5 ±0.4,P=0.001 and ∆SF left cheek 6.75 ±2.9, P=0.057; at 12months follow-up ∆SF right cheek 6.1 ±0.75, P=0.005,and ∆SF left cheek 7.6 ±2.6, P=0.037; at 24 months ∆SFright cheek 0.6 ±2.6, P=0.648 and ∆SF left cheek 0.07±0.9, P=0.880. In the three patients who underwent a sur-gical correction, ultrasound did not show a significantdecrease in SF thickness in cheeks.Conclusions: A clinical implication is that when autologousfat transplantation is chosen for facial atrophy treatment,the subcutaneous adipose graft site should not be the buf-falo hump area. Liposuction from the face after fat grafttransplant may be complicated by the development of afibrotic tissue and may have only limited aesthetic success.


Abstracts


HOMA-IR in the assessment of insulin resistancein HIV patients

E Álvarez García1, C Miralles Álvarez2, A RepárazAndrade1, D Rodríguez Pérez1 and A López Domínguez2

1 Clinical Laboratory, Universitary Hospital Complex of Vigo(CHUVI), Vigo, Spain; and 2 Internal Medicine, UniversitaryHospital Complex of Vigo (CHUVI), Vigo, Spain

Background: Hyperinsulinaemia and insulin resistancehave been well established among HIV-positive patientson therapy. Insulin resistance may have serious metabolicand cardiovascular implications. To evaluate insulin resis-tance, we used HOMA-IR values that correlate well withthe findings of more invasive techniques.Methods: Control group: 43 healthy volunteers (17 males,26 females), age range 23–63 years (mean 39.77), withnormal BMI (mean 22.4) and fasting glucose <100 mg/dl.Group A (HIV+ without treatment for at least 3 monthsbefore): 98 patients (53 males, 45 females), age range15–58 years (mean 37.69) with normal BMI (mean 22.0).Group B (HIV+ on HAART): 242 patients (155 males, 87females), age range 14–80 years (mean 40.22) with nor-mal BMI (mean 22.0).

Fasting glucose was assessed by hexokinase method(AU5400™ Olympus). Serum insulin was determined byan immunometric chemiluminescent assay (std WHONIBSC 1st IRP 66/304) (Immulite™ 2000 DPC).

HOMA-IR = (insulin × glucose)/22.5. The statisticalanalyses used SPPS 9.0. Insulin and HOMA-IR wereanalysed with values ln transformed. Results: Results are expressed as median and 5–95 per-centile. Control group: glucose: 89 mg/dl (71.4–98.0);basal insulin 6.02 µUI/ml (2.20–10.14); HOMA: 1.32(0.45–2.23). Group A: glucose: 89.5 mg/dl (75.0–108.5);basal insulin 7.91 µUI/ml (2.71–33.15); HOMA: 1.82(0.59–7.78). Group B: glucose: 92.0 mg/dl (76.0–112.0);basal insulin 8.42 µUI/ml (3.14–25.26); HOMA: 1.91(0.61–6.39) One-way ANOVA test shows reliable statisti-cal differences among groups in the HOMA index;F(82.380)= 9.586, P<0.0001.

Scheffe test: control group versus group A: P(α=0.05)<0.05; control group versus group B: P(α=0.05) <0.05;and group A versus group B: P(α=0.05)=0.547.Conclusion: HOMA-IR was significantly higher in theHIV-positive groups compared with the control group.These data suggest that the tendency towards insulin resis-tance may be more related to the HIV infectious processor to factors associated with immunological dysfunctionrather than the HAART. A deeper study including CD4cell counts and considering IP treatments should be donein order to confirm these results.


Abdominal obesity predicts insulin resistance butnot to fasting triglyceride concentration in malestreated with PI-containing HAART

RB Cavalcanti and S Walmsley

University of Toronto, Toronto, Ont., Canada

Background: Abdominal obesity (as defined by increasedwaist circumference) is correlated with elevated triglyc-erides and insulin resistance in non-HIV-infected subjectswith the metabolic syndrome. Increased waist circumfer-ence is also a predictor of cardiovascular risk. This studyaims to evaluate whether waist circumference correlateswith insulin resistance and triglyceride measurements inmale individuals treated with protease inhibitor (PI)-con-taining HAART regimens.Methods: HIV-infected, non-diabetic males with lipodys-trophy and treated with PI-containing HAART wereassessed for waist circumference (WC), fasting lipid pro-file, insulin and glucose. Homeostasis model assessment(HOMA-IR) and quantitative insulin sensitivity check(QUICKI) were calculated to assess for insulin resistance(IR) (defined as HOMA IR >3.8 or QUICKI <0.33).Pearson correlation coefficients were used to estimate cor-relation between WC and triglycerides, HDL-cholesterol,HOMA-IR, QUICKI and fasting glucose values. Subjectswere classified as having an abdominal obesity (AO)based on WC ≥102 cm. Mean HOMA-IR, fasting glucoseand triglyceride values were then compared between thosewith and without AO using two-tailed Student’s t-test.Results: Eighty-nine males were studied (mean age 42 yr,mean CD4 131, mean BMI 24.2), of which 12 (13.5%)had WC ≥102 cm; 15 (17%) had insulin resistance byHOMA-IR and 31 (35%) had IR by QUICKI.Correlations between WC and fasting glucose, HDL-cholesterol and triglycerides were weak and not signifi-cantly different from zero. Correlations between WC andmeasures of insulin resistance were significant for QUIC-KI (ρ=–0.42 P<0.001) and approached significance forHOMA-IR (ρ=0.20, P=0.05). Subjects with AO had sig-nificantly lower insulin sensitivity as measured by QUIC-KI (0.32 vs 0.34, P<0.019) compared with those withoutAO. However, the group with AO did not differ from thenon-AO group with regards to HOMA-IR, fasting glu-cose, HDL-cholesterol or triglyceride levels. Discussion: Similar to what is found other populations,AO is correlated with insulin resistance in this group ofmales treated with PI-containing HAART. However,fasting triglyceride levels were not correlated with AO,suggesting that mechanisms accounting for hypertriglyc-eridaemia in patients receiving PI differ from thoseobserved in the metabolic syndrome.




Myo-inositol/D-chiro-inositol metabolism in HIVlipodystrophy syndrome

J Placek, M Palmer, J Currier and G Vasquez

University of California Los Angeles AIDS Institute, Los Angeles,Calif., USA

Objectives: D-chiro-inositol is an important compoundconverted from myo-inositol that mediates the response toinsulin, and a deficiency may be an early marker for post-receptor insulin resistance. D-chiro-inositol deficiency canbe observed prior to the clinical manifestation of insulinresistance. In insulin resistance, an increased concentra-tion of myo-inositol and a decreased concentration of D-chiro-inositol is observed. We were interested in therelationship between myo-inositol and D-chiro-inositolmetabolism, ARV treatment and HIVLS. Methods: This was a cross-sectional pilot study to investi-gate the state of D-chiro-inositol metabolism in individu-als with HIV lipodystrophy syndrome (HIVLS). At entry,three groups of subjects with HIV were evaluated: GroupI: PI-treated patients with HIVLS (diagnosis of HIVLSwas based on self-reported body changes, waist/hip ratioof >0.95 for males and 0.85 for females or a waist cir-cumference >100 cm); Group II: patients using antiretro-viral therapy but who did not have HIVLS; and Group III:subjects who had neither HIVLS nor were using anti-retroviral therapy. All subjects had fasting insulin<15 iu/ml, fasting glucose <6.1 mmol/l and blood glucose2 h after a 75-gram oral glucose tolerance test<7.8 mmol/l. A 24-h urine sample was collected from eachpatient and analysed for levels of myo-inositol andD-chiro-inositol. The urine levels of myo-inositol andD-chiro-inositol were compared within and betweengroups.Results: 17 subjects participated in the study. Group I(HIVLS) had four patients, Group II (PI) had five sub-jects and Group III (no PI) had eight subjects. Meanmyo-inositol/D-chiro-inositol was 398.04, SD=436.61(HIVLS), 517.86, SD=399.06 (PI) and 511.24,SD=424.14 (no PI). Using ANOVA and Kruskal–Wallis (aparametric and nonparametric test, respectively) therewas no significant difference between the groups in anyvariable except WHR (which was an inclusion criteria forGroup I, HIVLS). Conclusion: The results demonstrate that there is proba-bly not a difference in myo-inositol/D-chiro-inositol whenpatients with HIVLS are compared with those on PIs butwithout HIVLS, and to those with HIV but not on a PI.However, the ratios are quite high for all three groupscompared with those seen in normal healthy controls in

the published literature. Perhaps insulin resistance isinvolved with HIV pathophysiology.


Resistin decreases after 3 months of rosiglitazonein HIV-infected patients with lipodystrophy

DS Kamin1, C Hadigan1, M Lehrke2, MA Lazar2 andS Grinspoon1

1 Program in Nutritional Metabolism, Massachusetts GeneralHospital, Harvard Medical School, Boston, Mass., USA; and2 Division of Endocrinology, Diabetes, and Metabolism and PennDiabetes Center, University of Pennsylvania School of Medicine,Philadelphia, Pa., USA

Background: HIV lipodystrophy is characterized by insulinresistance as well as abnormalities in circulating inflamma-tory markers and coagulation mediators. Resistin is aPPAR-γ responsive adipocyte-derived hormone that may belinked to insulin resistance and abnormalities in inflamma-tory tone.Aim: To determine whether improvements in insulin sensi-tivity induced by the PPAR-γ agonist rosiglitazone are relat-ed to changes in resistin among HIV-infected subjects withinsulin resistance and lipodystrophy. Methods: We administered rosiglitazone for 12 weeks to 25HIV-infected men and women (male/female 19/6; age 46±2 years; BMI 26 ±1 kg/m2; mean ±SD; nine subjectsreceived 8 mg/day, 16 subjects 4 mg/day) with insulin resis-tance (fasting insulin >15 µU/ml or 2-h insulin >75 µU/mlfollowing 75 g oral glucose challenge) and lipodystrophy(evidence of fat redistribution including lipoatrophy presentin the face, arms, hips or legs, by patient self-report and con-firmed on physical examination by a single investigator). Inaddition to fasting determination of adipocytokines andinflammatory markers, subjects completed hyperinsuli-naemic-euglycaemic clamp testing at baseline and 12 weeks. Results: Circulating resistin decreased significantly from6.37 ±0.55 ng/ml to 5.38 ±0.56 ng/ml (P=0.02), adiponectinincreased (2.8 ±0.4 µg/ml to 5.9 ±0.7 µg/ml; P=0.0001),whereas leptin (6.97 ±1.67 ng/ml vs 5.92 ±1.39 ng/ml), cir-culating markers of inflammation (TNF-α, CRP) and coag-ulation (PAI-1, tPA) did not change. There was nodifference in baseline resistin levels between subjectscurrently on a protease inhibitor (PI) and those not on a PI(P=0.33). Resistin and resistin response to rosiglitazone didnot correlate with levels of inflammatory markers.Circulating leptin was positively correlated with CRP(r=0.60; P<0.001), and negatively correlated with tPA(r= –0.44; P<0.05). Additionally, change in resistin levelsdid not correlate with changes in insulin sensitivity (mea-sured by hyperinsulinaemic-euglycaemic clamp testing)following 12 weeks of rosiglitazone.


Abstracts

Conclusion: Among HIV-infected patients with insulinresistance and lipodystrophy, PPAR-γ agonists administra-tion results in decreased resistin levels, a putative adipocy-tokine which may regulate insulin sensitivity. Furtherinvestigation into the mechanistic role of resistin in insulinresistance among HIV-infected patients with lipodystrophyis warranted.


