Subacromial impingement syndrome is the most frequent cause of shoulder problems which themselves affect 1 in 3 adults. Management commonly includes exercise and corticosteroid injection. However, the few existing trials of exercise or corticosteroid injection for subacromial impingement syndrome are mostly small, of poor quality, and focus only on short-term results. Exercise packages tend to be standardised rather than individualised and progressed. There has been much recent interest in improving outcome from corticosteroid injections by using musculoskeletal ultrasound to guide injections. However, there are no high-quality trials comparing ultrasound-guided and blind corticosteroid injection in subacromial impingement syndrome. This trial will investigate how to optimise the outcome of subacromial impingement syndrome from exercise (standardised advice and information leaflet versus physiotherapist-led exercise) and from subacromial corticosteroid injection (blind versus ultrasound-guided), and provide long-term follow-up data on clinical and cost-effectiveness.The study design is a 2x2 factorial randomised controlled trial. 252 adults with subacromial impingement syndrome will be recruited from two musculoskeletal Clinical Assessment and Treatment Services at the primary-secondary care interface in Staffordshire, UK. Participants will be randomised on a 1:1:1:1 basis to one of four treatment groups: (1) ultrasound-guided subacromial corticosteroid injection and a physiotherapist-led exercise programme, (2) ultrasound-guided subacromial corticosteroid injection and an advice and exercise leaflet, (3) blind subacromial corticosteroid injection and a physiotherapist-led exercise programme, or (4) blind subacromial corticosteroid injection and an advice and exercise leaflet. The primary intention-to-treat analysis will be the mean differences in Shoulder Pain and Disability Index (SPADI) scores at 6 weeks for the comparison between injection interventions and at 6 months for the comparison between exercise interventions. Although independence of treatment effects is assumed, the magnitude of any interaction effect will be examined . Secondary outcomes will include comparison of long-term outcomes (12 months) and cost-effectiveness. A secondary per protocol analysis will also be performed.This protocol paper presents detail of the rationale, design, methods and operational aspects of the SUPPORT trial.ISRCTN42399123.Current controlled trials  Painful shoulder problems are a common cause of impaired function and affect 1 in 3 adults , accountExercise aims to reduce pain and functional impairment by improving posture, muscle strength, scapular stability and scapulohumeral rhythm . It can Corticosteroid injections are commonly used to reduce pain and inflammation associated with SIS  althoughMusculoskeletal ultrasound (MSUS) has become an increasingly accepted tool for guidance of therapeutic interventions ,26. HoweThe evidence supports the need for a high quality trial to determine how to optimise treatments for SIS patients and to reflect the complementary ways in which these interventions are used clinically i.e. that exercise and injection are often combined within the same package of care. The primary objectives of the SUPPORT trial  are to assess whether (1) a physiotherapist-led intervention consisting of individualised, supervised and progressed exercise provides better improvements in pain and function than a standardised advice and exercise leaflet, and (2) US-guided subacromial corticosteroid injection provides better improvements in pain and function than unguided (blind) injection. Secondary objectives are to determine whether there is an interaction effect of combining US-guided injection and an individualised, supervised and progressed exercise programme and to conduct two separate cost-effectiveness analyses for the comparisons outlined in (1) and (2).The study will be a 2×2 factorial randomised controlled trial Figure . EthicalPatients will be recruited through musculoskeletal interface clinics in Staffordshire and Stoke-on-Trent Partnership Trust. These services incorporate early referral to, and rapid assessment by, specialist clinicians ) within a multidisciplinary team-working environment. Local family practices will be informed about the trial.Consecutive patients referred from primary care to interface shoulder clinics will be assessed for eligibility by interface clinicians.