Acute Coronary Syndromes account for 15% of deaths in the UK, and patients remain at significant risk of re-admission for future complications and death. Pathologically the underlying process of atherosclerosis is driven by inflammatory mechanisms, which are activated in ACS patients. Previous studies have investigated the role of inflammatory markers in this process, including interleukin 1 (IL-1) and C Reactive Protein (CRP). Pre-clinical studies indicate that IL-1 may be a primary driver of ACS and that the naturally occurring interleukin-1 receptor antagonist (IL-1ra) may inhibit the atherosclerotic process. This study will investigate the effects of IL-1ra on inflammatory markers in man.Three centres in the UK are planning to recruit 186 Non-ST elevation myocardial infarction patients to receive either interleukin-1 receptor antagonist (Anakinra) or matching placebo. Patients will receive a daily subcutaneous injection of either study drug or placebo over a 14 day period. The primary outcome is area under the curve of high sensitivity C-Reactive Protein (CRP) over the first 7 days.The MRC-ILA-HEART Study is a proof of concept clinical trial investigating the effects of IL-1ra upon markers of inflammation in patients with Non-ST elevation myocardial infarction. It is hoped this will provide new and exciting information in relation to an "anti-inflammatory" strategy for patients with acute coronary syndrome.ISRCTN89369318 Acute coronary syndromes (ACS), caused by coronary atherosclerotic plaque destabilisation and interruption of coronary flow, are common, serious conditions that account for 15% of all deaths in the UK and are associated with considerable morbidity among survivors. ACS are classified by the presenting ECG: patients with persistent ST elevation myocardial infarction (STEMI) generally require early reperfusion . There rThe past decade has seen a growth in treatment options available for ACS as a consequence of better understanding of the pathophysiology of this condition. At a biological level the process is driven by inflammatory mechanisms in the vessel wall where atherogenesis is initiated by lipids and other environmental agents ,5. InstaThe therapeutic implications of these pathogenic pathways are that primary and, to some extent, secondary prevention are targeted at mutable risk factors  and anti-thrombotic strategies for the treatment and prevention of intracoronary thrombus. Evaluation of therapeutic strategies designed to treat the inflammatory processes involved in acute or chronic coronary artery disease (CAD) is at an early stage. Some agents may potentially have anti-inflammatory properties (statins in particular and fish oils) but this is not their primary mechanism of action and proving beneficial effects consequent upon the anti-inflammatory properties of these agents has been difficult.Since the description of ACS as an inflammatory event potential sources of, and functions for, inflammatory cytokines in this process have been investigated ,6. IL-1 The suggestion that IL-1 is involved in the pathogenesis of CAD, and in particular in ACS, is derived from a number of sources as there is no animal model of this condition. IL-1 and IL-1ra are expressed in diseased human coronary arteries ,9. CoronIn mouse models of atherosclerosis IL-1ra inhibits the formation of intimal fatty streaks in apolipoprotein E-deficient mice , and IL-In a cohort of 63 NSTEMI ACS patients we examined the impact of IL-1 gene polymorphisms upon markers of endothelial cell activation and CRP . The intOverall, our preliminary data indicate that 1) IL-1 is a potent mediator of arterial inflammation in the context of atherosclerosis and vessel wall injury and 2) IL-1 inhibition restores endothelial function, reduces inflammatory vessel wall responses and reduces vascular oxidative stress. We wish now to translate these findings to man.ACS, as has been stated before, is an intensely inflammatory event at the level of the vessel wall. Data from angioscopy , angiogrIn the latter group  are the markers of inflammation, coagulation and of platelet activation. We believe that there are plausible links between IL-1 activation and these markers. The acute phase reactant C-reactive protein (CRP) is a strong predictor of cardiovascular events ,28. In NDuring NSTEMI, a rise in von Willebrand Factor (vWF) between 24 and 48 h predicts adverse clinical events , and an There are two agonistic cytokines (IL-1α and IL-1β) that signal via the Type 1 IL-1 receptor (IL-1R1) and a specific Interleukin-1 receptor antagonist (IL-1ra),  that inhibits IL-1 actions by binding to the IL-1R1, blocking the receptor and preventing IL-1 from binding thereby preventing receptor signalling .IL-1ra is available for the treatment of a number of inflammatory conditions as a recombinant, non-glycosylated, N-terminal methionine added protein . Although less successful than anti-TNF therapies in rheumatoid arthritis (RA), there appears to be considerable benefit in the management of Juvenile arthritis (Still's disease), and it is the treatment of choice in the rare autoinflammatory illnesses typified by Muckle-Wells syndrome. From all of the indications in which IL-1ra has been used it has emerged that IL-1ra is a very safe form of treatment and appears to be devoid of the side-effect of re-activation of infectious illness seen with anti-TNF therapies. The only problems encountered are mild, reversible injection site reactions and neutropaenia in <1% of patients after a month or more of treatment.Dosing with IL-1ra is simple. A single, daily, subcutaneous 100 mg injection of IL-1ra is the standard dose of IL-1ra for all individuals with normal renal function. In RA this causes a 35% reduction in CRP after approximately 1 week of treatment. Higher doses do not achieve any greater reduction in CRP of relief of RA symptoms. Single daily dosing schedules are used in the sucessful treatment of Juvenile arthritis and Muckle-Wells. The half-life of IL-1ra is 4–6 hours with maximum absorption from an s.c. injection at 3–7 hours. (Data on file supplied by Amgen).All of these data indicate that the IL-1 cytokine has the potential to act as a primary driver of ACS. Prolonged coronary activation is associated with worse outcome in ACS. It is therefore plausible that blockade of the IL-1 system could be of therapeutic benefit. We believe our pre-clinical data indicates that a purely anti-inflammatory approach based upon Interleukin-1 (IL-1) inhibition using IL-1 receptor antagonist (IL-1ra) is a feasible and logical therapy for ACS and this approach could open a new therapeutic paradigm for evaluation in further studies.The main objective is to assess whether treatment of NSTEMI ACS with IL-1ra alters the inflammatory process involved in this conditionRandomised, double blind, placebo-controlled multi-centre phase II clinical trial1. Acute cardiac chest pain consistent with an acute coronary syndrome2. <48 hours from onset of symptoms that led to hospital admission3. And at least one of the following:a) A raised troponin as defined by local parameters specified at each centreb) Other ECG changes consistent with acute myocardial ischaemia  and an elevated level of Troponin above local laboratory values indicating myocardial damage1. < 18 years of age2. Persistent ST elevation on the presenting ECG3. Intention to treat with an urgent reperfusion strategy (thrombolysis or primary percutaneous coronary intervention)Percutaneous coronary intervention within previous 3 months4. Previous coronary artery bypass grafting5. ECG showing paced rhythm6. Cardiogenic shock 7. Any serious co-morbidity which makes it unlikely that the patient will complete trial procedures and follow-up8. Treatment or under active follow-up for rheumatoid arthritis, other connective tissue diseases or inflammatory bowel disease9. End stage renal disease or a Creatinine > 220 μmol/L10. Pregnancy or suspected pregnancy 11. Eosinophilia12. Anti-TNF biologies13. Active infection14. MalignancyEligible patients will be randomised in equal proportions between IL-1ra and placebo, receiving either a once daily, subcutaneous (s.c). injection of IL-1ra (dose 100 mg per 24 h) for 14 days, or a daily s.c. injection of placebo for 14 days. The hospital pharmacist will issue the relevant drug package. (Please see appendices for pharmacy information).The study drug and placebo will be provided by Amgen Inc in its commercially available recombinant form which is in clinical use for the treatment of arthritic conditions. The study drug and placebo will be relabelled by Amgen, in collaboration with CTEU according to MHRA guidelines.The first dose of IL-1ra will be given within 24 h +2 h of the positive Troponin. Injections will be given at a standardised time (24 ± 2 h after the previous dose), immediately after blood sampling. IL-1ra or placebo will administered to the patient by the research nurse while the patient is in hospital. During the hospital stay, the patient will be taught to self-administer the injection by the research nurse and on discharge will continue at home. This has proven possible in other ACS trials that required self injection of subcutaneous heparin . Full wrAll patients will receive standardised additional treatments according to best practice including aspirin, clopidogrel, heparin (intravenous or low-molecular weight), a glycoprotein IIb/IIIa inhibitor and a statin. Other medication will be at the physicians' discretion. It is the intention that all patients will proceed to coronary angiography according to current guidelines, and subsequent revascularisation if necessary.Each vial contains 100 mg of anakinra in 1 ml (100 mg/ml). Anakinra is a human interleukin – 1 receptor antagonist (r -metHuIL -1ra) produced by recombinant DNA technology in an E coli expression system.Amgen Europe B.V., Minervum 7061, 4817 ZK Breda, The NetherlandsEach vial consists of sodium citrate, sodium chloride, disodium edentate, polysorbate 80, sodium hydroxide and water for injection.Participating centres and investigators have extensive experience of clinical trials and admit large numbers of patients with acute coronary syndromes. It is expected that screening for eligible patients will occur on a daily basis and potentially eligible patients will be approached for consent to participate. Screening for patients should occur routinely in ward areas that admit ACS patients. Centres are encouraged to screen and enrol at the weekends and out of hours depending on local resources. Each centre is requested to randomise and follow-up an average of 62 patients (30 per annum or 2–3 patients per month) over a 2 year period.A member of the research team will screen patients for eligibility as soon as possible after presentation. If patients meet the eligibility criteria they will be given the patient information sheet to consider. Patients will be given as much time as they require to consider taking part but due to the acute nature of this condition, patients will be recruited as soon as possible after the positive troponin is confirmed. A TIMI  and GRACE risk score will be determined for each patient at baseline ,37."Informed consent" requires individual discussion with