No evidence for reduced insulin-mediated glucoseuptake in skeletal muscle with 4 weeks ofindinavir in healthy men

SS Shankar1,2, MP Dubé1,3, RV Considine1,2, L Christner1,2 and HO Steinberg1,2

1 Indiana University School of Medicine, Indianapolis, Ind., USA;2 Division of Endocrinology, Indiana University School ofMedicine, Indianapolis, Ind., USA; and 3 Division of InfectiousDiseases, Indiana University School of Medicine, Indianapolis,Ind., USA

Background: Indinavir (IDV) has been reported to reduceinsulin sensitivity and impairs endothelial function andnearly abrogates basal nitric oxide-dependent tone inhealthy men. We hypothesized that IDV-induced insulinresistance is due to impaired GLUT4 activity at the levelof skeletal muscle. Methods: We assessed leg glucose uptake (LGU), whole-body insulin sensitivity and endothelial function in 16lean, healthy male subjects before and after 4 weeks ofIDV 800 mg thrice daily. Arteriovenous glucose difference(AVG∆), a measure of glucose extraction at the level ofskeletal muscle which depends on GLUT4 activity, wasmeasured after 4 h of steady-state (SS) euglycaemic hyper-insulinaemia and after lowering LBF with a superimposedarterial infusion of the NO synthase inhibitor, NG-monomethyl-L-arginine (LNMMA) at 16 mg/min for15 min. LGU was calculated as AVG × LBF. Results: There were no changes in body measurements,blood pressure, whole-body insulin sensitivity by hyperin-sulinaemic clamp, or lipids after IDV. Mean (±SEM) plas-ma adiponectin levels increased (16.4 ±2.2 µg/ml pre-IDVvs 19.1 ±2.3 µg/ml post-IDV, P<0.05). AVG at SS washigher after IDV (26 ±4 mg/dl pre-IDV vs 31 ±5 mg/dlpost-IDV, P=0.05). However, rates of LGU did not differ(91 ±1 mg/min pre-IDV vs 85 ±2 mg/min post-IDV, P=NS).LBF at SS was lower after IDV (0.35 ±0.03 l/min pre-IDVvs 0.29 ±0.02 l/min post-IDV, P<0.05) and decreased inresponse to LNMMA before and after IDV (–39 ±5% pre-IDV vs –21 ±11% post-IDV), with a resultant comparablefinal LBF value (0.21 ±0.021 l/min pre-IDV vs 0.225±0.025 l/min post-IDV, P=NS). The expected increase inAVG in response to LNMMA was robust and comparable

before and after IDV. Rates of LGU during LNMMA weresimilarly comparable (76 ±12 mg/min pre-IDV vs 69±11 mg/min post-IDV, P=NS). Conclusions: In healthy subjects, 4 weeks of IDV did notimpair glucose extraction at the level of skeletal muscle,suggesting no impairment of GLUT4 transport activity invivo, in spite of a marked impairment in endothelium-dependent vasodilation. This could imply that thedecrease in large vessel endothelial function primarilyaffected non-nutritive perfusion, while nutritive perfusionwas preserved. Alternatively, skeletal muscle glucoseuptake may be independent of large vessel endothelialfunction, or IDV-related increases in adiponectin may pro-tect against insulin resistance. Acknowledgements: This study was supported by thesegrants from the NIH: HL 72711, AI 052852, and GeneralClinical Research Center grant M01-RR00750.


HAART modulates liver lipid metabolism throughdirect effect on hepatocytes



Objective: HAART has been associated with overt hyper-lipidaemia, hepatic steatosis and hepatic insulin resistance.Whole body lipid regulation is mainly controlled at the levelof hepatocytes and adipocytes by nuclear transcription fac-tors which modulate the expression of lipogenic genes.Sterol regulatory element-binding proteins (SREBPs) havebeen previously identified as regulators of triglycerides,cholesterol and fatty acid metabolism. We aimed to checkthe hypotheses that NRTIs and protease inhibitors eventu-ally modulate SREBP-dependent genes in hepatocytes.Methods: The effects of indinavir (IDV) and zidovudine(AZT) on the inhibition/activation of SREBP-1-dependentgenes were measured by incubation of HUH7 cells withdifferent concentrations of the drugs (IDV 5–200 µM,AZT 6–1000 µM) for 24 h prior to mRNA isolation andreverse transcription. Transcription profiles of SREBP-1,FAS (fatty acid synthase), LDL-receptor (LDL-R) andGLUT2 expression were created using real-time PCR. Theeffects of ritonavir (RTV) on the inhibition/activation ofSREBP-1-dependent genes were measured by incubationof HepG2 cells with different concentrations (RTV 5–30 µM) of the drug for 24 h prior to mRNA isolationand reverse transcription.Results: IDV and AZT activated SREBP-1 and SREBP-dependent genes encoding FAS and LDL-R. AZTincreased the expression of GLUT2, whereas IDV did nothave any effect on this SREBP-independent gene. RTV



activated SREBP-1 and SREBP-dependent genes encodingFAS and acetyl-coenzyme A carboxylase (ACC). Theeffects were studied dose dependently and significantincrease versus control was observed at concentrations ofclinical relevance (IDV: SREBP-1 96%, P=0.0458, FAS61%, P=0.0041, LDL-R 75%, P=0.001; AZT: SREBP-1100%, P=0.0148, FAS 109%, P=0.0379, LDL-R 118%,P=0.001; RTV: SREBP1 50%, P=0.0339, FAS 200%,P=0.0206). Conclusions: IDV, RTV and AZT activate importanteffector genes of the SREBP-1 pathway. This is consistentwith major HAART-related adverse effects like hyperlipi-daemia and hepatic lipid accumulation. The short-termeffect of AZT on lipogenic gene expression attributesadditional mechanisms to this drug other than mitochon-drial toxicity.


Changes in nuclear gene expression resulting fromNRTI-induced inhibition of mitochondrialtranscription reveal links between mitochondrialdysfunction and lipid metabolism

PWG Mallon1–3, R Sedwell1,3, P Unemori1,3,M Rafferty4, K Williams4, D Chisholm5, K Samaras5,S Emery1, A Kelleher1–3, DA Cooper1–3 and A Carr2

1 National Centre in HIV Epidemiology and Clinical Research,University of New South Wales, Sydney, Australia; 2 HIVImmunology and Infectious Diseases Clinical Services Unit,St Vincent’s Hospital, Sydney, Australia; 3 HIV ImmunovirologyResearch Laboratory, St Vincent’s Hospital, Sydney, Australia;4 Clinical Trials Centre, St Vincent’s Hospital, Sydney, Australia;and 5 Garvan Institute of Medical Research, Sydney, Australia

Background: Use of nucleoside reverse transcriptaseinhibitors (NRTI) has been associated with both mito-chondrial dysfunction, detectable in vivo after 2 weeks asdown-regulation of mitochondrial RNA (mtRNA) tran-scription, and lipoatrophy. The mechanisms wherebymitochondrial dysfunction leads to lipoatrophy are poor-ly understood.

We examined changes in nuclear and mitochondrialgene expression in subcutaneous adipose tissue biopsiesfrom 20 HIV-negative healthy volunteers randomized to 6weeks of standard dose zidovudine and lamivudine(AZT/3TC) or stavudine and lamivudine (d4T/3TC),using real-time quantitative RT-PCR. Expression of genesof interest was presented as a ratio to β-actin expression.Simultaneous to the coordinated down-regulation ofmtRNA transcription [median% changes [IQR], COX1–63% (77), COX3 –88% (54) and cytochrome b –60%(62); all P≤0.005], expression of PPARγ, important foradipocyte differentiation, was also significantly decreased

[–50% (50), P=0.003]. In contrast, nuclear-encoded genesthat regulate mtRNA transcription were significantly up-regulated [PGC1 +83% (137), P=0.02; NRF1 +25% (116),P=0.01; and mtTFA +42% (131), P≤0.01). Genes responsi-ble for fatty acid oxidation were also up-regulated [PPARα+50% (102), P=0.002 and LPL +55% (153), P=0.01] aswas expression of leptin [+85% (180), P=0.02]. Thesechanges occurred in the absence of mitochondrial DNAdepletion. Many of the changes in nuclear gene expressioncorrelated with changes in PGC1 expression, suggesting acentral role for this transcriptional co-activator in mediat-ing nuclear responses to mitochondrial dysfunction.

These results suggest significant compensatory changesin nuclear gene expression of genes important for lipidmetabolism in response to NRTI-induced inhibition ofmtRNA expression in vivo. Decreases in PPARγ expression,previously described with protease inhibitor exposurenow seen with NRTI exposure, help explain how NRTI-induced mitochondrial dysfunction may lead to lipoatro-phy and offer a molecular explanation for the increasedseverity of lipodystrophy, observed clinically when NRTIsand PIs are used in combination.


Nucleoside reverse transcriptase inhibitors (NRTI)decrease adipocyte and monocyte mitochondrial(mt) messenger RNA transcription in the absenceof changes in mtDNA or cell morphology

PWG Mallon1–3, R Sedwell1,3, P Unemori 1,3,K Merlin3, C McGinley3, P Ammaranond1,3,M Peperias3, M Rafferty4, K Williams4, K Samaras6,AL Morey5, D Chisholm6, A Kelleher1–3, DA Cooper1–3

and A Carr2

1 National Centre in HIV Epidemiology and Clinical Research,University of New South Wales, Sydney, Australia; 2 HIVImmunology and Infectious Diseases Clinical Services Unit,St Vincent’s Hospital, Sydney, Australia; 3 HIV ImmunovirologyResearch Laboratory, St Vincent’s Hospital, Sydney, Australia;4 Clinical Trials Centre, St Vincent’s Hospital, Sydney, Australia;5 Department of Anatomical Pathology, St Vincent’s Hospital,Sydney, Australia; and 6 Garvan Institute of Medical Research,Sydney, Australia

Long-term therapy with nucleoside analogue reverse tran-scriptase inhibitors (NRTIs) can lead to lipoatrophy.Although NRTIs inhibit adipocyte DNA polymerase γ(DNA pol-γ) in vitro affecting mitochondrial (mt) DNAreplication, it is unclear if mtDNA depletion is the prima-ry defect in NRTI-induced mitochondrial toxicity.

We examined mtRNA expression from adipose tissueand peripheral blood monocytes, and mtDNA copynumber and cell morphology, in adipose tissue from 20


Abstracts

HIV-negative, healthy, adult volunteers randomized to 6weeks standard dose stavudine and lamivudine (d4T/3TC)or zidovudine and lamivudine (AZT/3TC), followed by6 weeks washout. Assessments included fasting lipids andglucose, and measurement of body composition. Fat biop-sies were performed at weeks 0 and 2 and monocytescollected at weeks 0, 6 and 12. RNA and DNA wereextracted and mtRNA expression and mtDNA copy num-ber measured by real-time RT-PCR.

Adipose tissue mtRNA expression decreased significant-ly by week 2 [median % change (IQR), COX1 –63% (77),COX3 –88% (54) and cytochrome b –60% [62], P=0.001–0.005] without evidence of mtDNA depletion (85(53) and 75 (102) ng/cell at week 0 and week 2 respective-ly, P=0.1) or changes in microscopic appearances of mito-chondria or adipocyte number [62 (23) and 59 (36) cellsper high power field at week 0 and week 2 respectively,P=0.6]. Changes in mtDNA and mtRNA did not correlate.