•18 years and over•No history of significant shoulder trauma, for example, fracture, clinically-suspected full thickness cuff tear•A clinical diagnosis of SIS .Accurate diagnosis of SIS is challenging but combination of the patients’ history and response to Neer and Hawkins-Kennedy tests (to rule SIS out with a negative test) and pain on shoulder abduction (to rule SIS in with a positive test) provides optimal sensitivity and specificity . Patient•Below 18 years old•Those whose main complaint is due to neck problems, acromioclavicular pathology, or other primary shoulder disorders including adhesive capsulitis or clinically-suspected full thickness cuff tear•Potentially serious pathology  or ipsilateral shoulder surgery/replacement•Those already on a surgical waiting list for shoulder surgery•Contra-indications to local corticosteroid injection •Participation in a shoulder-focused exercise programme or shoulder injection in the last month•Inability to provide informed consent, complete written questionnaires, or read instruction leaflets written in EnglishAll patients referred to musculoskeletal interface services at each site with shoulder pain, identified from the referral letter from primary care, will be triaged to an interface service shoulder clinic. One to two weeks prior to their appointment in the interface service shoulder clinic all patients will be sent a letter of invitation and a Participant Information Sheet (PIS).Patients will attend for their routine clinic appointment, according to normal NHS clinical attendance procedures. The interface clinician will undertake the clinical consultation according to usual practice. Patients who fulfil the criteria for a diagnosis of SIS will be asked by the interface clinician if they wish to be considered for trial participation. Patients who are interested in trial participation will be invited by the interface clinician to see a Research Nurse, who will further confirm eligibility and explain the trial in full. Ineligible patients and those who do not wish to participate in the trial will receive normal clinical care delivered by the interface clinician according to usual practice. Anonymised age and gender will be collected for those patients who decline to take part, in order to assess the external validity of the recruited sample of patients.Eligible patients who are interested in the trial will be asked by the Research Nurse to provide written informed consent to participate. The Research Nurse will allocate the participant a unique study number. The participant will then complete a baseline questionnaire Table . The ResAs the Research Nurse will remain blinded to treatment allocation, a trial administrator in the interface service shoulder clinic will organise the randomisation, using Keele University’s Clinical Trial Unit’s telephone randomisation service, and then inform participants of their treatment allocation. Participants will be randomly allocated in a 1:1:1:1 ratio to 1 of 4 treatment groups using block randomisation to ensure that participants from each interface clinic have an equal chance of receiving any of the interventions:•US-guided subacromial corticosteroid injection and physiotherapist-led individualised, supervised and progressed exercise•US-guided subacromial corticosteroid injection and advice and exercise leaflet•Unguided (blind) subacromial corticosteroid injection and physiotherapist-led individualised, supervised and progressed exercise•Unguided (blind) subacromial corticosteroid injection and advice and exercise leafletThe injection will be delivered in the same interface service shoulder clinic. Those randomised to receive the advice and exercise leaflet will receive this immediately following their injection. For participants randomised to receive the physiotherapist-led individualised, supervised and progressed exercise programme, the trial administrator will arrange their first physiotherapy appointment within 3 weeks of the shoulder injection, thereby meeting recommendations that recommendations that resistance exercise should be avoided for 2 weeks following steroid injection .An evaluation of the success of Research Nurse blinding procedures will be completed and a procedure for reporting incidents where blinding has been compromised will be in place, through case report forms.Clinicians within the interface service shoulder clinics will deliver US-guided subacromial injection using a standard technique. Interface clinicians performing US-guided injections will comprise three rheumatologists, three extended scope physiotherapists, one musculoskeletal sonographer, one GPwSI, and one senior rheumatology nurse. Ultrasound examinations will be performed using the LOGIQe system with a 12 MHz transducer. The skin and transducer will be cleaned with chlorhexidine 0.5% solution and sterile gel will be applied to the transducer. The participant will be seated with the shoulder internally rotated and the ipsilateral hand placed on the buttock to allow greatest access to the subacromial bursa. The transducer will be placed anterolaterally and the hypoechoic subacromial bursa visualised. A 21G needle will be inserted under real-time US control until the needle-tip is seen to enter the bursa. A commercially available pre-mixed solution of methylprednisolone 40 mg and 1% lidocaine 1 ml will be injected in accordance with recent best-practice recommendations [Different interface clinicians to those providing the US-guided subacromial injections will deliver unguided (blind) subacromial injection using a standard technique. Unguided (blind) injectors will comprise two rheumatologists, one rehabilitation medicine specialist, four extended scope physiotherapists and a GPwSI. The participant will be seated with their arms hanging their by sides with elbow bent and forearm resting on lap. The skin will be cleaned with chlorhexidine solution 0.5%. A 21G needle will