the patient about the nature of the procedures to be conducted in a language that is easy to comprehend. The patient should fully understand that he/she might be allocated to either the IL-1ra or the placebo group and the potential adverse effects of participation. The patient should also understand that his/her refusal to participate in the study will not affect the quality of subsequent medical care and if they do consent to participate they may withdraw at any point without affecting their care.Before any trial-related procedures may be performed, informed consent must be obtained from the patient by the investigator by means of a signed declaration. The investigator must sign in the CRF to confirm that informed consent was obtained and store the original of the signed declaration of consent in the patient's notes. A copy should be given to the patient and a copy filed in the CRF.Randomisation will be performed using a 24-hour voice interactive system set up the trial co-ordinating centre  of the Royal Brompton Hospital, London). Centres will be asked for a few simple details about the patient including initials, date of birth and eligibility criteria. The caller will be given a randomisation number, corresponding to blinded study treatment, and a fax or e mail will be sent to the centre confirming this. Randomisation will be stratified by centre and by whether glycoprotein IIb/IIIa antagonists are being used or planned. Patients entered into the study in this way will be considered as part of the intention to treat cohort irrespective of whether they actually receive treatment or not, or if they subsequently with draw from the study.Patients will have blood taken every day for seven days for hsCRP , Troponin (baseline and days 1–7 and 14), full blood count , creatinine, electrolytes, liver function tests , Interleukin-6 and von Willebrand factor . Blood will be taken prior to administration of either IL-1ra or placebo. A summary of samples and study related procedures to be undertaken is shown in Table All patients will be followed up for MACE at 30 days (at clinic), three months and one year by telephone call with the Research Nurse. Patients will also be followed up for up to five years by the ONS.Common genetic variations in the IL-1β gene have been shown to influence IL-1 β production and been implicated in the causation of premature coronary artery disease. IL-1β effects are mediated via the IL-1 receptor that is antagonised by IL-1ra. As with other receptors, stimulation may result in down-regulation and inhibition in up-regulation. We aim to demonstrate this by measurement of IL-1r mRNA expression. Genomic DNA and mRNA samples will be collected, stored and studied. These will be used to study the genetic substrate of observed differences in inflammatory markers and also effects of IL-1ra treatment – with initial experimental questions being as outlined here. Two additional blood samples will be taken at base-line and follow up at day 30 (1) 6 ml blood in EDTA tube for DNA extraction using the 'Versagene' DNA purification system (2) 5 ml blood in 'PAXgene' vacuum tube.There are three sub studies proposed for the study (please see appendix 1.2. for protocols) including:1. Cardiac Magnetic Resonance (CMR) – Leeds Patients only2. ST-Holter segment analysis – Sheffield patients only3. Forearm Endothelial Cell response – Edinburgh patients onlyArea under the curve of serum high sensitivity CRP over the first 7 days1. Mean hsCRP at 7, 14 and 30 days2. Area under the curve of Troponin-I3. vWF and IL-64. ST segment depression on Holter monitor5. Myocardial injury as determined by Gadolinium enhanced CMR scan6. Forearm endothelial cell response7. Incidence of major adverse cardiovascular events (MACE) at 30-days, three months and at one year.8. Flagging with Office of National Statistics (ONS) for up to 5 years.To provide the study with an 80% power to detect a relative reduction of one-third (the reduction seen in IL-1ra treatment of rheumatoid arthritis at this dose (data on file [Amgen]) between the treatment and the placebo groups at a 5% level of significance will require approximately 80 patients in each group . This is based upon the log-transformed value of the area under the curve and a standardised difference (difference/standard deviation [D]) of approximately 0.45. The standardised difference had been used since it is very difficult to estimate what the values of the log-transformed AUC will be. A change of 0.45 on the standardised normal will correspond to a one-third change in the primary outcome, regardless of the scale of the measurement, assuming the outcome measure is normally distributed. As the sample size is sufficiently powered to detect a change equivalent to one-third on the standard normal, it will also be sufficient to detect the same level of change on the primary outcome measure. It is anticipated that follow-up will be near complete in this study. However, allowing for data loss, for whatever reason, of 10% in each arm we have increased the required sample size to approximately 180 patients (60/centre). Furthermore, the sample size needs to be adjusted for the 2 interim analyses that will be conducted during the trial. Using the method of O'Brien and Flemming (1979) gives an increased sample size of 184 patients.).A limited number of interim analyses will be performed by the trial statistician as specified by the Data Monitoring Committee (DMC). The accumulating results will not be available to the trialists or other principal investigators. The final, definitive analysis of the trial data will be conducted 25 months after the date of commencement of the trial. The interim analysis will test for efficacy, safety and futility. The method of O'Brien and Fleming suggests that stopping for efficacy will only occur if the first interim analysis shows a treatment effect equivalent to p < 0.0005 or if the second interim analysis shows a treatment effect equivalent to p < 0.014.Primary analysis will be of log-transformed AUC of 7 day CRP. The primary analysis will include all patients with at least 4 follow-up readings, on an intention-to-treat basis, and a secondary analysis will include only those with all follow-up readings on a per-protocol basis. ANOVA  will be used to analyse the primary outcome measure and to estimate the treatment effect. For patients with at least 4 readings, any missing measurements will be imputed using the last observation carried forward method. After adjustment for the interim analyses, based on the method of O'Brien and Fleming, the primary endpoint will be considered to be statistically significant if it shows a treatment effect equivalent to p < 0.045.The main role of the TSC is to monitor and supervise the progress of the trial. The TSC membership is listed in Table Representatives from the sponsor organisation, University of Sheffield, and funding organisation, Medical Research Council, will attend the Trial Steering Committee as observers.The main role of the DMC is to consider the data from any interim analyses and specifically to assess any safety issues (such as unexpected serious adverse events) that occur and report back to the TSC. The DMC membership is listed in Table The Clinical Event Review Committee will review adverse events during the study and adjudicate them to ensure the events meet the definitions given. Each event will be independently adjudicated by two committee members. A third committee member will be called to adjudicate an event if agreement is not reached by two members. The adjudications will be blind to the knowledge of which arm of the trial the patients are in.The study will be co-ordinated and managed by the Clinical Trials and Evaluation Unit (CTEU) a dedicated clinical trials department within the Royal Brompton Hospital. In addition to providing overall project co-ordination, the CTEU will assist in preparing the final protocol, the investigators' manuals, design the Case Report Forms (CRF), provide the randomisation service and design and instigate the data management system. The CTEU will ensure that the trial runs according to the pre-agreed timetable, recruitment targets are met, CRFs are completed accurately, compliance with relevant ethical and regulatory standards, and that all aspects of the study are performed to the highest quality. The CTEU will also assist in the training of investigators and co-ordinators at the start-up of the study and for performing monitoring procedures throughout. A smaller trial management team consisting of lead investigators at each site and CTEU will meet weekly by conference call.Support for patient identification, study administration, data collection and follow-up will be provided to each participating site. Study site co-ordinators will be responsible for screening patients (and recording the data on a screening log), enrolling patients into the trial, providing a contact point for patients, liaising with CTEU, completing CRFs, arranging all follow up visits and measurements, recording adverse events, ensuring forms are sent to CTEU and that all edit queries are resolved.Each centre will be provided with a Protocol, Manual of Operations, questionnaires and patient CRFs. Data will be recorded onto two part NCR CRFs and the top copy sent to the CTEU at the times specified.Investigators must ensure that Local Ethics Committee approval has been obtained as well as Agreements signed off by their Institution prior to the start of the study. Investigators are responsible for performing the study in accordance with the European Clinical Trials Directive, the Medicines for Human Use  Regulations 2004, NHS Research Governance, MRC Guidelines for Good Clinical Practice in Clinical Trials (1998) and the Declaration of Helsinki guidelines. Investigators are required to ensure compliance to the protocol, CRFs and Manual of Operations. Investigators are required to allow access to study documentation or source data on request for monitoring visits and audits performed by the CTEU or any regulatory authorities.Before the study commences each centre will receive a training visit by CTEU. These visits will ensure that personnel at each site  fully understand the protocol, CRF and the practical procedures for the study.At regular intervals during the study CTEU will perform monitoring visits to each centre. The purpose of these visits is to ensure compliance to the protocol and that ethical and regulatory guidelines are met. Source data verification and checking of essential documents will be performed. Monitoring visits also provide an opportunity for further training if required (eg new staff). Central review of study data will also be performed throughout the study.At the end of the study each centre will receive a site visit from CTEU to resolve any outstanding edit queries or adverse events and to verify the correct storage of study documentation.This study is a randomised trial of a medicinal product in a new indication. The IL1-receptor antagonist is commercially available and approved for use in specific inflammatory conditions. As such the study will need to comply with the European Clinical Trials Directive and the Medicines for Human Use  Regulations 