Monocyte mtRNA expression (COX1) also decreased toweek 6 [–38% (36), P=0.01], an effect that persisted to week12 [–42% (101), P=0.005], 6 weeks after stopping studydrug. Changes in adipose COX1 at week 2 did not correlatewith changes in monocyte COX1 expression at week 6.

Independent of HIV infection, mtRNA expression inadipose tissue and monocytes decreases early after expo-sure to AZT/3TC or d4T/3TC. In adipose tissue, thiseffect occurs without evidence of mtDNA depletion orchange in ultrastructure and in monocytes persists 6weeks after stopping drug. These data suggest that NRTIsinhibit mtRNA transcription by a means other thanthrough mtDNA depletion mediated through DNA pol-γinhibition, and that monocyte mtRNA expression may bea useful peripheral blood surrogate to monitor NRTI-induced mitochondrial toxicity.


Favourable increases in high-density lipoproteincholesterol (HDL-C) in HIV-infected therapy naivesubjects receiving fosamprenavir/ritonavir in theSOLO/APV30005 Study — 120 week analysis

J Flamm1, M Lorber2, D Thomas3, N Givens3 andT Stark3

1 Kaiser Permanente Medical Group Sacramento, Calif., USA;2 Institute Clinical Immunology, Allergy and AIDS, RAMBAMMedical Center, Haifa, Israel; and 3 GlaxoSmithKline R&D US andEurope

Background: Chronic HIV infection is associated withdecreases in total cholesterol (TC), low-density lipoproteincholesterol (LDL-C), and high-density lipoprotein choles-terol (HDL-C). In HIV-negative subjects, low HDL-C(<40 mg/dl) and high TC/HDL-C ratios are associated with

an increased risk for cardiovascular disease. Proteaseinhibitors have often been associated with increases in TCand LDL-C but little change in HDL-C. Method: 120-week lipid data for antiretroviral treatment-naive male (M) and female (F) subjects who completed ≥48weeks on a fosamprenavir/ritonavir 1400 mg/200 mg once-daily (FPV/r once daily; Lexiva®; Telzir®) regimen in SOLOand continued FPV/r once daily (n=211) in APV30005, arepresented. Samples taken in fasted state were analysed.Results: 174 (F: 49; M: 125) subjects provided fasted lipidat baseline (BL). At BL, median HDL-C level was 36 mg/dl(F: 43 mg/dl; M: 34 mg/dl). After starting FPV/r once daily,HDL-C increased over 48 weeks by a median of 8 mg/dl(+21%; F: +26%; M: +19%) with further median increasesfrom BL of 12 mg/dl (+36%; F: +28%; M: +41%) at week96 and 13 mg/dl (+35%; F: +32%; M: +35%) at week 120.The interquartile ranges of the median HDL-C increases atweek 120 were: F +7% to +80% and M +9% to +59%.Overall, TC increased by a median of 53 mg/dl (+31%) atweek 48, 47 mg/dl (+30%) at week 96 and 51 mg/dl(+29%) at week 120. Median changes in the TC/HDL-Cratio were +0.37 at week 48. Notably, after week 48, themedian change from BL in the TC/HDL-C ratio becamenegative (–0.01 at week 96 and –0.07 at week 120). Theproportion of subjects with low (<40 mg/dl) HDL-Cdecreased from 62% (108/174) at BL to 36% (64/177) atweek 48 and 27% (40/149) at week 96 and then remainedunchanged (31% (47/154) at week 120). The proportion ofsubjects with a TC/HDL-C ratio >6.5 did not change overtime [11% (14/130) at BL, 9% (12/130) at week 120].Conclusion: In chronically HIV-infected ART-naive patientswith overall low BL HDL-C, substantial increases in HDL-C combined with minimal changes in the TC/HDL-C ratiowere observed following long-term treatment with FPV/ronce daily over 120 weeks.


Long-term efficacy of nelfinavir, adverse eventsand lipodystrophy

E Daniels1, B Clotet2, P Clax1, C Artega1 and M Nelson3

1 Pfizer, Inc., New York, NY, USA; 2 IrsiCaixa Foundation,Germans Trias i Pujol University Hospital, Barcelona, Spain; and 3Chelsea and Westminster Hospital, London, UK, for the AG1343-1260 Study Team

Objective: Nelfinavir (nelfinavir mesylate) is a potent andwell-tolerated twice-daily protease inhibitor, approved in1997. The objective of AG1343-1260 was to assess thelong-term efficacy and safety of nelfinavir beyond72 weeks.



Methods: Patients, initially naive to antiretroviral therapy,who completed at least 72 weeks of nelfinavir (1250 mgtwice daily)-based antiretroviral therapy with plasmaHIV-1 RNA <50 copies/ml were eligible for continuedtreatment for 48 weeks on 1250 mg twice daily nelfinavirand two nucleoside reverse transcriptase inhibitors in thisprospective, multicentre, open-label study. The followingassessments were conducted: HIV-RNA levels, CD4+ cellcounts, measurements of body fat, self-assessments ofbody habitus and safety (adverse events, haematology andchemistry laboratory tests including lipid parameters). Results: Sixty patients (88% male) with median age of 39years were enrolled and treated; the median cumulativetime on nelfinavir-based highly active antiretroviral thera-py (HAART) was 257 weeks (119–439 weeks). MedianCD4+ cell count was >500 at baseline and was maintainedthroughout the study. In this study, no patient discontin-ued due to adverse experiences. Only two serious adverseevents occurred (gastroenteritis and lymphoma-like reac-tion) and both were unrelated to nelfinavir. Fifty-sevenpatients had viral load suppression <50 copies/ml (threepatients had 80 or fewer copies) at the time of study ter-mination. Eight patients had Grade 3 elevations in totalcholesterol at baseline and an additional two people hadGrade 3 elevations during the study. There were no Grade4 laboratory abnormalities. There was no incidence ofmarked change (shift from a baseline grade 0 to an on-treatment Grade 3 or 4, or from a baseline grade 1 to anon-treatment Grade 4) in any laboratory test; that is, totalbilirubin, transaminases, alkaline phosphatase, calcium,creatinine, lactic dehydrogenase, amylase, bicarbonate,glucose, potassium, uric acid, total cholesterol, triglyc-erides, haematology and urinalysis. Mean body fat mea-surements and self-assessed body fat habitus showed nosignificant change from baseline at 48 weeks. Conclusions: Extended nelfinavir-based HAART was safeand resulted in sustained suppression of HIV and sustainedCD4+ cell count >500 for 2–8 years (median=4.9 years).


Simplified Protease Inhibitor Trial (SPRINT):metabolic effects of once daily saquinavir SGCplus ritonavir (SQV/r) vs twice-daily indinavirplus ritonavir (IDV/r)

M Harris1, N Press1, A Thorne1, C Zala2, P Cahn2, C Ochoa2, S Schneider3, B Hanna4, J Singer1, J Montaner1 and the CTN 161 (SPRINT) study team

1 Canadian HIV Trials Network, Vancouver, BC, Canada;2 Fundacion Huesped, University of Buenos Aires, Buenos Aires,Argentina; 3 Living Hope Clinical Trials, Inc., Long Beach, Calif.,USA; and 4 Health Services Center, Hobson City, Ala., USA

Objective: To compare changes in metabolic laboratoryparameters (fasting lipids and glucose) in patients ran-domized to either once daily SQV/r or twice daily IDV/r,each with two RTIs. Methods: 164 HIV+ adults were randomized to receiveeither open-label SQV/r 1600 mg/100 mg once daily orIDV/r 800 mg/100 mg twice daily, with either two NRTIsor one NRTI plus one NNRTI, as selected by their treat-ing physician. Viral load (VL), CD4, fasting lipids [totalcholesterol (TC), HDL, LDL, triglycerides (TG)], glucoseand anthropometric measurements (height, weight andthigh/arm/waist/hip circumference) were assessed atscreening and weeks 0, 4, 8, 12, 16, 24, 36 and 48. Results: The analysis includes 89 patients with availablecholesterol data at baseline and week 24. At baseline, theSQV/r (n=43) and IDV/r (n=46) groups were similar(P>0.05) with respect to the following characteristics: age40.5 years, 80% male, 92% PI naive, 70% VL >5.0 log10

copies/ml, CD4 140 cells/mm3, weight 70 kg, BMI23.3 kg/m2, WHR 0.9, LDL 2.5 mmol/l, HDL 0.9mmol/l, TC/HDL 4.5 (medians). The IDV/r group hadstatistically higher TC and TG than the SQV/r group[median TC 4.4 and 3.7 mmol/l, respectively (P=0.03)and median TG 1.3 and 1.0 mmol/l, respectively(P=0.01)] and tended to have higher glucose [median 5.1and 4.9 mmol/l, respectively (P=0.08)] at baseline; how-ever, the median values are within the normal range andthe differences may not be clinically significant.Furthermore, similar proportions of patients in the twoarms had TC >5.2 mmol/l, TG >2.3 mmol/l, and glucose>6.1 mmol/l (P>0.1) at baseline. Background regimensincluded an NNRTI in three patients (two efavirenz, onenevirapine) on IDV/r and none on SQV/r. No differenceswere observed between arms with respect to changes inTC, HDL, LDL, TC/HDL, TG or glucose from baselineto either week 24 or week 48 (P>0.1). Week 24 VL andCD4 responses were similar between the two arms(n=147, P>0.05). Conclusions: A once-daily regimen of SQV/r with twoRTIs and a twice-daily regimen of IDV/r with two RTIshave similar effects on fasting lipids and glucose after 24and 48 weeks of treatment.


Improvement in dyslipidaemias among patientsreceiving HAART after substitution of tenofovirDF (TDF) for stavudine (d4T) with or without useof a concomittant lipid lowering agent (LLA)

J Gilmore1, B Guyer2 and C Barbour1

1 Desert AIDS Project, Palm Springs, Calif., USA; and 2 GileadSciences, Foster City, Calif., USA


Abstracts

Background: In a large, prospective, comparative trial,d4T-based HAART was associated with higher rates ofdyslipidaemias than TDF-based HAART. We evaluatedthe effect on serum lipids after replacement of d4T withTDF with or without use of an LLA, among patients inour clinic who were experiencing HAART dyslipidaemias. Methods: Ongoing, prospective, observational study.Patients were included if they were 1) receiving HAARTwith HIV RNA <400 copies/ml for >6 months, and 2) hadfasting serum cholesterol (CH) >240 mg/dl or low-densitylipoprotein (LDL) >130 mg/dl or triglycerides (TG)>300 mg/dl. The primary analysis was change from base-line (BL) in CH and TG at weeks 24 and 48 post-switch.Results: Data from 19 male patients were collected (fivereceiving LLA; 14 not receiving LLA). Out of 19 patients,10 were also receiving a PI(s), eight were receiving anNNRTI and one patient was receiving triple NRTIsincluding TDF. Average age, years HIV positive, years onHAART and number of prior ART regimens were 50, 11,9 and 5, respectively. For patients not receiving an LLA,BL CH, LDL and TG were 230 mg/dl, 107 mg/dl and389 mg/dl, respectively. After week 48 (n=4), median(min, max) change in CH, LDL and TG were –5 (–75,+46), +22 (+16, +28) and –206 (–537, +6). For patientswho received an LLA, BL CH, LDL and TG were230 mg/dl, 138 mg/dl and 200.5 mg/dl, respectively. Afterweek 48 (n=11), median (min, max) change in CH, LDLand TG were –18 (–92, +5), –16 (–74, +11) and –57(–289, +373), respectively. All patients remained virologi-cally suppressed throughout 48 weeks. Conclusions: For patients switched from d4T to TDF, theneed for an LLA should be individualized and may not berequired for every patient. Switching to TDF representsanother treatment option for patients who experienceHAART-associated dyslipidaemias.

ABSTRACT 103

Antiviral Therapy 2004; 9:L59

Inhibition of reverse cholesterol transport inritonavir-treated hamsters

PV Nerurkar, YK Lee, J Frank and VR Nerurkar

University of Hawaii at Manoa, Honolulu, HI, USA

Protease inhibitors (PI) form the cornerstone of antiretro-viral treatment for HIV-infected patients. Ritonavir(RTV)-treated seronegative and HIV-positive patientsdemonstrate a significant increase in serum triglyceridesand cholesterol levels with a decrease in HDL-cholesterol.Excess of total cholesterol with a decrease in HDL-cholesterol can contribute to atherosclerosis and gallstoneformation. Although RTV has been demonstrated toincrease hepatic synthesis and secretion of cholesterol, no

studies have been conducted to test the effects of RTV onreverse cholesterol transport involving HDL-mediatedtransport of extrahepatic cholesterol to the liver and itsconversion to bile acids. The aim of our study was toinvestigate reverse cholesterol transport in RTV-treatedhamsters. Our data indicate that treatment of hamsterswith 45 mg/kg of RTV for 8 weeks significantly increasedserum triglycerides (400%; P<0.01), total cholesterol(150%; P<0.05), LDL-cholesterol (135%, P<0.05), LDL-cholesterol:HDL ratio (130%; P<0.04) and plasmaapolipoprotein B (apoB; 400%; P<0.001), with a simulta-neous decrease in plasma apoA1 (25%, P<0.05). RTValso significantly inhibited the mRNA expression ofcytochrome P450 7A1 (CYP7A1) in the liver, an enzymethat catalyses the first, rate-limiting step in the conversionof cholesterol to bile acids and represents a major path-way for eliminating cholesterol. RTV also inhibited ATP-binding cassette transporter (ABCA1) gene expression,which transports intracellular cholesterol and phospho-lipids to cell surface-bound apolipoproteins and formsnascent HDL. Increased expression of ABCA1 has beendemonstrated to increase hepatic cholesterol efflux,decrease cellular cholesterol accumulation as well asincreasing plasma apoA1 levels in animals. Based on ourdata, it is highly possible that decreased hepatic ABCA1expression may contribute to RTV-associated increases inliver and plasma cholesterol and reduced apoA1 in HIV-infected patients on PI-containing regimens. BothCYP7A1 and ABCA1 are negatively regulated by thenuclear orphan receptor farensoid X-receptor (FXR). Inour studies, RTV significantly increased hepatic FXRmRNA expression (P<0.05). Overall, our data suggestthat inhibition of reverse cholesterol transport may con-tribute towards RTV-associated dyslipidaemia.

Partially supported by a grant (G12RR003061) fromthe RCMI Program NCRR, NIH.


NRTI Sparing Trial: metabolic effects of nevirapine(NVP) + lopinavir/ritonavir (LPV/r) vszidovudine/lamivudine (AZT/3TC) + NVP vsAZT/3TC + LPV/r

M Harris1, C Ochoa2, C Allavena3, E Negredo4, A Thorne1, P Cahn2, C Zala2, F Raffi3, B Clotet4, J Singer1, J Montaner1 and the CTN 177 study team

1 Canadian HIV Trials Network, Vancouver, BC, Canada; 2 FundacionHuesped, University of Buenos Aires, Buenos Aires, Argentina; and3 Hôtel Dieu, Nantes, France; and 4 Lluita contra la SIDA Foundation,Germans Trias i Pujol University Hospital, Barcelona, Spain

Objective: To compare changes in body mass index (BMI),waist/hip ratios (WHR), fasting lipids, glucose and lactate



in patients randomized to an NRTI-sparing regimen(NVP/LPV/r) or one of two NRTI-based regimens(AZT/3TC with either NVP or LPV/r). Methods: 78 antiretroviral-naive HIV+ adults were ran-domized to one of three open-label treatment arms. Viralload (VL), CD4, mitochondrial/nuclear DNA ratio (mt/nDNA), height, weight, waist/hip/chest circumference, fast-ing lipids [total cholesterol (TC), HDL, LDL and triglyc-erides (TG)], glucose and lactate were assessed at baselineand weeks 12, 24 and 48. VL, CD4 and mt/n DNA will beassessed at 48 weeks. Results: Baseline characteristics of the 52 evaluable patientscompleting week 24 were similar (P>0.10) for NVP/LPV/r(n=14), AZT/3TC/NVP (n=18) and AZT/3TC/LPV/r(n=20): 79% male, age 37 years, VL >4.9 log10 copies/ml,CD4 220 cells/mm3, BMI 22.2 kg/m2, WHR 0.9, TC 3.9mmol/l, LDL 2.3 mmol/l, HDL 0.9 mmol/l, TC/HDL 4.4,TG 1.0 mmol/l, glucose 4.9 mmol/l, lactate 0.9 mmol/l(medians). Differences between arms were observed atweeks 12 and 24 for TC (P=0.04, 0.06 respectively) and TG(P<0.01, <0.01), but not for other metabolic parameters.TC: NVP/LPV/r > AZT/3TC/LPV/r > AZT/3TC/NVP. TG:NVP/LPV/r > AZT/3TC/LPV/r > AZT/3TC/NVP. TCincreased from baseline to week 24 by 1.5 mmol/l (median)for NVP/LPV/r, 1.3 for AZT/3TC/LPV/r and 0.7 forAZT/3TC/NVP (P=0.24 for comparison of changesbetween arms). TG increased by 1.1 and 0.9 mmol/l forNVP/LPV/r and AZT/3TC/LPV/r respectively, anddecreased by 0.1 mmol/L for AZT/3TC/NVP (P<0.01). Atweeks 12 and 24, more NVP/LPV/r patients had TC>5.2 mmol/l (73%, 73%) than AZT/3TC/LPV/r (39%,39%) or AZT/3TC/NVP patients (22,24%) (Chi-squareP=0.03 at weeks 12 and 24). No other differences wereobserved between arms in proportions of patients crossingclinically relevant thresholds for lipids, glucose, lactate,BMI or WHR. No patients received lipid-lowering agents. Conclusions: After 24 weeks of treatment in antiretroviral-naive patients, NVP/LPV/r and AZT/3TC/LPV/r had simi-lar effects on lipid profiles, increasing both TC and TG. TCand TG were lowest in patients receiving AZT/3TC/NVP.The three arms did not differ with respect to impact onLDL, HDL, TC/HDL, glucose, lactate, BMI or WHR.


Study of lipodystrophy in treated HIV patientsand evaluation of their psychological problems

F Jalali and NP Moghadam

Mashad University of Medical Sciences, Mashad, Iran

Objectives: Highly active antiretroviral therapy (HAART)has dramatically reduced the morbidity and mortality ofHIV-infected patients. However, several metabolic side

effects have been described to be associated with long-term HAART. These include hypertriglyceridaemia,hypercholesterinaemia, insulin resistance, impaired glu-cose tolerance and lactic acidaemia. Lipodystrophy (LD)may produce psychological problems. This report aims tostudy the prevalence of LD and psychological effects intreated HIV patients.Methods: Currently, 351 HIV-infected patients (276 maleand 75 female, age 27–63), admitted in Emam Reza HIVcentre for medical treatment for their disease. A cohort ofpatients treated with different regimens of HAART forperiods from 1–4 years was assessed for clinical LD. Bloodanalysis included plasma viral load, fasting glucose, cho-lesterol and triglicerydes. LD is characterized by a redistri-bution of fat tissue, consisting of peripheral fat loss(particularly in the face and extremities) and central fatstorage (particularly intra-abdominal).

In addition, in these patients, a questionnaire wasspecifically designed to measure anxiety, depressive symp-toms, social distress and anxiety, social support, quality oflife and body image alteration.Results: The results show there is a high correlationbetween patient and physician quantification of LD or fataccumulation (r=0.52–0.8, P<0.001). LD syndrome wasobserved in 97 patients (27%). Central obesity was notedin 40 patients. High total cholesterol level was observed in46 patients, high TG level in 73 and high glucose levels infive of them. Overall, there was a close correlation amongthe severity of LD, social distress and depression (r=0.6,P<0.001).Conclusions: LD is a major health problem in HIV-infect-ed patients who are treated with highly active antiretrovi-ral therapy (HAART). LD is often accompanied bycardiovascular risk factors like insulin resistance, diabetesmellitus, hyperlipidaemia and a disturbed endothelial func-tion. Severe LD causes depressive symptoms and anxiety.


Association of lipid profiles in HIV-seropositivepatients with antiretroviral therapy in a realworld scenario

S Mauss1, F Berger1, G Schmutz1, S Holm2, B Kuhlmann2, E Wolf3 and WO Richter4

1 Centre for HIV and Hepatogastroenterology, Düsseldorf,Germany; 2 HIV-Specialised Practice, Hannover, Germany;3 MUC-research, Munich, Germany; and 4 Institute for LipidResearch, Windach, Germany

Background: Dyslipidaemia is a frequently observed phe-nomenon in HIV patients with or without antiretroviraltherapy. Recent data indicate different lipid profiles associ-ated with antiretroviral drugs. However, in-depth analysis


Abstracts

of lipid profiles were not performed in pivotal studies formost of the presently used antiretroviral combinations. Inaddition lipids are often not corrected for patient charac-teristics associated with lipid changes, thereby confoundingthe analysis of the data. Methods: Lipid profiles in patients with (n=264) and with-out (n=107) antiretroviral therapy from two HIV outpa-tient clinics were assessed. A high proportion of patientswere female 105 (29%). Patients on lipid-lowering drugswere not included. Total-, LDL-, HDL-, VLDL-cholesterol(C), triglycerides, VLDL-triglycerides, VLDL-, LDL-Apo Band lipoprotein a were measured in fasted state. Only anti-retrovirals present in more than 10 patients were consid-ered for analysis. ANOVA and regression analysis wereused for statistical computation . The multivariate analyseswere adjusted for sex, lipoatrophy, lipohypertrophy, BMIand the use of ART.Results: Lipid changes were associated with (P<0.05): Age:total-C↑, LDL-C↑, VLDL-C↑, LDL-ApoB↑; BMI: total-C↑, LDL-C↑, VLDL-C↑, TG↑, VLDL-ApoB↑, LDL-Apo B↑,VLDL-Apo B; lipoatrophy: HDL-C↓; male sex: total-C↓,HDL-C↓, VLDL-C↑, TG↑.

After adjustment for patient characteristics in a regres-sion analysis the following antiretrovirals were associatedwith lipid changes (P<0.05): tenofovir: total-C↓, LDL-C↓,LDL-Apo B↓, VLDL-C↓, VLDL-TG↓; efavirenz: total C↑,LDL-C↑; nevirapine: total-C↑, HDL-C↑, LDL-C↑;lopinavir/r: total-C↑, VLDL-C↑, TG↑; saquinavir/r: VLDL-C↑, TG↑, VLDL-TG↑. No significant changes were foundfor zidovudine, lamivudine, abacavir, stavudine, didanosineand nelfinavir. Conclusion: After adjustment for a variety of patient char-acteristics, different antiretroviral drugs in a variety of anti-retroviral combinations are characterized by distinct lipidprofiles, which may have implications for the cardiovascu-lar risk management in HIV-positive patients.