2004. An application will be made for a clinical trials authorisation (CTA) to the Medicines and Healthcare Regulatory Agency (MHRA) prior to starting the study. The study has been registered in the European Community with a Eudract number.The grant has been awarded to the University of Sheffield who will act as the main Sponsor of the study. The Sponsor's role is clearly set out in the European Clinical Trials Directive and NHS Research Governance documents. Research agreements will be held with the 4 collaborating groups: University of Edinburgh/Royal Infirmary of Edinburgh, University Hospitals of Leeds, Sheffield Hospitals NHS Trust and Royal Brompton and Harefield NHS Trust. The Clinical Trials and Evaluation Unit of the Royal Brompton will be responsible, on behalf of the University of Sheffield, for ensuring that regulatory compliance is carried according to the high standards expected.The University of Sheffield (Sponsor) has delegated responsibility for pharmacovigilance to the trial co-ordinating centre  of the Royal Brompton Hospital, London).The CTEU will be responsible for recording all reported serious adverse events from investigational trial sites, reporting all serious adverse events to a constituted serious adverse events committee (SAEC) for review and expedited reporting of suspected unexpected serious adverse reactions (SUSARs) in accordance with statutory regulations. The SAEC will consist of one nominated investigator from each trial site who can provide clinical expertise to adjudicate serious adverse events and determine if they are SUSARs, and advise the CTEU whether expedited reporting is required.An adverse reaction of an investigational medicinal product (AR) can be defined as all untoward and unintended responses to an investigational medicinal product related to any dose administered.In the event an AR is reported during the trial, investigators will assess the severity of the adverse event using the following criteria, detailed on the adverse event report form in the case report form (CRF):Mild: Awareness of signs or symptoms, but easily tolerated; are of minor irritant type; causing no loss of time from normal activities; symptoms would not require medication.Moderate: Discomfort severe enough to cause interference with usual activities.Severe: Inability to do work or usual activities; signs and symptoms may be of systemic nature or require medical evaluation and/or treatment.In all placebo-controlled trials conducted by Amgen, injection site reactions (ISRs) were frequently reported in the majority of patients (95% of which were reported to be mild to moderate in severity). ISRs included erythema, ecchymosis, inflammation and pain. On a dose of 100 mg/day, 71% of patients developed an ISR (n = 1565) compared to 28% of placebo treated patients (n = 733). The median duration of typical symptoms was 14 to 28 days. Other adverse events reported by patients administered with IL-1ra were, URI, headache, nausea, diarrhoea, sinusitis, arthralgia, flu like symptoms and abdominal pain. Allergic reactions reported in patients were rare.Reported ISRs and other drug related adverse reactions will be reported on the adverse event report in the CRF. Patients reporting ISRs will be required to identify another injection site in which to administer the injection. Investigators will submit all adverse events reports for each patient to the CTEU at days 7, 14 and 30, three months and one year. The CTEU will maintain a log of all adverse reactions.Serious adverse events (SAE) or reactions can be defined as any untoward medical occurrence or effect that at any dose• results in death,• is life-threatening,• requires hospitalisation or prolongation of existing inpatients hospitalisation,• results in persistent or significant disability or incapacity,• is a congenital anomaly or birth defect.• Myocardial Infarction (MI)• Stroke• Revascularisation• Major Bleed• Death• Serious Infections – Previous trials conducted by Amgen demonstrated an incidence of serious infections in 1.8% of IL-1ra treated patients compared to 0.7% in placebo group. There were no on-study deaths due to serious infectious episodes.9/I) in 2.4% of patients compared with 0.4% of placebo patients. None of these patients had serious infections associated with the neutropenia.• Neutropenia – Administration of IL-1ra was associated with neutropenia  will be marked "possible SUSAR" on the SAE report. The investigator will also attach a discharge/medical summary and fax to the CTEU with the SAE report within 24 hours. Any other SAE not fitting the criteria above will be reported on the "other SAE" report in the CRF.The CTEU will review all serious adverse event/reaction reports before sending an electronic copy to the SAEC for adjudication. All reported SAE/Rs will be adjudicated by the SAEC to assess the seriousness and causality between the IMP and/or concomitant therapy and serious adverse event/reaction. This assessment must take place within two working days upon receipt of the electronic copy of the form. If the SAEC judge the SAE/R to constitute a SUSAR, the CTEU will ensure expedited reporting procedures are followed.A SAR can be considered unexpected when the adverse reaction, the nature or severity of which is not consistent with the applicable product information. All suspected unexpected serious adverse reactions related to an IMP, which occur during the trial, are subject to expedited reporting.The CTEU will unblind the SUSAR report before sending it electronically to the SAEC with the medical summary attached. The SAEC will review the report and summary within two working days and adjudicate whether the event constitutes a SUSAR. The SAEC will inform the CTEU of their decision to ensure expedited reporting procedures are followed if necessary.All SUSARs will be reported to the MHRA and NHS REC, by the trial co-ordinating site (CTEU). All SUSAR reports will be unblinded by the CTEU prior to submission. A SUSAR, which is fatal, or life threatening will be reported to the MHRA and the main REC by the CTEU as soon as possible and within 7 days after the CTEU become aware of the event. A SUSAR, which is not fatal, or life threatening must be reported to the MHRA and main Research Ethics Committee (REC) as soon as possible and within15 days after the CTEU become aware of the event. The CTEU will inform all trial investigators of all reported SUSARs within 15 working days.The CTEU will submit annual safety reports of all suspected serious adverse reactions in accordance with regulatory requirements to the MHRA and Main REC. Annual safety reports will be submitted to the MHRA on the date of the original clinical trials authorisation. Annual progress reports will also be submitted to the main REC on the same date. Annual safety reports will also be submitted to the Data Monitoring Committee for review.Investigators must ascertain the reasons for the withdrawal due to adverse events, failure to attend, non-compliance, withdrawal of consent or other reasons. The withdrawal form must be faxed to the CTEU within five working days, unless withdrawal is due to a SAE, in which case the investigator will follow SAE reporting procedures.The treatment code must not be broken except in medical emergencies when the appropriate management of the patient necessitates knowledge of the treatment. Each pharmacy will keep a copy of the randomisation allocation list, in case unblinding is necessary. Should a SUSAR occur, the CTEU will unblind the patient and follow expedited reporting procedures.Patients that are enrolled into the study are covered by indemnity for negligent harm through the standard NHS Indemnity arrangements. The University of Sheffield has insurance to cover for non-negligent harm associated with the protocol. IL1-ra is commercially available for the treatment of inflammatory arthritic conditions. The rate of serious side effects attributable to treatment is <1%. This will include cover for additional health care, compensation or damages whether awarded voluntarily by the Sponsor, or by claims pursued through the courts. Incidences judged to arise from negligence (including those due to major protocol violations) will not be covered by study insurance policies. The liability of the manufacturer of IL1RA (Amgen Corporation) is strictly limited to those claims arising from faulty manufacturing of the commercial product and not to any aspects of the conduct of the study.This study will conform with the Declaration of Helsinki guidelines on research involving human subjects. The study protocol will be submitted to the Multi-centre Research Ethics Review Committee, and Ethics Committees at each centre participating in the study and approval obtained before the study commences.The results from the trial will be submitted for publication in a major journal irrespective of the outcome. The Trial Steering Committee will be responsible for approval of all manuscripts arising from the study prior to submission for publication. Sub-studies of centre-specific data may only be carried out with the knowledge and approval of the Trial Steering Committee.Any individual authorship of presentations and reports related to the study will be on behalf of the collaborative group. The final follow-up study results paper will name local co-ordinators as well as those involved in central co-ordination and trial management.At the end of the study, patients will be able to request a copy of the results of the study from the investigator at that site.March 2006 – Grant awarded by MRCJan 2007 – Recruitment startsDec 2009 – Recruitment completedJan 2010 – Last patient completes 30 day follow up2010 – Publication of dataCRP – C Reactive Protein. IL-1 – Interleukin 1. IL-1ra – Interleukin 1 receptor antagonist. ACS – Acute Coronary Syndrome. ECG – Electrocardiogram. AUC – Area under the curve. REC – Research Ethics Committee. IMP – Investigational Medicinal Product.Professor David Crossman declares holding limited stock in "IL-Genetica". There would be no direct benefit to this company from the trial.All other co-author(s) declare that they have no competing interests.DC: Conceived of the study, participated in the design and co-ordination and helped to draft the manuscript. AM: Participated in the design and co-ordination and helped to draft the manuscript. JG: Participated in the design and co-ordination and helped to draft the manuscript. JGr: Participated in the design and co-ordination and helped to draft the manuscript. AH: Participated in the design and co-ordination and helped to draft the manuscript. KF: Participated in the design and co-ordination and helped to draft the manuscript. AL: Participated in the design and co-ordination and helped to draft the manuscript. MF: Participated in the design and helped to draft the manuscript. BL: Participated in the design and co-ordination and helped to draft the manuscript. CF: Participated in the design and co-ordination and helped to draft the manuscript.All authors read and approved the final manuscript. Each author is supported directly by their host institution.Dr David SmithPharmacy Information Services ManagerNorthern General HospitalHerries RoadSheffieldS5 7AUProf Norman LanniganHead of PharmacyRoyal Infirmary of Edinburgh51 Little France CrescentEdinburghEH16 4SADr Caroline BedfordClinical Trials and Unlicensed Medicines ManagerLeeds Teaching Hospitals NHS TrustLeedsExamples of the labelling can be found appended to the Summary of Product Characteristics.Store in a refrigerator (2°C – 8°C).EU/1/02/203/001-003Samples will be stored in -20°C freezers at each centre. All participating centres have research freezers available for use. FedEx will then send samples in batches to the core labs.The Leeds cohort of patients will undergo baseline and follow-up Gadolinium enhanced CMR as a quantitative measurement of extent of myocardial injury. We wish to evaluate the possible mechanisms of any potential benefit and, therefore, feel that investigation of myocardial function, infarct size and oedema is justified. CMR is an accurate and reproducible technique for making these assessments. CMR can distinguish between scar tissue  and normal (viable) myocardium using the late gadolinium hyper-enhancement technique ,39. CMR 1) Localisers and breath-hold plan scans to define LV short axis. 2) Resting wall-motion sequence. Multiphase HLA, VLA and short axis cine stack (10–12 slices) using a fast gradient echo sequence to assess wall motion. Then in identical scan planes; Breath hold, black blood, T2-weighted triple inversion recovery sequence. 