Metabolic complication and fat changes in ThaiHIV-infected patients

M Homsanit1, J Cofrancesco Jr 2 and KE Nelson3

1 Mahidol University, Bangkok, Thailand; 2 Johns HopkinsUniversity School of Medicine, MD, USA; and 3 Johns HopkinsUniversity Bloomberg School of Public Health, Md., USA

Background: Antiretroviral therapy (ART) has been asso-ciated with metabolic abnormalities (hyperglycaemia, dys-lipidaemia and insulin resistance) and changes in fat(visceral fat accumulation and subcutaneous loss). Thereare reports of increase risk for cardiovascular disease asso-ciated with these changes. Most evidence comes from west-ern populations and has not been well studied in Asia.

Objective: To evaluate metabolic abnormality and fatchanges in an HIV-infected Thai population.Methods: A total of 247 Thai HIV-infected patients wereevaluated; 126 were ART-naive, 121 ART-treated: 60 onnon-PI regimens and 61 PI-based regimens. All underwentclinical evaluation of fat changes and biochemical investi-gation for fasting plasma glucose (FPG), total cholesterol(TC), triglyceride (TG), HDL cholesterol (HDL-c) andLDL cholesterol (LDL-c). Dual-energy X-ray absorptiome-try (DEXA) was performed to measure visceral to subcu-taneous fat ratio (VT ratio).Results: Clinical lipodystrophy features were found in10.32%, 41.67% and 83.61% of ARV-naive, non-PI treat-ed, and PI-treated patients, respectively (P<0.001). Ofthese, fat loss was found in 10.32%, 36.67% and 78.69%(P<0.001); fat gain in 0.79%, 6.67% and 24.59%(P<0.001), respectively. Mean FPG were 87.75 ±8.62,93.50 ±8.47, 94.77 ±16.43; TC 172.65 ±36.31, 213.85±43.40, 280.89 ±379.81, TG 119.63 ±61.74, 167.95±93.86, 256.03 ±165.31; HDL-c 44.11 ±13.92, 53.08±15.56, 46.04 ±11.55; and LDL-c 105.22 ±32.52, 128.17±36.64, 140.56 ±41.35 mg/dl (P value <0.05 for all); meanVT ratios were 1.11 ±0.03, 1.45 ±0.06 and 1.93 ±0.08(P<0.001) in ARV-naive, non-PI treated and PI-treatedpatients, respectively. After adjusting for age, sex andincome level, odds ratios of lipodystrophy were 6.41 (95%CI: 2.83–14.50) for ARV-naive compared with non-PItreated, 51.23 (95% CI: 18.05–145.38) for ARV-naivecompared with PI-treated, and 8.38 (95%CI: 3.37–20.82)for non-PI treated compared with PI-treated.Conclusion: Fat changes and metabolic abnormalities arecommon in Thai HIV-infected patients, similar to westernpopulations. Antiretroviral therapy, especially PI-based reg-imens, are significantly associated with these derangements.


Dietary evaluation and metabolic alterations inHIV-related lipodystrophy

G Orlando1, G Guaraldi1, F Giuricin1, G Grisendi1, F Carubbi1, S Galetti2, I Zini1, V Borghi1 andR Esposito1

1 University of Modena and Reggio Emilia, Modena and ReggioEmilia, Italy; and 2 Dietary Service, Azienda Policlinico, Modena, Italy

Objectives: Metabolic alterations are frequently observedin people with HIV-related lipodystrophy on HAART.Diet is supposed to influence metabolic alteration in thispopulation. The aim of this cross-sectional study was toanalyse the role of diet on metabolic alterations in patientswith clinical diagnosis of lipodystrophy.Methods: Patients underwent a 1 week dietary question-naire evaluating food intake. Nutritional assessment



(carbohydrates, proteins, saturated fats) was performedby a trained dietologist with Winfood software.

The sample was divided according to metabolic alter-ations (diabetes = fasting glucose >126 mg/dl), hyper-triglyceridaemia (>175 mg/dl), hypercholesterolaemia(total >200 mg/dl or LDL >130 mg/dl) and according tosex and current antiretroviral regimen (PI vs non PI, D4Tvs others NRTIs).Results: 42 patients were evaluated, 45% were female.The metabolic baseline characteristics were: mean bloodglucose 94 ±16 mg/dl, mean triglycerides 184 ±175 mg/dl,mean total cholesterol 191 ±48 mg/dl, mean HDL-choles-terol 42 ±14 mg/dl and mean LDL-cholesterol 119 ±39mg/dl. Main nutrients consumed with the diet wereexpressed as percentage of total daily calories: proteins 15±2%, lipids 32 ±5%, saturated lipids 3.9 ±0.9% and car-bohydrates 17 ±4%.

32.5% of patients had high level of triglycerides,15.6% had high cholesterol, 20.5% had high LDL-cho-lesterol and 5% of the sample had diabetes. 42.5% ofpatients were assuming PI and 12.5% stavudine at base-line. No difference in carbohydrates, proteins and saturat-ed fats daily intake was found in patients with or withoutmetabolic alterations, taking into account sex and currentantiretroviral regimen.Conclusions: A direct relationship between metabolicalterations and dietary habits has been observed inplurimetabolic syndrome. This study, however, wasunable to detect a direct influence of diet on metabolicalteration in HIV-related lipodystrophy, supporting a mul-tifactorial hypothesis. A follow-up study will allow inves-tigation of the role of nutritional counselling in themanagement of metabolic alterations in these patients.


Increased CD36 expression on circulating monocytes during HIV infection

L Meroni1, A Riva1, P Morelli1, M Galazzi1,D Mologni1, F Adorni2 and M Galli1

1 Institute of Infectious Diseases and Tropical Medicine, LuigiSacco Hospital, University of Milan, Milan, Italy; and 2 NationalResearch Council, ITBA, Milan, Italy

Background: Metabolic alterations and abnormalities offat distribution are common findings in antiretroviral-treated HIV-1-infected patients. CD36 is a multifunction-al receptor with a wide tissue distribution that plays acrucial role in lipid cellular uptake and metabolism. Objectives: To define the level of CD36 expression on cir-culating monocytes from HIV-1-infected patients andhealthy controls and to correlate CD36 expression levelswith metabolic and immunovirological parameters.

Methods: CD36 expression on peripheral blood mono-cytes was measured by means of flow cytometry in 165HIV-1-infected patients and in 35 healthy volunteers. Ofthe 165 HIV-1-infected patients, 38 were naive for anti-retroviral therapy; 50 patients were on a PI-based regi-men, 48 were on an NNRTI based regimen and 27 weretaking only NRTIs. Statistical analyses were performed bymeans of univariate and multivariate analysis of variance(ANOVA) models. Results: CD36 expression was significantly higher in HIV-1-infected patients compared with healthy controls(P<0.0001). HIV-1 infection was the only variable associ-ated with CD36 expression on a multivariate analysis,while no correlation was found between CD36 levels andage, sex, body mass index, lipids serum levels, HIV-RNAlevels, time on antiretroviral therapy and kind of anti-retroviral regimen.

On an univariate analysis, CD4 cell count was nega-tively correlated to CD36 MFI on monocytes (P<0.05;Pearson coefficient: –0.179). On a multivariate analysis,CD4 cell count maintained a statistically significant corre-lation with CD36 levels (P<0.05; R2=0.074).Conclusions: HIV-1 infection is associated with anincreased expression of CD36 on circulating monocytesand antiretroviral drugs play only a minor role in its com-plex homeostasis. Being related to HIV infection itself,CD36 over-regulation is a permanent condition in infect-ed patients, resulting in the potential for continued lipidaccumulation and foam cell formation. Thus, CD36 over-expression might act in accordance with other well-known proatherogenic conditions associated with HIVinfection, such as pro-inflammatory effects on endotheli-um, insulin resistance and dyslipidaemia.


The reduction in serum adiponectin levels in HIVpatients correlates with apolipoprotein-B clearance

M Shahmanesh1, S Das1, F Shojaee-Moradie2,M Stolinski2, W Jefferson2, N Jackson2, G Gilleran2,NJ Crabtree1, P Nightingale1, R Cramb1 andM Umpleby2

1 University Hospital, Birmingham, UK; and 2 Guy’s Kings Collegeand St Thomas’ Hospitals’ Schools of Medicine, London, UK

Objective: Serum adiponectin levels are reduced inpatients with lipodystrophy. Little information is availableon its relationship with lipoprotein metabolism. Methods: We compared plasma adiponectin levels and anumber of serum cytokines in uninfected controls (n=12)with three HIV-positive groups including treatment-naive(n=15), on triple antiretroviral therapy containing pro-tease inhibitors (n=15) or non-nucleoside reverse


Abstracts

transcriptor inhibitors (n=25), and related this to VLDL,IDL and LDL apolipoprotein B (apo-B) kinetics using a 9-h 13C leucine infusion. Insulin resistance was calculated bythe homeostatic model (HOMAIR). Subjects underwent awhole body DEXA scan. Results: Plasma adiponectin levels [(median ±interquartilerange (IRQ)] were lower in treatment-naive (5.4 µg/ml,IRQ 4.7–8.5), PI (5.0 µg/ml, IRQ 3.3–6.4) and NNRTI(5.0 µg/l, IRQ 3.2–6.3) groups compared with controls(9.7 µg/ml, IRQ 6.9–13.3, P=0.003). There were nodifferences between the treatment groups. Adiponectincorrelated negatively to HOMA (r= –0.43, P<0.001) andpositively to VLDL apo-B fractional catabolic rate (FCR,r=0.43, P<0.001), IDL FCR (r=0.5, P<0.001), LDL FCR(r=0.28, P=0.023) and peripheral fat/BMI (r=0.61,P<0.001) but not VLDL, IDL or LDL absolute secretionrates (ASR) or trunk fat/BMI. In a multivariate regressionmodel, adiponectin levels were highly predictive of VLDLand IDL FCR, HDL cholesterol and total triglyceride levels,while in the same model HOMA only predicted IDL ASRand total triglyceride levels. Serum interleukin-8 (IL-8) andIL-10 were significantly higher in HIV treatment-naivepatients compared with controls and in the case of IL-8higher than HIV-infected patients on antiretroviral therapy.There were no correlations between serum cytokine levelsand apoB metabolism or body fat changes.Conclusion: Adiponectin levels are significantly lower inHIV-infected patients including those who are treatmentnaive. The strong correlation with VLDL and IDL, andLDL apoB FCR and plasma lipid levels suggests an effecton lipoprotein and hepatic lipase activity that may beindependent of insulin.


Lipoprotein (a) as cardiovascular risk factor inHIV-infected patients

WO Richter1, E Issler2, A Schaffert2, E Schnaitmann2

and A Trein2

1 Institute for Lipid Metabolism, Windach, Germany; and2 Centre for HIV Patients, Stuttgart, Germany

A very recent paper (N Rifal et al. Clinical Chemistry2004; 50:1364) reported a strongly increased risk forfuture angina pectoris in individuals with lipoprotein (a)>30 mg/dl and LDL-cholesterol >160 mg/dl (relative risk11.9) or in individuals with lipoprotein (a) >70 mg/dl (rel-ative risk elevated fourfold). We evaluated lipoprotein (a)concentrations in HIV-infected patients to estimate car-diovascular risk due to this risk factor. Methods: 300 patients (34 women, 266 men, mean age40.4 ±9.2 years, 61% on an antiviral drug regimen, 46%smokers, 6% on a lipid lowering diet, mean cholesterol

215.6 ±53.3 mg/dl, triglycerides 222.7 ±200.8 mg/dl, HDL-cholesterol 42.7 ±12.9 mg/dl and LDL-cholesterol 128.6±38.0 mg/dl) were investigated. Lipoprotein (a) was deter-mined by turbimetry (Technoclone, Vienna, Austria) andlipid parameters by enzymatic methods. LDL-cholesterolwas measured in the infranatant after ultracentrifugation.Results: Median lipoprotein (a) concentration was 13 mg/dl[interquartile range (IQR) 4–48 mg/dl]; 92 patients had alipoprotein (a) concentration >30 mg/dl, 18 of them (6%of all patients) had an additionally elevated LDL-choles-terol >160 mg/dl. A further 41 patients had lipoprotein(a) above 70 mg/dl. In total, 59 (19.7%) patients had astrongly increased cardiovascular risk due to lipoprotein(a). In patients without antiviral drug treatment, meanlipoprotein (a) was 13 mg/dl (IQR 4–42 mg/dl). The meanlipoprotein (a) in patients treated with, for example,lopinavir/ritonavir (n=55, six women, 49 men) was14 mg/dl (IQR 6–47 mg/dl), which did not differ signifi-cantly from patients without therapy. Sixteen of thesepatients had lipoprotein (a) above 30 mg/dl; four of themalso had LDL-cholesterol above 160 mg/dl. In addition,six individuals showed lipoprotein (a) concentrationsabove 70 mg/dl. Ten patients (18.1%) treated withlopinavir/ritonavir had an increased risk for cardiovascu-lar disease, which did not differ significantly from the per-centage in the whole or untreated group. Conclusion: About one-fifth of HIV-infected patients,whether on antiviral treatment or not, had a stronglyincreased risk for cardiovascular disease due to elevatedlipoprotein (a).


Investigation of cytokine promoter polymorphismsin the tumour necrosis factor (TNF)-α, and interleukin (IL)-10 genes in HIV-infected patientswith lipodystrophy

A Farajallah1, D Chew1, E Albanese1, W Chen1, J Rodrigues1, J Greytok2 and H Fernandes1

1 University of Medicine and Dentistry of NJ / NJ MedicalSchool, Newark, NJ, USA; and 2 Bristol-Myers Squibb, NJ, USA

Objectives: Cytokines have been implicated as an underly-ing pathogenetic mechanism in the development of HIV-associated lipodystrophy. Single nucleotide polymorphisms(SNPs) in the promoter region of the TNF-α and IL-10genes affect the level of cytokine that is produced. We stud-ied the association of cytokine promoter SNPs in TNF-αand IL-10 genes with HIV-associated lipodystrophy. Methods: A case-controlled study of 25 African-American(AA) HIV patients with lipodystrophy (HIV L+) wasundertaken and compared with 25 AA HIV patients with-out lipodystrophy (HIV L–) and 25 HIV-negative AA



controls (NC). HIV L+ cases were provider-identified withone or more physical features of lipodystrophy with orwithout metabolic abnormalities. HIV L+ and L– patientswere matched by age, gender and years of HIV infection.Individuals were genotyped for the –238 and –308 SNPsin the TNF-α gene and the –819 and –1082 SNPs in theIL-10 gene. Genotyping was performed using realtimePCR with hybridization probes and melting curve analy-sis. Allele frequencies and haplotypes were determined inthe patient groups.Results: HIV L+ and L– patients were similar in terms ofdemographic data, baseline CD4 and antiretroviral histo-ry. The –819 and –1082 polymorphism was higher in HIVpatients compared with NC. Only 6% of the NC werehomozygous for the –819 TT polymorphism, comparedwith 33% of HIV L– and 21% of HIV L+. The incidenceof the –1082G−>A genotype was also higher in the HIVL+ group. Haplotype analysis of the IL-10 gene showedthat more HIV L– patients (73%) had the low and inter-mediate phenotype than the HIV L+ patients (60%). Nosignificant difference was observed in the occurrence ofthe –238 or the –308 TNF-α polymorphism. Conclusion: The results demonstrate an increase of thelow and intermediate IL-10 phenotype in HIV-infectedindividuals compared with HIV-negative controls,suggesting a link between IL-10 polymorphisms and HIVinfection. The phenotypic variation between the HIVpatients with and without lipodystrophy points to a pos-sible difference between the two groups. The IL-10 pro-moter haplotype should be further investigated as apossible risk factor in HIV patients with lipodystrophy.


Evaluation of blood lipid disorders andatherosclerosis measured by the intima mediathickness in a cohort of HIV-positive patients

F David-Ouaknine1, D Lagasse1, S Marque2 and JRivargoda1

1 Centre Hospitalier de Lagny, Lagny Sur Marne, France; and2 3Es, Paris, France

Objective: To find a link between antiretroviral treat-ments, dyslipidaemias and the carotid intima media thick-ness.Methods: Data were recorded from a cohort of HIV-1seropositive patients, followed up in a general hospital.The data were clinical (age, sex, body mass index, durationof HIV infection, CDC stage, antiretrovirals and number ofmonths of treatment, previous cardiovascular disease,blood pressure, ECG, treatment for hypertension, dyslipi-daemia or diabetes), biochemical (total cholesterol, HDLcholesterol, LDL cholesterol, triglycerides, fasting blood

glucose, CD4 and nadir CD4) and morphological (Dopplermeasurement of the carotid intima media thickness).Results: 120 patients (101 treated, 28 untreated) wereanalysed; the mean age was 42.9 years old, the sex ratiowas 1, the duration of HIV infection was 71.6 months,68% of the patients did not smoke, 17 had hypertension,the mean duration of antiretroviral treatment was 51.6months, the mean CD4 at inclusion was 440/mm3, andthe nadir CD4 was 226. The mean total cholesterol, HDLand LDL cholesterol, triglycerides and blood glucose werewithin normal limits; total cholesterol, LDL cholesteroland triglycerides tended to increase in the treated patients.There was no significant difference in the mean and max-imum intima media thickness between the untreated andtreated patients – 27 patients had carotid stenoses greaterthan 70%, three had plaques, 11 had calcifications; nolink was made between stenosis or plaques and antiretro-viral treatment.Conclusion: In our cohort, the cardiovascular risk factorsusually described in HIV-seropositive patients (heavysmoking, low HDL cholesterol and hypertension) wereonly found in a small percentage of patients. A link wasfound between classical cardiovascular risk factors andthe intima media thickness, but there was no significantincrease in the intima media thickness in the treatedpatients compared with the untreated ones. The start ofantiretroviral treatment is a good time to screen for and tocorrect cardiovascular risk factors.


Impaired activity of pravastatin on carotid intima-media thickness in HIV-infected patients

F Boccara1, T Simon2, P Boutouyrie1, PM Girard3,P Jaillon2, B Laloux1, E Bozec1 and S Laurent1

1 Unité de Pharmacologie Clinique and EMI-U 0107 INSERM,Hôpital Européen Georges Pompidou, Paris, France; 2 Unité deRecherche Clinique-Est, CHU Saint Antoine, Paris, France; and3 Infectious Diseases Department, CHU Saint Antoine, Paris,France

Background: Accelerated atherosclerosis and coronaryartery disease (CAD) are emerging complications in HIV-infected patients (HIV+) under highly active antiretroviraltherapy (HAART).Objective: To evaluate the effect of pravastatin on carotidintima-media thickness (IMT) in dyslipidaemic HIV+.Materialandmethods:Usingacross-sectional study,27dys-lipidaemic (LDL cholesterol ≥1.6 g/l) HIV+ under HAART(≥12 months) and pravastatin (≥12 months) were comparedwith 27 dyslipidaemic HIV+ under HAART but withoutlipid-lowering treatment (controls), matched for age, sexand tobacco. Internal diameter, IMT, circumferential wall


Abstracts

stress and elastic modulus were determined at the site ofthe common carotid artery using a non-invasive high res-olution echotracking device and applanar tonometry.Central pulse wave velocity (PWV) was measured(Complior®). Exclusion criteria were age <30 years andprevious history of CAD or stroke.Results: The cohort’s mean age was 49.5 ±8 years. Bothgroups were similar for clinical characteristics, includingcardiovascular risk factors. None of the arterial parame-ters determined were significantly different between thetwo groups. IMT of HIV+ under pravastatin (mean dura-tion: 1.8 ±0.8 years) was similar to HIV+ without pravas-tatin (692 ±129 µm versus 716 ±46 µm; P=0.52).

Using logistic regression, determinants of IMT wereage and carotid pulse pressure. Pravastatin did not influ-ence IMT. The determinants of aortic stiffness (carotido-femoral PWV) were age, duration of HIV infection,abdominal perimeter and lypodystrophic syndrome.Conclusion: The use of pravastatin was not associatedwith a significantly lower carotid IMT in HIV+. The roleof lipodystrophic syndrome and associated metabolic dis-orders in accelerated atherosclerosis need to be furtherinvestigated.


Coronary artery bypass graft in HIV-infectedpatients. A multicenter case control study

F Boccara1, A Cohen1, G Odi1, E Di Angelantonio1,E Teiger2, G Barbarini3 and G Barbaro4; on behalfthe French Italian Study on Coronary Artery Diseasein AIDS Patients (FRISCA-2)

1 Department of Cardiology, Saint Antoine University Hospital,Assistance Publique-Hôpitaux de Paris and Université Paris VI,Paris, France; 2 Department of Physiopathology, Henri MondorUniversity Hospital, Assistance Publique-Hôpitaux de Paris andUniversité Paris XII, Creteil, France; 3 Department of Infectious andParasitic Diseases, University of Pavia, Italy; and 4 Department ofMedical Pathophysiology, University La Sapienza, Rome, Italy

Background: Coronary artery disease is an emerging com-plication in HIV-infected patients under highly active anti-retroviral therapy. Immediate results and long-termoutcome after coronary artery bypass graft (CABG) hasnot been evaluated in this population. Methods: Between January 1997 and December 2003, wecompared baseline characteristics, immediate results andclinical outcome (Major Adverse Cardiac Events: deathfrom any cause, myocardial infarction, re-interventionand/or PCI) at 34 months between 22 consecutive HIV+and 42 HIV– control patients matched for age and gender(mean age, 49±8 years; 95% male) who underwentCABG. Cardiovascular risk factors were well-balanced

and nearly identical in both groups with a higher rate ofhypercholesterolaemia (96% vs 74%, P=0.045) andhypertriglyceridaemia (82% vs 45%, P=0.005) in HIV+patients, whereas obesity was more frequent in the controlgroup (33% vs 0%, P=0.001). In the HIV+ group, meanpre-operative CD4 was 503 ±200/mm3 compared with427 ±162/mm3 in post-operative (P=0.004) without clini-cal significance in the follow-up. Coronary multivesseldisease (≥2 vessel disease) was present in 21 (96%) HIV+and 39 (93%) HIV– (P=0.683). LVEF and mean numberof grafts was similar in the two groups (55%±10 vs 50%±14, respectively, P=0.179; 2.7 ±0.6 vs 2.7 ±1.0, respec-tively, P=0.923). At 30 days, the rate of post-operativedeath, MI, stroke, mediastinitis and re-intervention wasidentical in both groups. At 34.3 ±20.5 months follow-up,rate of occurrence of first MACE is higher in the HIV+group compared with the HIV– group. Using a Cox pro-portional hazards model, the only predictor of MACE atfollow-up was HIV infection itself with a hazard ratio of6.3 (95% CI 2.2–17.9, P=0.001).Conclusion: CABG is a feasible and safe revascularizationprocedure in HIV-infected patients. Immediate postopera-tive outcome demonstrate no difference between HIV+and HIV– patients. At long-term follow-up, the rate ofMACE was significantly higher in HIV+ patients com-pared with HIV– due to an increased rate of repeat revas-cularization procedure (reCABG and PCI).