3) Late gadolinium hyper-enhancement CMR: T1-weighted, segmented inversion-recovery sequence 15 minutes after intravenous injection of Gadolinium (0.2 mmol/kg). This protocol will be piloted on 20 patients before the start of the main clinical trial.As soon as possible after recruitment, between days 2 and 4 of treatment, Sheffield patients will be identified as suitable for ST segment monitoring. Exclusion criteria will include bundle branch block, significant conduction abnormality, digoxin therapy or serious electrolyte disturbance. The presence of other potentially confounding features, such as mitral valve prolapse, posture change, changes due to body habitus and temperature change will be assumed to be similar in the two groups. A Holter monitor will be attached using the 3 standard leads for 24 h. If catheterisation be performed during the period of the recording, an appropriate entry will be made on the time log prior to the monitor being removed and replaced afterwards. If revascularisation be undertaken, the monitor will not be replaced. The monitor will be removed by one of the ECG technicians after 24 h (or as just stated) and analysed by Ms Gomersall (an experienced ECG technician who works in Sheffield), using the standard Reynolds protocol for ST segment analysis. She will be blinded to the identity of the treatment. The number of ischemic events, their frequency and total duration will be compared in both treatment groups.in vivo effects of cytokines in patients with IHD on endothelial vasomotor and fibrinolytic function are unknown. As acute coronary syndromes are associated with both impaired fibrinolytic function and transient elevation of acute phase proteins we wish to establish whether IL-1ra affects endothelial function.This study will examine the effect of Interleukin-1 receptor antagonist (IL-1ra) on vasomotor and fibrinolytic function in the forearm circulation of patients with acute coronary syndromes. The pro-inflammatory cytokines Interleukin-1 (IL-1) and tumour necrosis alpha-α (TNF-α) have been implicated in the initiation and amplification of local and systemic inflammatory responses leading to the development of, and progression of atherosclerosis. However the direct Patients will be recruited for the main study. Those recruited in Edinburgh (n = 62) will be asked if they are also prepared to participate in this sub-study. Recruited subjects will attend on two occasions. A comparison of IL-1ra versus placebo on endothelial function will be undertaken. The first study will be performed on days 2–4 following recruitment, at a standardised time following administration of either IL-1ra or placebo . The second study will be performed on day 14 at the same time of day as the first study. Volunteers will have fasted and avoided caffeine for at least four hours and avoided alcohol for 24 hours prior to each study. The subjects will undergo brachial artery cannulation with a 27 standard wire gauge under controlled conditions. Forearm blood flow will be measured in the infused and non-infused arms by venous occlusion plethysmography using mercury-in-silastic strain gauges. Blood samples will be obtained from 17-gauge venous cannulae inserted into the antecubital fossae of both forearms.Selective infusions into the brachial artery of the non-dominant arm will be undertaken. Substance P will be infused at 2, 4, 8 pmol/min, acetylcholine at 5, 10 and 20 μg/min (endothelium-dependent vasodilator that does not release t-PA), and sodium nitroprusside at 2, 4 and 8 μg/min (endothelium-independent vasodilator that does not release t-PA) will be infused for 5 min at each dose. Blood samples will be taken simultaneously from each forearm at baseline, immediately following, and at 4 further time points after the TNF-α infusion . These time points have been derived using the results of previous work to minimise non-contributory venous samples. Blood will be taken to assess stimulated coagulation factors and cytokines. Samples for plasma cotinine, haematocrit, white cell count will also be taken.


Knowledge of the HBV genotype with which a patient is infected is crucial information for a physician to have when planning clinical treatment for that patient. Previous studies have suggested that there are possible differences in the pathogenicity and therapeutic response of different HBV genotypes. However, the prevalence of the various HBV genotypes and Precore and Core mutations is unknown in the UAE. Therefore, we sought to determine the prevalence of the different HBV genotypes in the UAE population.A total of 88 HBsAg-positive patients were included in the study.A method for genotyping and subtyping HBV by partial HBsAg gene sequencing using primers that are complementary to all known genotypes was used. Precore and core region of these viruses were also sequenced in 88 patients.HBV genotype D was the most prevalent (79.5%) genotype identified in our study population, followed by genotypes A (18.2%) and C (2.3%). The following subtypes were isolated: ayw2 (80.7%), adw2 (14.8%), and adw (2.3%). The HBV-DNA viral load was higher in HBeAg-positive patients than it was in patients who were HBeAg-negative. Precore mutants were found in 51 (58.0%) of 88 patients. Mutations in the basal core promotor were found in 22 (25.3%) of 88 patients.HBV infection is a major health problem in the UAE, and while genotypes B and C are the most prevalent HBV genotypes in the Asian population, our study reveals that genotype D is the predominant genotype that is present in the UAE. More patients were HBeAg-negative than were HBeAg-positive in our study sample, which could be due to the duration of infection of the included patients. Additionally, the viral loads of the HBeAg-positive patients were higher those of the HBeAg-negative patients. Analysis of nucleotide 1858 showed presence of thymine in all patients with genotypes C, and D and in a few patients with genotypes A. This nucleotide was closely related to the presence of precore mutants. Mutations in the basal core promoter were found in 22 of 88 (25.3%) samples. These mutations were more frequent in patients infected with genotype A (37.5%) and not found in patients infected with genotype C. Hepatitis B Virus (HBV) is a well-known agent of acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Around 400 million people worldwide carry the virus of which more than 250 million reside in Asia.The course of the disease can vary from a subclinical self-limited illness to chronic active hepatitis, which can either lead to death after many years or to fulminant hepatitis. The chrHBV was formerly classified into four different subtypes that were afterward subdivided according to the antigenic determinants of HBsAg in adw (adw2 and adw4), ayw , adr (adrq adrq), and ayr. Subtype a is common to the majority of viruses and is related to a neutralizing epitope. Divergence of the complete genome in a same subtype is ca. 8%, similar to the one found between different subtypes.Genotypically, HBV is divided into eight groups, A-H. These groups were identified based on an intergroup divergence of 8% or 4% inIn the natural course of chronic HBV infection, the loss of HBeAg expression and the appearance of antibodies directed against it (Anti-HBe) are usually accompanied by cessation of viral replication. However such a serology profile may also be seen in individuals who harbor pre- core (PC) and basal core promoter (BCP) mutants where replicative infection continues. The frequent genomic mutation that leads to HBeAg negativity is the mutation of the nucleotide (nt) 1896 from G to A (G-A). This mutation converts codon 28 of the precore sequence to a termination codon (TGG→TAG) and thus prevents HBeAg from being expressed. PC variA second group of mutations affect the basal core promoter region and result in a transcriptional reduction of precore but not pregenomic and core mRNA. These HIn the present study, we sought to determine the prevalence of the various HBV genotypes in the UAE as this information was previously unknown. We determined the HBV genotype, subtype, viral load, and HBeAg antibody status and examined those and other clinical characteristics (including age and gender) of the patients included in our study to determine if there was a correlation between the molecular characteristics of the HBV virus with which a patient was infected and their clinical characteristics. We also verified the frequency of precore and BCP mutations in the UAE patients.A total of 88 consecutive serum samples from HBsAg-positive patients between the period of January 2008 till December 2009 were evaluated in this study. Most of samples collection was done with no relation of symptoms appearance, but as a routine check up or for follow up. These samples were derived from 74 males and 14 females with a mean age of 35.33 ± 11.5 years (range: 18 to 70 years). All of these patients were UAE citizens. Serum samples were stored at -20°C and thawed immediately before use. This work has been approved by the Zayed military hospital. No written consent was needed for this work since no additional sample was taken for the study. The samples were evaluated for the presence of several serological markers of HBV infection  using the bioMérieux ELISA kit according to the manufacturer's instructions.The extraction and amplification of HBV-DNA was carried out by nested PCR using the methods described by Kaneko et al..V00866) to which the primers corresponded were as follows: 1) HBS1F (positions 244 to 267), 2) HBS2F (positions 255 to 278), 3) HBS2R (positions 648 to 671), and 4) HBS1R (positions 668 to 691). Serum samples were treated as described above and subjected to two rounds of amplification sequentially with outer (FHBS1 and RHBS1) and inner (FHBS2 and RHBS2) primers. The amplification