Evaluation of coronary artery calcification byelectron beam computed tomography (EBCT) inHIV-infected men receiving prolonged proteaseinhibitor (PI) therapy

DM Parenti, A Klouj, MM Rice, AD Roberts, APLiappis, N Bisby, SZ Schuck, J Ehrlich, AGWasserman and GL Simon

The George Washington University Medical Center, Washington,DC, USA

Prolonged therapy with highly active antiretroviral ther-apy containing PIs has been associated with hyperlipi-daemia and some studies have suggested an increased riskof myocardial infarction. The presence of coronary arterycalcium (CAC) as detected by EBCT correlates with coro-nary artery atherosclerosis and is an independent predic-tor of cardiovascular morbidity. 70 HIV-positive menwithout cardiac symptoms receiving >24 months of PItherapy underwent evaluation with EBCT. The mean agewas 48 years (range 30–72); mean CD4 was 546. Patientshad received a mean 55 months of PI therapy, including39% that received boosted PI therapy for a mean 27months. The mean/median CAC scores were compared



with age-range matched published norms for asympto-matic men. 60% of HIV+ patients had a positive CACscore (>0). 48% of those patients on a single PI regimenhad a CAC score >0 compared with 75% of those on aboosted PI regimen, OR=3.3 (1.2–9.5, P=0.03). Therewas no significant difference in age between the single PIand boosted PI groups. There was also no significant cor-relation between CAC score and the duration of eithersingle PI or boosted PI therapy. A positive calcium scorewas associated with elevation of serum triglyceride,with an OR=1.01 for every unit increase in triglyceride(1.000–1.01, P=0.035), and a trend in association withtotal VLDL, OR=1.01 for every unit increase in VLDL(0.998–1.049, P=0.076). There was no association withtotal cholesterol, total LDL cholesterol, LDL particlenumber, intermediate VLDL, small HDL (HDL 1,2) orsmall LDL (LDL 1). There appears to be no differencein CAC scores when PI-treated patients are comparedwith age-range matched published norms for asympto-matic men, but there appears to be an associationbetween boosted PI use and coronary artery calcifica-tion. These findings suggest that further studies be doneexamining the effects of boosted PI regimens on accel-erated atherosclerosis.


Endothelial dysfunction, adiponectin plasma levelsand lipodystrophy in patients on antiretroviraltherapy

V Estrada, JL Zamorano, T Sainz, S Serrano, L de Isla, MT Martinez-Larrad, JL Gonzalez and M Serrano Rios

Hospital Clinico San Carlos, Universidad Complutense, Madrid,Spain

Objectives: Low adiponectin (AD) plasma levels areobserved in patients on antiretroviral therapy (ART) with fatredistribution abnormalities (FRA). Hypoadiponectinaemiahas been associated with impaired vasoreactivity in the gen-eral population. Previous studies found a significant rela-tionship between use of protease inhibitors (PI) andendothelial dysfunction. The purpose of our study is toanalyse the relationships between plasma AD, FRA andendothelial function as measured by high resolution ultra-sound (HRUs). Methods: 61 HIV-infected male patients (mean age 43.1 ±9.9 yrs) on ART (mean accumulated time on treat-ment 40.2 months) were studied. 44.2% of them presentedFRA, most of them lipoatrophy. Median CD4 was478/mm3 and 76% presented HIV-1 viral load<50 copies/ml. 17 HIV-infected naive patients with similarage and BMI were used as controls. HRUs was used to

assess endothelium-dependent brachial artery flow-mediat-ed vasodilation (FMD). Longitudinal images of the brachialartery were obtained before arm cuff inflation to200 mmHg for 5 min, and 10 min after deflation. Inpatients with a reduced vasodilator response (<10% vesseldiameter increase from baseline), a third artery scan wasobtained after the sublingual administration of 400 µg glyc-eryl trinitrate via spray. ED was defined when FMD was<5%. AD plasma levels were measured by RIA.Results: Mean FMD of patients on ART was 11.6% (IC95%, 8.3–14.9) similar to control group 11.7% (IC 95%,7.4–16), P=NS. The proportion of patients who presentedED was similar between treated 11/61 (18%) and naivegroups 6/17 (35.3%), P=NS. There was a significant corre-lation between FMD and vasodilator response to nitrates(r=0.48, P=0.001). Plasma adiponectin, leptin, lipoproteins,insulin, CD4 lymphocyte count and HIV-1 viral load, didnot correlate with FMD. Presence of fat distributionchanges did not influence FMD values. Patients on PI orNNRTI also showed similar FMD values. In multivariatelinear regression analyses, only basal artery diameter signif-icantly contributed to FMD.Conclusions: Endothelial dysfunction is observed in 18% ofpatients on ART, in a similar proportion to naive patients.Its presence is independent of fat redistribution abnormali-ties, plasma adipokines, lipoproteins, immune status or useof PI or NNRTI.


Comparison of Framingham and PROCAM scoresfor risk assessment of cardiovascular disease inHIV-positive adults with metabolic complications

L Normén1, B Yip2, JSG Montaner1–3, M Harris1,2, J Frohlich3, G Bondy3 and RS Hogg2,3

1 Canadian HIV Trials Network, Vancouver, BC, Canada; 2 BCCentre for Excellence of HIV/AIDS, Vancouver, BC, Canada; and3 University of British Columbia, Vancouver, BC, Canada

Objectives: 1) To calculate Framingham and PROCAM riskscores of coronary heart disease (CHD) in HIV-infectedadults referred to a specialized clinic for metabolic compli-cations, 2) to assess if the two methods give similar risk pre-dictions and 3) to compare risk scores between users andnon-users of protease inhibitors (PI).Methods: From 1999–2003, 337 HIV-positive adults wereseen in a specialized clinic for elevated blood lipid concen-trations, insulin resistance and/or lipodystrophy. CHD riskscores at the initial visit were calculated using theFramingham and PROCAM methods. A sub-analysis ofpatients with complete history of antiretroviral (ARV) ther-apy was performed. PI users were categorized as such if PIswere part of their latest ARV regimen prior to baseline.


Abstracts

Risk was assessed as categories of low to very high risk.CHD risk was compared between participants with andwithout PI treatment. Results: The studied group was mainly male (95%) with anaverage age of 47 (SD=8) years. Current smoking wasreported by 31%, diagnosed diabetes by 13% and hyper-tension by 8%. Mean blood lipid concentrations were: TC6.57 (SD=1.92) mmol/l, HDL-C 0.97 (SD=0.41) mmol/l;and TG 5.93 (SD=4.84) mmol/l. The Framingham 10-yearrisk of CHD was 15% (SD=9%, n=267). The PROCAMrisk score was 12.1% (SD=10.2%, n=258). In the sub-analysis (n=237), 32% had low 10-year risk of CHDaccording to Framingham, 33% had moderate risk, 7%had a high risk and 28% had a very high (>30%) risk. TheSpearman correlation between the scores was 0.84(P<0.01). There was no difference in CHD risk between PIand non-PI users using either method (P>0.05).Conclusions: The average CHD risk in the studied HIV-posi-tive adults with metabolic complications was moderate usingboth Framingham and PROCAM scores. A substantial num-ber had a very high, predicted 10-year risk of developingCHD. Both risk scores gave similar results in this particularpopulation, whether on PIs or not. However, the predictivevalue of both methods on actual CHD development still needsto be established in the general HIV-positive population.Acknowledgements: This study was funded by theCanadian HIV Trials Network and Bristol-Myers SquibbCompany.


Cardiovascular risk assessment with carotidultrasound and coronary calcium score in a cohortof HIV-infected subjects

A Mangili1,2, J Gerrior1, A Tang1, DH O’Leary3, JF Polak3, EJ Schaefer1, SL Gorbach1,2 and CA Wanke1,2

1 Infection/Nutrition Division, Tufts University School ofMedicine, Boston Mass., USA; 2 Division of Geographic Medicineand Infectious Diseases, Tufts University-New England MedicalCenter, Boston, Mass., USA; and 3 Diseases and Division ofRadiology, Tufts University-New England Medical Center, Boston,Mass., USA

Background: There is concern that HIV infection and useof HAART lead to accelerated atherosclerosis (AS) andincreased risk of cardiovascular disease (CVD). We mea-sured two surrogate markers of CVD, carotid intima-media thickness (IMT) by ultrasound and coronarycalcium score (CCS) by computed tomography, in HIV-infected adults from Nutrition for Healthy Living(NFHL), a longitudinal study of nutritional and metabol-ic parameters.

Methods: Cross-sectional analysis in 300 participants. 74(25%) were female, 137 (46%) were not white, 225(75%) were on HAART, 138 (46%) used proteaseinhibitors (PI), 142 (47%) smoked and 24 (8%) hadIVDU history. Mean age was 44 yrs (±7), BP 118/76(±16/10), BMI 27 (±5), waist 93 cm (±12) and tricepsskinfold 12.8 mm (±10.4). t-tests were used for compari-son of means and correlation coefficients were calculated. Results: For the common carotid artery (CCA), meanIMT was 0.61 mm (±0.18) and 25 (8%) had IMT>0.8 mm; for the internal carotid artery (ICA), mean IMTwas 0.74 mm (±0.49) and 67 (22%) had IMT >0.8 mm.For CCS, 133 (45%) had a score of 0, 141 (47%) of <100(moderate), 24 (8%) of >100 (high). Mean total choles-terol (TC) was 189 (±59), triglycerides (TG) 216 (±510),HDL 41 (±18), glucose 85 (±22), CD4 455 (±302) and logHIV RNA 3.1 (±1.1). TC, TG, ApoB and ApoE werehigher on HAART and PI regimens (P<0.05). However,IMT and CCS were not different by HAART and by PIuse (P>0.05). CD4 correlated with CCA IMT (P=0.03)and there was a trend for association of duration of HIV.Age, BP, BMI, waist, ApoE and Lp(a) correlated withCCA IMT; age, BMI, ApoB and ApoE correlated withCCS (P<0.05). Results for ICA IMT were similar. Conclusions: Of participants, 8–22% had abnormalsurrogate markers of CVD, but those were not associatedwith HAART or PI use. There were more significantcorrelations with IMT than with CCS, but CCS mightmeasure more advanced CVD. At the current time, thecorrelations were primarily with traditional CVD risk fac-tors. Some HIV-specific (not treatment-specific) factorswere observed; they may become more evident with pro-longed HIV infection and treatment.


HS-CRP is elevated in HIV-positive patients with atrend to increased levels in patients prior tocoronary events

I Woolley1, H Schneider2, A Dunne3, T Middleton4,V Sundararajan5, V Carter6, A Dart7 and A Mijch1

1 Department of Infectious Diseases, Alfred Hospital, Prahran,Vic., Australia; 2 Department of Biochemistry, Alfred Hospital,Prahran, Vic., Australia; 3 Macfarlane Burnet Institute for MedicalResearch and Public Health, Melbourne, Vic., Australia; 4 VictorianInfectious Diseases Reference Laboratory, North Melbourne, Vic.,Australia; 5 Department of Human Services, Victoria, Australia;6 Department of Nutrition, Alfred Hospital, Prahran, Vic.,Australia; and 7 Department of Cardiology, Alfred Hospital,Prahran, Vic., Australia

Cardiac events occurring in HIV-infected individuals maybe related to traditionally recognised risks, HIV treatments



or other factors. A case control examination of HIV-relat-ed factors was undertaken in those with and withoutdefined symptomatic cardiac disease treated at the AlfredHospital.

Cases were defined as having a documented myocar-dial infarct, an angiogram demonstrating vascular disease,a positive nuclear medicine scan or exercise test, or clini-cal disease with or without ECG changes treated as angi-na. Controls were selected from those individuals with noCVD matched age (±2 years), era and gender (before andafter HAART 1996), without documented cardiac events.Thirty-three cases and 66 controls were identified.