conditions for the two rounds of the nested PCR were as follows: initial denaturation at 94°C for 20 s, followed by 30 cycles of amplification at 94°C for 20 s, 56°C for 20 s, and 72°C for 30 s, followed by a final extension step at 72°C for 1 min in a PTC-200 Thermocycler .We used selected primers that have been described previously and thatFor the detection of BCP and precore mutants, HBV-DNA-positive samples were amplified by using the primers described by Takahashi et al..PCR products were subjected to cycle sequencing reactions as described previously using thAF046996) was used as an outgroup.Genotyping, BCP, and precore mutant analysis were carried out by sequence comparison with known sequences from different HBV genotypes that have been previously described and were aligned as described above. The Geneious program  was used for genotyping as well as for phylogenetic and molecular evolutionary analyses. The DNA sequences obtained from the PCR analyses were aligned using Geneious and then analyzed using the neighbor-joining method via a distance matrix that was calculated using the Kimura two-parameter model. Woolly All samples were submitted to HBV DNA quantification using the commercial TaqMan Amplicor HBV assay (Roche Diagnostics), which has a lower limit of detection of 12 IU/L.2 test with the Yates correction or Fischer's exact test was used to analyze quantitative data and to compare proportions. All calculated P-values were two-tailed and all P-values < 0.05 were considered to be statistically significant.For statistical analysis, we used the PASW Statistics software package, version 18.0. Either the χGU594063-GU594150.Sequences from the S gene that were acquired during this study were deposited in the GenBank under numbers ayw2 (80.7%), adw2 (14.8%), and adw (2.3%). Two patients infected with HBV genotype C could not be subtyped. Within genotype A, subtypes adw and adw2 were detected, while within genotype D, only subtype ayw2 was detected.The baseline characteristics of the study population as well as the frequencies with which the various HBV genotypes and subtypes were observed are shown in Table The prevalence of each of the HBV genotypes that were isolated from the included patients was also assessed with respect to patient age Table . There wWe compared the mean alanine aminotransferase (ALT) levels of patients infected with each of the three genotypes that were detected. The highest mean ALT level (51.30 ± 18.32 U/L) was found in the individuals infected with HBV genotype D  than it was in HBeAg-negative patients . Additionally, HBeAg-positive patients were more likely to have chronic hepatitis than HBeAg-negative patients (P = 0.048).In our study, 83 (94.3%) of patients were HBeAg-negative and 5 (5.7%) were HBeAg-positive .Mutations in the basal core promoter were found in 22 of 88 (25.3%) samples. These mutations were more frequent in patients infected with genotype A (37.5%), less frequent among genotype D-infected patients (23.2%) and not found in patients infected with genotype C, but no statistical significant difference was found.HBV infection is an important global health problem that places a continuously increasing burden on developing countries like the UAE. About 400 million people worldwide are chronic carriers of the virus. In addiDifferences in the distribution and clinical characteristics of the eight HBV genotypes have been studied extensively around the world. Better responses to treatment have been reported for genotypes A and B than genotypes D and C-30. On tThe 88 samples that were genotyped indicated that genotype D had the highest prevalence in our population, followed by genotype A. Genotypes E and F were not isolated from any of our patients, indicating that these genotypes are not present in this region. Interestingly, the initial studies on HBV genotype distribution in other parts of Asia found that genotypes B and C were the most prevalent genotypes in this region. However, almost all of these studies were performed in Japan and China, which are geographically distant from the UAE. Later studies revealed that seven of the eight HBV genotypes are present in Asia. For insGenotypes E, G, and H were not found in our study population. Genotype E has only been isolated in certain regions of Africa and in oIn this study, the most important predictor of an elevated ALT level and a high HBV-DNA level was HBeAg status, as HBeAg-positive patients were more likely to have higher ALT levels and HBV-DNA levels than HBeAg-negative patients.Among the HBeAg-positive patients, we found that 40% were inactive carriers, compared to 60% of patients with chronic hepatitis B Table . Among 8Precore mutants had an intermediate frequency in our population (58%). Such mutants were found in all patients infected with genotype C, with high frequency in patients infected with genotypes D, and at a very low frequency in genotype A-infected patients. The occurrence of this mutation is dependent upon the nucleotide (cytosine or thymine) at position 1858, which forms a base pair with nt 1896 in the pregenomic RNA loop at the ε encapsidation sign. A thymine at position 1858 is particularly common in genotype D viruses. The presence of a cytosine at position 1858 precludes the G-to-A mutation at nt 1896, since this would destabilize the stem-loop structure of the RNA encapsidation signal. InteracThe frequency of this mutation varies widely around the world. Castro et al. studied The BCP mutation was found in 25.3% of our patients and was present in A and D genotypes. BCP mutants frequency ranged from 23.2% in genotype D to 37.5% in genotype A. On the other hand, other authors have reported that the presence of these mutants is not related to the presence of precore mutants. This poHBV genotype influences the severity of liver disease that patients experience as well as their response to interferon and antiviral therapy. It is also thought to influence the emergence of resistant strains. Therefore, patients infected with certain genotypes of HBV that are known to be resistant to common treatment regimens can be counseled to seek alternative therapeutic options to spare them the cost and burden of treatment. The knowledge of the prevalence of HBV genotypes in a certain region is thus of immense importance to allow the proper and effective management of HBV patients in that region. As we have reported for the first time, HBV genotype D appears to be the predominant genotype in the UAE. In this study, the viral loads of HBeAg-positive patients were higher than those of HBeAg-negative individuals. Therefore, it should be considered worthwhile for clinicians to adopt better strategies to prevent and cure HBV infection. Precore mutants are more common among genotype C and D-infected patients, whereas BCP mutants were present in A and D genotypes. These types of studies are thus important both for epidemiological reasons and because they can help promote efforts to develop effective treatments for HBV.The authors declare that they have no competing interests.MS: conceived and designed the experiments, participated in experiments, analyzed data, wrote the paper, AE: participated in experiments, HA; participated in experiments, AA: participated in experiments, AI: contributed to data analysis.All authors have read and approved the final manuscript.


Bone metastases from prostate carcinoma are predominantly osteoblastic. Recently, urinary pyridinoline (Pyr) and deoxypyridinoline (Dpyr) have been employed as indicators of bone resorption. In this study, we evaluated urinary Pyr and Dpyr levels in 19 prostate carcinoma patients, of whom 12 had bone metastasis and seven had not, and 11 age-matched control subjects. There was a significant difference in Pyr levels between the control group and the patients with metastasis . The mean level of Dpyr in the patients with metastasis (10.8 +/- 8.0 nmol mmol-1 creatinine) was significantly higher than that in the control group , and also higher than that in the patients without metastasis . There was no significant difference in Pyr and Dpyr levels between the control group and the patients without metastasis. These results suggest that bone resorption is also accelerated in prostate carcinoma patients with bone metastasis.


In this study, using a ‘trapped’ non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease.Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist  Cells interacting with pathogens in the vicinity of an infection or injury produce small pro-inflammatory molecules called chemokines, which attract and coordinate the movement of specific leukocytes towards these sites Recruitment of circulating neutrophils to the site of infection is the first line in host defense. Such a response should be immediate and robust, yet controlled. Mechanisms that spatially and temporally regulate neutrophil levels must exist in order to minimize any collateral damage to healthy tissue. If this process is not properly regulated, the infiltrating neutrophils not only kill pathogens but also destroy host tissue, a hallmark of inflammatory diseases CXCL8 , one of the best-characterized members of the chemokine family, recruits neutrophils under such conditions as bacterial infection and tissue injury More than 40 chemokines have been identified in humans, and structure determination and solution characterization have shown that dimerization is a fundamental shared property by most, if not all, chemokines. Most interestingly, chemokines show novel and complex dimerization properties that set them apart from all known proteins of similar size (MW<10 kDa). However, investigating the activities of CXCL8 (or any chemokine) monomer and dimer is not trivial as the very phenomenon of monomer-dimer equilibrium prevents studying one species without interference from the other. We circumvent this bottleneck by designing a ‘trapped’ non-associating CXCL8 monomer and a trapped non-dissociating dimer, and have shown that the structures of the trapped CXCL8 monomer and dimer are indistinguishable from those of the native monomer and dimer; therefore their functions should reflect those of the native monomer and dimer in vivo data show that WT exists as both monomers and dimers, and that all three variants show distinctly different recruitment profiles. At the highest dose tested, the dimer shows the highest recruitment and was the most competent in quickly mobilizing large quantities of neutrophils. On the other hand, the monomer was less active and actually showed lower recruitment at higher doses, but was active over longer time periods. In contrast, recruitment by the WT was context dependent – it showed properties resembling monomer, dimer, or distinctly unique that could not be described as either solely due to either monomer or dimer. The latter case was particularly evident at later time points, where WT was most active, indicating synergy between monomers and dimers. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and to GAGs on endothelial cells. Our in vivo data reveal that differences in recruitment profiles cannot be explained on the basis of receptor affinities, suggesting that differences in GAG-binding and gradient formation, in the context of the in vivo milieu, dictate the overall recruitment. Our in vitro studies using a microfluidic device do show that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient. Our observation that both CXCL8 monomer and dimer play distinctly different in vivo roles is novel. We propose that CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease, and dysregulation in this process such as permanently tipping the balance towards the dimeric or monomeric form could trigger a ‘runaway’ inflammatory response resulting in severe tissue injury or failed resolution of bacterial infection leading to conditions such as sepsis.In this study, we have characterized neutrophil recruitment profiles of the monomer, dimer, and wild type (WT) CXCL8 in a mouse lung model. Our in vivo. The mouse lung is a well-studied model for inflammation and infection. In order to simulate the time course of an inflammatory response, we instilled increasing doses of CXCL8 WT, trapped monomer, or trapped dimer into the lung of BALB/c mice, and measured neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) at various times. We initially characterized recruitment at 6 h, as the lifetime of neutrophils is ∼6 h after which they start to undergo apoptosis and/or phagocytosed by macrophages. Our data showed that the monomer and dimer have distinct neutrophil recruitment profiles, and that their relative activities at various dosages can vary by many orders of magnitude . However, significant expression of KC was observed for the 10 µg dose at 2 and 6 h time point for the dimer, and unexpectedly, also for the R6K mutant . As the The observation that there is no correlation between endogenous mouse cytokine/chemokine levels and neutrophil recruitment suggest that CXCL8 continues to play a role in recruitment though the actual CXCL8 levels are barely detectable at 6 h. This could mean that CXCL8 is mostly immobilized and so could not be detected and/or that CXCL8 levels that were present at early time points has set the molecular machinery in place for subsequent overall recruitment. For instance, we observe significant levels of neutrophils in the lung tissue at 6 hrs that would eventually travel into the alveolar spaces and be detected in BALF at later time points.Unlike monocytes, neutrophils once in the tissue are not known to traffic back into circulation and are believed to undergo apoptosis and/or phagocytosed by monocytes. The relatively higher levels of neutrophils for the monomer and WT at 12 h indicate that the rate of neutrophil trafficking from vasculature to the tissue are different for the monomer and dimer. The dimer seems to mobilize neutrophils quickly and efficiently whereas mobilization by the monomer is more persistent though at lower levels over a longer period of time.To understand the consequence of increased neutrophil recruitment into the lung, we compared the histopathology of lung tissue of CXCL8 dimer-treated mice after 6 h and those infected with human metapneumovirus (hMPV), a respiratory virus that induces lung injury in infected mice after 24 h We also measured total protein levels in BAL, as acute lung injury and tissue damage causes influx of protein into the air spaces as a consequence of increased permeability of the alveolar–capillary barrier. CXCL8-treated mice showed ∼2-fold increase in total protein for all variants at 6 h which remained essentially the same over 18 h . These dTo understand how inflammation is resolved, we characterized neutrophil and monocyte levels over a period of 72 h in mice treated with 10 µg of CXCL8 dimer. We observed that neutrophil levels are maximal at 6 h and then drop dramatically. On the other hand, the alveolar monocyte numbers initially decreased up to 6 h and then continued to increase up to 72 h, and showed extensive phagocytosis of neutrophils at 24–48 h indicating resolution of inflammation . It is win vivo and in vitro activities of our variants. Nevertheless, we observe that our in vitro chemotaxis measurements of monomer and dimer correlate with some, but not all, of our in vivo observations. The most important observation from both studies is that higher chemokine concentrations do not translate to more robust chemotaxis. Low levels of chemotaxis and lack of recruitment for the dimer observed for shallow gradient and low concentration reflect the in vivo response to the 0.1 µg dose. Robust chemotaxis observed for the dimer at steep gradient and high concentration reflect recruitment observed for the 10 µg dimer at the 6 hr time point.To understand the mechanisms underlying how monomers and dimers mediate recruitment, we measured the chemotactic activity of our trapped monomer, dimer, and the WT for CXCR2 expressed in HL60 cells using a microfluidic chamber that allows control of both the steepness of the chemokine gradient and protein concentration in vivo is dictated by many factors, and most importantly, will be modulated by variations in its concentrations. Under conditions of active neutrophil recruitment, local in vivo CXCL8 concentrations will vary by many orders of magnitude, and so at any given time and place, it could exist as a monomer, dimer, or both. CXCL8 was the first chemokine to be discovered and characterized in vitro functional studies and dimeric at millimolar concentrations used in structural studies CXCL8 function To better understand the role of monomers and dimers in receptor activation, we recently characterized the functional responses of our trapped monomer, trapped dimer, and WT in mammalian cells individually expressing CXCR1 or CXCR2 in vitro activity to both human and mouse CXCR2 expressed in mammalian cell lines. Interestingly, a mouse equivalent of CXCL8 does not exist as none of the mouse ELR-chemokines show robust activity for either human or mouse CXCR1, and it has been proposed that GCP-2/CXCL6 fulfils this role in mice Sequence and functional analysis indicate that both human and mouse express a number of neutrophil-activating chemokines. All share considerable sequence homology, have the characteristic ELR motif in the N-terminus, and show robust in vitro and knockout animal model studies that GAG-binding promotes and facilitates formation of a concentration gradient, which is essential for directional neutrophil recruitment in vitro and ex vivo studies have shown that GAG binding and dimerization are coupled, and that the dimer binds GAGs with higher affinity In vitro functional measurements are carried out under controlled and steady state conditions using defined concentrations, whereas in vivo conditions are more complex with concentrations that could vary by many order of magnitude spatially and temporally, and so correlating in vitro observations to in vivo directional neutrophil recruitment is not trivial. Indeed, experiments using microfluidic device that allow varying CXCL8 gradients and concentrations show complex chemotaxis profiles  of UTMB, in accordance with the NIH and UTMB institutional guidelines for animal care. Cages, bedding, food and water are sterilized before use. All animal work was approved by Institutional Animal Care and Use Committee .The trapped human CXCL8 monomer (L25NMe) was designed by substituting the dimer interface residue Leu-25 with N-Methyl Leu, which disrupts H-bonding interactions and introduces steric bulk about the two-fold symmetry point Female, 8- to 10-week-old BALB/c mice purchased from Harlan  were housed under specific pathogen-free conditions in the animal research facility of University of Texas Medical Branch (UTMB), in accordance with the National Institutes of Health and UTMB guidelines for animal care. Under light anesthesia, mice were inoculated intranasally with 0.1, 1 or 10 µg of CXCL8 variants in Dulbecco's phosphate-buffered saline (D-PBS) in a 50 µl volume For estimating neutrophils by FACS, cells were stained with a neutrophil-specific fluorescently labeled antibody against the murine myeloid differentiation antigen Gr-1 , and analyzed with a FACScan flow cytometer equipped with CellQuest software (Becton Dickinson).2O2 as substrates. The change in absorbance at A460 was measured at 1 min intervals for 7–8 min. One unit of MPO activity is defined as the amount of enzyme which degrades 1 µmol of H2O2/min at 25°C.For estimating neutrophils by myeloperoxidase (MPO) assay, cells from BAL fluid were resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (HTAB), sonicated, centrifuged, and the supernatants were tested for MPO activity using o-dianisidine (Sigma) and HTissue trafficking of CXCL8 WT, trapped monomer, and trapped dimer were analyzed in the lung model. BAL, lung, and serum samples were analyzed using ELISA. CXCL8 levels in BAL, serum, and lung tissue samples were measured immediately after thawing and appropriate dilution in HBSS, using the IL-8/CXCL8 DuoSet ELISA Kit . CXCL8 levels were normalized to initial levels  for each CXCL8 variant, and plotted as a function of time.The cell-free supernatants, collected and stored at −70°C as described above, were thawed to room temperature and tested for various mouse cytokines and chemokines using the Bio-Plex Mouse Cytokine 23-Plex panel , according to the manufacturer's instructions. All of the cytokines/chemokines were assayed using BALF from at least two sets of mice each containing three to four different animals for any given dose and time point. The reported values are the average of all the values.Total protein in BAL supernatants was estimated by the BCA assay using the BCA protein assay kit  following the manufacturer's instructions.Human promyelocytic HL-60 cells stably transfected with the CXCR2 plasmid were used for the chemotaxis assays. The HL-60 cells were differentiated by adding 1.25% dimethylsulfoxide (DMSO) to the cell suspension, and differentiation was evaluated by observing morphological changes evident 6–7 days after DMSO addition. The microfluidic chemotaxis device used in this study was especially designed to establish a stable and uniform chemokine concentration gradient All results unless specified otherwise are shown as mean and standard deviation from the mean. Differences were considered significant at p<0.05. All experiments were repeated at least three times. Statistical analyses were carried out using ANOVA (Graph Pad prism 4).Figure S1Estimation of neutrophil levels in the lung Broncheo-alveolar lavage fluid (BALF). BAL neutrophils (N) and macrophages (M) as seen in cytospin slides stained with hematoxylin and eosin (H&E), obtained from mice treated with PBS (control), CXCL8 wild type (WT), trapped monomer and trapped dimer. Scale Bar, 20 µm.(1.85 MB DOC)Click here for additional data file.Figure S2Estimation of neutrophil levels in the BALF using MPO assay. MPO is a neutrophil granule enzyme and its activity has been shown to correlate with neutrophil levels. Measured neutrophil levels are similar to those measured from differential counting . P is at(0.18 MB DOC)Click here for additional data file.Video S1A video file showing neutrophil chemotaxis for the monomer.(5.81 MB MOV)Click here for additional data file.Video S2A video file showing neutrophil chemotaxis for the dimer.(2.57 MB MOV)Click here for additional data file.


Clustering of related haplotypes in haplotype-based association mapping has the potential to improve power by reducing the degrees of freedom without sacrificing important information about the underlying genetic structure. We have modified a generalized linear model approach for association analysis by incorporating a density-based clustering algorithm to reduce the number of coefficients in the model. Using the GAW 15 Problem 3 simulated data, we show that our novel method can substantially enhance power to detect association with the binary rheumatoid arthritis (RA) phenotype at the HLA-DRB1 locus on chromosome 6. In contrast, clustering did not appreciably improve performance at locus D, perhaps a consequence of a rare susceptibility allele and of the overwhelming effect of HLA-DRB1/locus C, 5 cM distal. Optimization of parameters governing the clustering algorithm identified a set of parameters that delivered nearly ideal performance in a variety of situations. The cluster-based score test was valid over a wide range of haplotype diversity, and was robust to severe departures from Hardy-Weinberg equilibrium encountered near HLA-DRB1 in RA case-control samples. Haplotypes generally contain more information than individual single-nucleotide polymorphisms (SNPs) about the underlying genetic architecture, and therefore offer greater power to detect association between markers and traits. However, the power of haplotype-based methods for association mapping, like that of other approaches, is diminished in studies of complex traits by the presence of both allelic heterogeneity  and locus heterogeneity. One approach to ameliorate the effect of allelic heterogeneity is to cluster similar haplotypes, under the assumption that these may have diverged more recently in a population's history than the occurrence of a disease-causing mutation.We combined the density-based clustering algorithm of Li and Jiang  with theIn this report, we compare the performance of the haplotype- and cluster-based methods in detecting association with RA, and assess the type I error of both methods in the presence and absence of dHWE.All analyses were carried out with knowledge of the true location of susceptibility loci.N" will be denoted as "SNP N". We tested the markers flanking the HLA-DRB1 locus  and locus D  for redundancy using BEST [Marker names from the chromosome 6 dense SNP scan are abbreviated here such that "denseSNP6_2 tests as implemented in the R genetics library package, version 1.2.0. Analyses were performed on sets of 1500 cases – one affected sib chosen at random from each affected-sib pair (ASP) family – and 2000 unrelated controls. All cases and controls in each set were from the same replicate.Testing for dHWE was carried out using the exact test in HaploView, and, for confirmation, the exact and χWe used the ASSOC program in S.A.G.E.  for caseMinPts, is located within a given distance ε from it. Haplotypes within this ε neighborhood are clustered together. We modified HapMiner such that very common haplotypes, defined by the parameter pmin, are never clustered together. This prevents improper grouping of ancient haplotypes. For the analyses presented here, we selected a value for pmin of 1/(2k), where k is the number of haplotypes present with a frequency large enough to include in the GLM (see below).We have extended the regression-based approach for association testing of Schaid et al.  to incor2 random variable with degrees of freedom equal to the rank of the variance matrix for the score statistic. In our cluster-based approach, parameter estimates are obtained for clusters, rather than for haplotypes. We calculated the variance of the score statistic as per the generalized score test of Boos [Cluster assignments for all possible haplotypes are imported into the haplo.score function in HaploStats ,3. This  of Boos  as imple of Boos  because  of Boos  inflatedWe carried out studies of power and type I error of association mapping methods on subsets of all 100 replicates. Cases were randomly selected from the offspring of ASP families, such that no sample contained both sibs from any family; controls were chosen at random from the unrelated controls. All individuals within a sample were taken from the same replicate. We adjusted the sample size for each analysis to yield moderate (40–60%) power from the haplotype-only test. Where necessary, we included sex and the number of DRB1*04 alleles in the model as covariates, to reduce the signal strength to a level useful for power comparisons; this adjustment was not part of the analysis. Type I error in association-positive regions was assessed by randomizing case/control status  relative to genotype data by permutation.A second null-model analysis was performed at locations far removed from the HLA region and locus D. The mean haplotype diversity, measured as the number of unique haplotypes present in the phased data, for all sets of six and eight consecutive markers was estimated over replicates 1–50 in two large regions on chromosome 6q comprising SNPs 11001–12000 and 15001–16000. Four levels of diversity were defined: low , medium (50th), high (90th) and very high (99th). Samples were extracted at selected locations at each diversity level, and score tests for association were carried out as above (without permutation).p < 0.001 in at least five of the 100 replicates. Six consecutive markers flanking DRB1 gave p-values below 0.001 in every replicate and median p-values of less than 10-6 from HaploView over all replicates. The three methods for testing HWE produced nearly identical results (data not shown). As expected, there was marked loss of heterozygosity in these markers. Because the score test for association assumes HWE, we took care to assess type I error for the two methods within this region (see below).We observed extensive dHWE in the neighborhood of HLA-DRB1  was low when the TDT had unusually low power. Specifically, of the eight markers with median p-values greater than 0.1, six were within the top three quintiles of the percentage of informative families . We expected a uniformly strong correlation between the available information on transmission and power of the TDT, and we were unable to explain this surprising result.As a preliminary step toward haplotype association analysis, we performed exploratory single-marker scans on case-control samples and trios. An initial scan with ASSOC on case-control samples gave evidence of an extremely strong genetic signal in the HLA region (data not shown). Evidence for association of most SNPs near DRB1 with RA was, overall, highly significant under both TDTEX and FBAT Fig. . Neverthp < 0.05 without clustering in samples of 150 each cases and controls  was observed between SNP 3437 at DRB1 and SNP 3917, 1.6 kb from locus D (data not shown). Given the overwhelming effect of DRB1 on RA, this small level of LD may explain the association between RA and haplotypes at locus D.Ability to detect association at locus D was markedly reduced with adjustment for sex and number of DRB1*04 alleles, with power at the 0.01 significance level falling almost to background Table . This obFalse-positive rates measured at HLA-DRB1 in the absence of covariates matched expectation when trait values were permuted relative to genotypes, both with and without haplotype clustering, as did those measured at locus D. With adjustment for sex and DRB1*04 alleles, cluster-based score tests again yielded proper type I error, whereas the test was somewhat conservative without clustering, returning a false-positive rate of about 0.025 at a significance level of 0.05. Results were similar for all six- and eight-marker haplotypes examined in power analyses (data not shown). Thus, our cluster-based score test appears to be robust to the severe dHWE encountered within the HLA region.To further assess the validity of our approach, we performed score tests of association at four levels of haplotype diversity in regions distant from the HLA region (see Methods). Both the haplotype- and cluster-based approaches were valid at all diversity levels for sets of six and eight adjacent SNPs Table . HoweverIn summary, incorporation of haplotype clustering by the procedure of Li and Jiang  noticeabMinPts affected performance very little at the relatively large range of ε displayed here. At small values of ε, however, MinPts may significantly affect the degree of clustering (data not shown). Limiting the extent of clustering by setting pmin relatively small prevents "overclustering," in which haplotypes not recently diverged are grouped together, but also reduces the potential advantage of clustering. In practice, a reduction in power on clustering haplotypes is indicative of overclustering . The Shannon information criterion employed by Tzeng et al. [Comparisons of the two approaches at these loci suggest guidelines for selecting operating parameters for HapMiner. Although performance was optimized at several values of ε, setting ε to 0.5 provided near-optimal results in all situations. The choice of g et al. ,17 for dg et al.  in that Our work provides evidence that the GLM framework for association mapping is robust to severe departures from HWE under the null model. However, the GLM approach appears to be sensitive to adjustment with a covariate that is very tightly correlated with the trait, in that it may lose power in the presence of more extensive haplotype diversity, although clustering decreased this sensitivity, most likely by reducing the number of coefficients. It is possible that removing the multiplicative effect of HLA-DRB1*04 alleles on the odds of RA also extracted most of the trait information, perhaps changing the null distribution of the score statistic.The apparent association at locus D appears to be largely due to HLA-DRB1 and locus C. The Problem 3 data appear to be unusual in that one locus exerts such a strong effect on the disease of interest that association is clearly discernible from a distance of over 5 cM. It is not surprising that our ability to detect locus D was marginal. The association study design is predicated on the "common disease-common variant" hypothesis , which pThe author(s) declare that they have no competing interests.