CD4 at admission and nadir, HIV RNA at event/matching date and prior peak, antivirals and cardiovascu-lar risks were analysed. There was no difference in record-ed smoking history, diabetes mellitus, cholesterol,triglycerides, hypertension, HAART therapy, HIV viralload or days of protease inhibitor therapy.

As a substudy of this study we examined the highlysensitive C reactive protein (HS-CRP) levels in our casesand controls prior to censoring from stored viral loadsamples usually taken at routine outpatient visits. TheCRP level has been shown to be one of the stronger pre-dictors of a cardiac event in an HIV-negative populationand postulated to be a direct player in the pathogenesis ofcoronary disease. Whereas the normal levels in an HIV-negative population are less than 5, the levels in our studywere a mean of 6.70 (n=49, SD=10.41) for controls and10.92 (n=23, SD=18.92) for cases. The difference betweencases and controls did not reach statistical significance byunivariate analysis.

These data in a small population suggest further exam-ination in a larger population is warranted.


Percutaneous coronary intervention in HIV-infected patients: immediate results and long-termprognosis

F Boccara1, E Teiger2, A Cohen1, G Odi1, E DiAngelantonio1, G Barbarini3 and G Barbaro4; onbehalf the French Italian Study on Coronary arterydisease in AIDS patients (FRISCA-1)

1 Department of Cardiology, Saint Antoine University Hospital,Assistance Publique-Hôpitaux de Paris and Université Paris VI,Paris, France; 2 Department of Physiopathology, Henri MondorUniversity Hospital, Assistance Publique-Hôpitaux de Paris andUniversité Paris XII, Creteil, France; 3 Department of Infectiousand Parasitic Diseases, University of Pavia, Italy; and 4Department of Medical Pathophysiology, University La Sapienza,Rome, Italy

Background: Acute coronary syndromes and coronaryartery disease are emerging complications in HIV-infectedpatients (HIV+) under highly active antiretroviral treat-ment. Immediate results and long-term prognosis of per-cutaneous coronary intervention (PCI) remains unknownin this population. Methods and results: Between January 2001 andDecember 2003, we compared baseline characteristics,rate of procedural success and clinical outcome at 12months (Major Adverse Cardiac Events: death from anycause, myocardial infarction, target lesion or vessel revas-cularization) between 50 consecutive HIV+ and 50 HIV–control patients matched for age and gender (mean age,44±5 years; 89% male) who underwent PCI.Cardiovascular risk factors were well-balanced and near-ly identical in both groups with a higher rate of hyper-triglyceridaemia (78% vs 46%, P=0.001) and lowerHDLc (88% vs 42%, P<0.001) in HIV+ patients. ST seg-ment elevation MI was more frequent in HIV– patientscompared with HIV+ patients (48% vs 22%, P<0.01)whereas NSTEMI and late-diagnosed MI (1–30 days) wasmore frequent in HIV+ patients (74% vs 38%, P<0.001and 20% vs 8%, P=0.08). Procedural success rate wasachieved in 98% of cases and in-hospital course wasuneventful in both groups. Rates of occurrence of firstMACE and clinical restenosis at 12 months revealed nostatistical difference between HIV+ and HIV– patients(20% vs 16%, P=0.64 and 14% vs 8%, P=0.34). The useof protease inhibitor was not predictive of any cardiovas-cular events (MACE, TLR and TVR). In a multivariateanalysis, the initial clinical presentation with late diag-nosed MI (1–30 days) was the only independent predictorof MACE at 1-year follow-up (OR 4.54; 95% CI1.25–16.38, P=0.02). Conclusions: PCI is feasible and safe in HIV+ patients. At1-year follow-up after PCI, no differences in MACE andrestenosis rates were observed in either HIV+ or HIV–patients.


Antiretroviral exposure and carotid intimal medialthickness in a multicentre Canadian HIV cohort

M Smieja1, E Lonn1, FM Smaill1, L Kelleher1,S Schmidt1, S Smith1, S Holmes1, W Cai1,K Gough2, S Trottier3, J Gill4 and M Harris5 for theCanadian HIV Vascular Study Investigators

1 McMaster University, Hamilton, Ont., Canada; 2 University ofToronto, Toronto, Ont., Canada; 3 Universite de Laval, Quebec,Que., Canada; 4 University of Calgary, Calgary, Alb., Canada; and5 University of British Columbia, Vancouver, BC, Canada


Abstracts

Objectives: Treatment of HIV is associated with a pro-atherogenic lipid and glucose profile, but whether treat-ment accelerates atherosclerosis remains unclear. We areinvestigating the determinants of atherosclerosis, using awell-validated and reproducible measure of carotid arterythickening.Methods: HIV subjects aged 35 years or older, attendinguniversity-affiliated clinics in five Canadian centres, arebeing recruited into a prospective study of cardiovascularrisk. Clinical risk factors, fasting lipids and glucose,immunological parameters and medication exposure arerecorded. Subjects undergo yearly high-resolution carotidartery ultrasound, according to a standardized and quali-ty-controlled protocol. Videotaped images are read bycomputer-assisted algorithms to determine 12-segmentmean maximal intimal medial thickness (IMT). We soughtan association between carotid IMT and clinical andmetabolic variables, and with a history of exposure tozidovudine, stavudine, efavirenz or to protease inhibitors,by simple and multiple linear regression. Of 162 subjectsstudied at baseline, 59 have undergone 1-year follow-up.Results: Mean (SD) age of patients was 45.8 (8.0) years,93.2% were men and 41.4% were current smokers. MeanCD4-lymphocyte count was 479 with a mean log viralload of 2.26. A history of exposure to zidovudine, stavu-dine, efavirenz or protease inhibitors was present in79.6%, 60.5%, 60.5% and 70.4%, respectively, for amean of 41.9, 37.7, 25.9 and 46.1 months, respectively.Carotid IMT was 0.82 (0.25) mm at baseline andincreased by 0.04 (0.09) mm/year. Carotid IMT was asso-ciated with multiple clinical (age, smoking, history ofhypertension or diabetes, systolic and diastolic bloodpressure), and metabolic variables (total cholesterol, LDL-cholesterol, triglycerides and glucose). Exposure tozidovudine (P=0.03), stavudine (0.01) or proteaseinhibitors (0.02), but not to efavirenz (0.17), was associ-ated with greater carotid IMT. In multivariable linearregression, only age, current smoking, systolic blood pres-sure, total cholesterol and glucose remained significantpredictors, whereas antiretrovirals and triglycerides werenot associated. One year carotid IMT was not associatedwith antiretroviral exposure.Conclusions: Age, gender, smoking, cholesterol, glucoseand systolic blood pressure, but not antiretroviral expo-sure, were the main determinants of atherosclerosis inHIV subjects. Longer term follow-up is needed to deter-mine more precisely whether antiretrovirals accelerateatherosclerosis.Acknowledgement: Funded by the Ontario HIVTreatment Network.


HIV viral protein R causes atrial cardiomyocytemitosis, mesenchymal tumour, dysrhythmia, andheart failure

W Lewis, YK Miller, CP Haase, T Ludaway, J McNaught, R Russ, J Steltzer, A Folpe, R Long andJ Oshinski

Emory University School of Medicine, Atlanta, Ga., USA

Background: HIV viral protein R (Vpr) affects the immuno-cyte cell cycle, circulates as free polypeptide in plasma ofAIDS patients and is implicated in cardiac disease in AIDS.Paracrine effects of circulating Vpr may occur in the heartsof AIDS patients. Aims: Direct effects of Vpr on cardiomyocytes wereexplored using transgenic mice (TG). Methods: HIV Vpr was targeted to cardiomyocytes by cou-pling the Vpr gene to the a-MyHC promoter. TG and WTlittermate hearts were evaluated histopathologically, ultra-structurally, molecularly via RNA microarray analysis andquantitative RT-PCR, and functionally by cardiac MRI andelectrocardiograms (ECG). Results: Six lines were created (Vpra, b, c, d, e and h). VprRNA was expressed exclusively in myocardium. The Vprbline exhibited highest expression and those TGs developedcongestive heart failure (approximately 8 weeks), abnormalcardiomyocyte nuclei and mitoses (approximately 12weeks) and became moribund (approximately 20 weeks)with atrial mesenchymal tumours. Median survival waslowest in Vprb (median approximately 200 days), andhigher in other TG lines with lower Vpr expression (greaterthan 300 days). Cardiac MRI performed on Vprb heartsrevealed four chamber dilation, defective contraction andintracavitary atrial masses. Pathologically, cardiomegalyand intracavitary atrial mesenchymal tumours occurred(approximately 16–20 weeks). ECGs on TGs revealed pro-longed R-R, Q-T and P-R intervals (approximately 12weeks). Using microarray techniques, RNA encoding colla-gen and bone morphogenic protein 4, 6 and 7 wereincreased and were confirmed with RT-PCR. Conclusions: These results indicate that Vpr targeted to car-diomyocytes caused defective contractility and developmentof proliferative atrial tumours. Despite the absence of suchcardiac tumours in humans with AIDS, Vpr cardiomyocyt-ic effects resemble some of those found in terminally differ-entiated immunocytes. Findings with this TG model ofVpr-induced cell dysfunction may mechanistically linkAIDS CM to other cellular defects in AIDS through sharedcellular events. Supported by R01 HL072707.




Blood pressure elevation and hypertension inpaediatric patients with or without humanimmunodeficiency virus-associated lipodystrophy

S Benavides1, KI Koranyi2 and MC Nahata3

1 University of Texas, Pan American, Edinburg, Tex., USA;2 Children’s Hospital, Columbus, OH, USA; and 3 Ohio StateUniversity, Columbus, OH, USA

Objectives: Elevated blood pressure (EBP) and hyperten-sion (HTN) have been described in adults with humanimmunodeficiency virus (HIV) and metabolic disorders.The prevalence of EBP or HTN has not been reported inpaediatric HIV patients. The objective of the study was todetermine the prevalence of EBP and HTN in patientswith or without a clinical diagnosis of HIV-associatedlipodystrophy.Methods: Medical records of all paediatric patients seen atthe Immunodeficiency Clinic at Children’s Hospital,Columbus, OH from 1 July 1999 to 31 August 2003 werereviewed. Demographic data and evidence of lipodystro-

phy, dyslipidaemia, glucose intolerance and elevated bloodpressure were collected. EBP and HTN were defined by theFourth Report on the Diagnosis, Evaluation, and Treatmentof High Blood Pressure in Children and Adolescents.Descriptive statistics were compiled and patients were strat-ified into two groups (hypertensive or normotensive). Crosstabulations were used to determine if hypertension wasassociated with lipodystrophy. Results: Thirty-one patients (M=17, F=14), ages 1.5–16.6years including 12 (38.7%) Caucasian, 16 (51.6%) AfricanAmerican, two (6.5%) Latino and one (3.2%) biracial werereviewed. Of these patients, six (19.4%) patients hadlipodystrophy, 15 (48.4%) had hypercholesterolaemia andone (3.2%) had increased glucose concentrations. Inpatients with clinically diagnosed HIV-associated lipodys-trophy, three (50%) met criteria for diagnosis of HTN andthree (50%) had EBP. In patients without clinically diag-nosed HIV-associated lipodystrophy, 10 (40%) met criteriafor diagnosis of HTN and 14 (56%) had EBP. Conclusion: EBP and HTN are prevalent in paediatricpatients with HIV with and without lipodystrophy. Clinicalsignificance of this finding, particularly in the presence ofother metabolic abnormalities and their management, needto be prospectively studied.

abstracts presented at the6th international workshop on

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