Staphylococcus aureus (MRSA) with intermediate susceptibility to vancomycin and reduced susceptibility to daptomycin. The strain was isolated from the bone of a 56-year-old man with lumbar osteomyelitis after a 6-week treatment course of vancomycin for catheter-associated septic thrombophlebitis.We describe a case of treatment failure caused by a strain of USA300 community-associated methicillin-resistant  Staphylococcus aureus (MRSA) that was susceptible to tetracycline and trimethoprim-sulfamethoxazole. The patient was treated with oral trimethoprim-sulfamethoxazole and discharged to home to complete a 10-day course. His right upper extremity cellulitis subsequently resolved, but he returned to the hospital in January 2006 with volume overload and symptoms consistent with uremia. A tunneled right internal jugular hemodialysis catheter was placed on January 11, hemodialysis was initiated, and he was discharged to home.A 56-year-old man with a history of type 2 diabetes and chronic kidney disease was seen at San Francisco General Hospital in November 2005 because of hyperkalemia and volume overload. On day 4 of hospitalization, a fever of 39°C and cellulitis in the right arm associated with a peripheral intravenous line developed. Two blood cultures were drawn, the line was removed, and therapy was initiated with oral cephalexin. One of the 2 blood cultures subsequently grew methicillin-resistant On February 20, he was seen in the emergency department of another facility with nausea and altered mental status. He was afebrile but hypotensive, and tenderness at the entrance site of the hemodialysis catheter was noted. Two blood cultures were positive for MRSA. The patient’s catheter was removed, and intravenous vancomycin was started. Transthoracic and subsequent transesophageal echocardiograms were negative for endocarditis, but evidence of thrombosis in the superior vena cava was seen. Multiple blood cultures were positive for MRSA through March 1, after which they became negative. The vancomycin MIC for serial isolates remained unchanged. The patient was treated with vancomycin for a 6-week course, beginning March 1. Anticoagulation with coumadin was also initiated. After clearance of his blood cultures, a right subclavian tunneled hemodialysis catheter and a left upper extremity arteriovenous fistula were placed. Vancomycin trough levels were assayed on multiple occasions during the 6-week course; all were ≥20 μg/mL.S. aureus (VISA) isolate, with a vancomycin MIC of 8 µg/mL.On April 24, the patient was seen by his primary care provider for worsening bilateral knee and back pain and difficulty walking. A lumbar spine radiograph demonstrated cortical irregularity at L4-L5, indicative of discitis. He was admitted to the hospital, and vancomycin was initiated. Magnetic resonance imaging of the lumbar spine on April 25 demonstrated findings consistent with osteomyelitis and discitis at L4-L5. A needle biopsy of the L4-L5 lesion was performed on April 27. That evening, the patient was noted to be febrile and had an episode of emesis. Later in the evening, he was apneic and without a pulse, with fixed and dilated pupils. Cardiopulmonary resuscitation was performed. When neurologic function did not return during the next 4 days, supportive care was withdrawn, and the patient died May 1. Multiple blood cultures from this hospitalization remained negative, but results of a culture of lumbar fluid from a biopsy specimen on April 27 were positive for a vancomycin-intermediate S. aureus, intermediate susceptibility to vancomycin was lost, and daptomycin susceptibility was regained.The antimicrobial susceptibility profiles of the blood isolates from November and February and the lumbar isolate from April are shown in the SmaI digestion was performed on the 2 blood isolates from November and February and the lumbar isolate from April. All 3 isolates shared an identical pattern with the USA300-0114 control strain (spa typing) , and Panton-Valentine leukocidin genes and negative for the presence of the vanA gene, when primers and sequences previously described were used  group II genotype present in clonal cluster 5 strains has been speculated to predispose to emergence of the VISA phenotype. USA300 is an agr group III clone (agr.The first report of clinical VISA infection was from Japan in 1997 (S. aureus that contain subpopulations of daughter cells displaying intermediate sensitivity to vancomycin but for which the MICs for vancomycin fall within the susceptible range, can be difficult to detect in the microbiology laboratory because the phenotypes are unstable and can be lost on subsequent passages (The mechanism of reduced vancomycin susceptibility in VISA is thought to be mediated by an increase in the number of false targets because of a thickened cell wall (S. aureus infections are successfully treated with vancomycin, particularly in patients receiving hemodialysis. While most VISA isolates reported in the United States have been isolated from patients receiving hemodialysis, chronic renal disease and hemodialysis have not been definitively identified as risk factors for infections caused by VISA or hVISA (Our patient experienced clinical failure of vancomycin therapy despite high serum drug levels, which speaks to the difficulty with which highly invasive


Analysis of within-patient HIV evolution under anti-HIV therapy is crucial to a better understanding the possible mechanisms of HIV drug-resistance acquisition. The high evolutionary rate of HIV allows us to trace its evolutionary process in real time by analyzing virus samples serially collected from the same patient. However, such studies are still uncommon due to the lack of powerful computational methods designed for serial virus samples. In this study, we develop a computational method, vSPA , which groups viral sequences from the same sampling time into clusters and traces the evolution between clusters over sampling times. The method makes use of information of different sampling times and traces the evolution of important amino acid mutations. Second, a permutation test at the codon level is conducted to determine the threshold of the correlation coefficient for clustering viral quasispecies. We applied vSPA to four large data sets of HIV-1 protease and reverse transcriptase genes serially collected from two AIDS patients undergoing anti-HIV therapy over several years.The results show that vSPA can trace within-patient HIV evolution by detecting many amino acid changes, including important drug-resistant mutations, and by classifying different viral quasispecies coexisting during different periods of the therapy.Given that many new anti-HIV drugs will be available in the near future, vSPA may be useful for quickly providing information on the acquisition of HIV drug-resistant mutations by monitoring the within-patient HIV evolution under anti-HIV therapy as a computational approach. HIV is one of the fastest evolving viruses ever known. Its high evolutionary rate is the main reason for rapid acquisition of drug resistance and the difficulty of vaccine development. However, the fast rate can also be utilized in evolutionary analysis, enabling us to trace the evolutionary process of HIV in real time by analyzing virus samples serially collected from infected individuals.Many previous studies have examined HIV population dynamics, and revealed many unique and important features of viral evolution within an infected individual -6. NumerIn contrast to the rapid accumulation of viral data, few powerful methods for inferring the within-patient viral evolution are available. One reason appears to be the difficulty to deal with serial virus samples ,14. TradWe propose a new method, vSPA , for analyzing within-patient HIV evolution. We calculate the genetic distances between virus samples, and use Pearson correlation coefficient to classify viral variants at the same sampling time into different clusters. We further identify the evolutionary pathways between viral clusters across the sampling times. A permutation test at the codon level is used to determine the threshold correlation coefficient for the clustering. We applied the vSPA method to four large data sets of HIV-1 protease (PR) and reverse transcriptase (RT) genes serially collected from two Japanese AIDS patients, and compared the results with those from traditional tree-building methods and Sliding MinPD.The vSPA method was applied to four large data sets of PR and RT genes serially collected from Patients 1 and 2. Below, we refer to these data sets as P1PR, P1RT, P2PR and P2RT, respectively.The results of the permutation test for P1PR are shown in Additional file The clusters and their evolutionary dynamics inference for the 273 P1PR sequences are shown in Figure From the amino acid mutations that each cluster possessed  that had been administered to Patient 2 before the sampling started. The M184VI mutation occurred between time points B and E. The five subpopulations were mainly observed from time point F, before which no sample was taken for more than three years. Briefly, the two subpopulations with T69 insertion and back mutation at site 184 (SP69+184 M1 and SP69+184 M2) were observed, but they were not dominant except at time point E. The SP69ins-184I had been dominant for only a short period as well. In contrast, the SP69ins-184 M1, which lost T69 insertion and acquired back mutation at site 184 in the same period, diversified and had been a major subpopulation for a long period. This observation raises a possibility that there might be interaction between the lost T69 insertion and the back mutation at site 184.The SP69ins-184 M2 subpopulation included only one sequence k5 and had neither T69 insertion nor M184V mutation, but it was linked with a very early cluster B1. We investigated the difference between the clusters of time point K and found that only sequence k5 possessed the M230L mutation, a drug-resistant mutation to Enfuvirtide (EFV). Also, another mutation, K103R, was observed in the k5 alone. It is likely that sequence k5 was a memory variant having resistant mutations to EFV, given that this drug had not been administered for more than one year.All mutations existing at the first time point and newly acquired on the pathways can be found in Additional file We applied two representative methods, Neighbor-joining (NJ)  and maxiThe NJ and ML methods produced very similar trees  were selected for this study. Patient 1 is a 46-year-old male with hemophilia. His treatment history and clinical course were monitored by National Institute of Infectious Diseases (NIID) in Tokyo from 1996 to 2001  are grouped into the same cluster. Some variants could be grouped into more than one cluster because of the high similarity of the viral sequences; thus, clusters sharing more than half of their variants were merged to form a larger cluster Figure . The merAs the diversity of viral variants depends on the sampling time point, it may not be appropriate to use the same correlation coefficient threshold for all time points to cluster viral variants. We implemented a permutation test to determine the correlation coefficient to be used for each time point. Sequences in a viral cluster not only are highly similar but also possess characteristic drug-resistant mutations. To present the coding structure and to accommodate the co-occurring and interesting amino acid mutations, we conduct the permutation test at the codon level.Tn and Tn-1, were used in the permutation, with each codon rearranged randomly among the viral sequences to generate the data at the codon in the permutation data set  cluster over all the viral variants, and then chose the Tn-m cluster with the minimum average distance as the ancestor of the Tn cluster. When only a few sequences are included in a cluster, the calculated average distance may involve large sampling errors. However, this procedure of linking clusters of sequences appears to be more adequate for clustering highly similar sequences than linking sequences of shortest distances between clusters, as we do not know which single viral clone can be used as the representative of a cluster. By repeating this procedure, the ancestral cluster of each Tn cluster was identified. Then, we connected the ancestral clusters over the entire observation period to construct the evolutionary pathway. The graph-drawing tool Graphviz (version 10) was used to visualize the pathways and to output the results of vSPA automatically.In vSPA, the ancestor-descendant relationships were inferred at the cluster level and all variants grouped into the same cluster were assumed to have a common ancestor (cluster) in one of the previous time points. Since the ancestor of viral variants at time point .Four datasets analyzed in this study and the programs developed to carry out the vSPA algorithm are included in Additional file NH, HT and FR conceived and designed the method. NH wrote the program and analyzed the data. FR performed the MinPD. WS, JS and MM carried out the viral experiments. FR and NH drafted the manuscript. All authors read and approved the final manuscript.The results of permutation test of P1PR. This file includes 15 figures which show the results of permutation test for each sampling time point of P1PR.Click here for fileAcquired mutations inferred from PR and RT genes of Patients 1 and 2. This file includes four tables in which all mutations inferred by vSPA from P1PR, P1RT, P2PR and P2RT are listed.Click here for fileReconstructed phylogenetic trees by using the NJ and ML methods. The eight phylogenetic trees reconstructed from P1PR, P1RT, P2PR and P2RT are shown in this file.Click here for fileThe evolutionary network constructed using Sliding MinPD method. This file shows the evolutionary network constructed from the 273 PR genes of Patient 1 by Sliding MinPD.Click here for fileThe vSPA programs and four datasets analyzed in this study. The programs developed to carry out the vSPA algorithm and four datasets analyzed in this study and are included in this ZIP file.Click here for file