be inserted through the deltoid under the acromium process laterally and the same pre-mixed solution of methylprednisolone 40 mg and 1% lidocaine 1 ml will be injected.Participants receiving either US-guided or unguided (blind) subacromial injection will be advised not to drive immediately after receiving the injection and to observe a period of relative rest for the following two weeks, avoiding pushing or pulling movements with the affected arm and heavy or repetitive tasks. Protocols for both US-guided and unguided injection will allow the injection to be repeated at the discretion of the treating interface clinician in event of partial response. In line with recommendations, a maximum of 2 injections will be permitted during follow-up per participant , and theThe individualised, supervised and progressed exercise programme will be delivered by community-based, musculoskeletal physiotherapists and commenced within 3 weeks of injection. Exercise type, dose and progression will be individualised, supervised and progressed in 6 to 8 sessions over a 12 to 16 weeks. Based on available guidelines , a surveStage one will include postural correction, scapular stability control and proprioceptive exercises. Stage two will progress to range of movement exercises with scapular control in pain-free range, including for example shoulder joint forward flexion, abduction and rotation with progression from assisted to full active exercise. In stage two, stretches will be added if required and resistance muscle strengthening commenced. Stage three will progress the resistance through range to encourage rotator cuff muscle strengthening through all ranges of movement including overhead activity, adding resistance through the use of Theraband (elastic exercise bands) and simple weights. Therapists will encourage exercise behaviour using approaches such as self-monitoring and goal setting. These plans meet recommendations  that resParticipants who fail to attend for any physiotherapy appointment will be offered up to two further appointments. After failing to attend the third appointment, no further appointments will be offered.Attendance at the physiotherapy-led individualised, supervised and progressed exercise sessions will be monitored, and all non-attended sessions will be recorded on the appropriate case report form. The frequency and duration of home exercise will be assessed in self-complete follow-up questionnaires.Clinicians in the interface service shoulder clinics will provide the advice and exercise leaflet following the injection. The leaflet includes information about shoulder anatomy and SIS, plus simple self-help messages about pain relief (including the application of cold packs) and activities. It includes a small number of standardised exercises, including specific muscle strengthening and range of motion exercises . ExercisPrior to recruitment of participants into the trial, all interface clinicians and physiotherapists delivering treatment will be trained to deliver the trial interventions according to the trial intervention protocols and to record the interventions on standardised case report forms.Interface clinicians will attend a half-day group training session prior to the study commencing which will include training regarding study procedures, eligibility assessment, protocols for US-guided or blind injections, and the delivery of the advice and exercise leaflet. Further refresher sessions will be provided during the course of the trial.Interface clinicians performing blind (unguided) injections will have extensive clinical experience performing subacromial injections. Since musculoskeletal ultrasound is highly operator-dependent and relies on robust training with direct supervision to gain clinical competency, interface clinicians performing US-guided injections will either have extensive clinical experience performing US-guided injections or be required to complete a Consortium for the Accreditation of Sonographic Education (CASE)  accreditPhysiotherapists will attend a two-day group training session prior to the trial commencing which will cover information regarding trial procedures, anatomy and physiology relevant to SIS, current best practice guidelines , treatmeTreatment sessions will be audited at regular intervals throughout the trial through case report forms completed by both interface clinicians and physiotherapists at clinic, case-note reviews and observation of physiotherapy sessions by a member of the research team to ensure the interventions are delivered in accordance with the intervention protocols. Video recordings of US-guided injections will be taken throughout the study for audit and training purposes.The occurrence of all potential adverse events from all interventions will be monitored and assessed, using case report forms, contact with the trial co-ordinator, family practitioner report, and follow-up questionnaires. If patients suspect that they have suffered an adverse event as a result of any of the trial interventions, they will be asked to notify the trial co-ordinator by telephone, and to seek medical advice from their family practitioner. In our letter informing participants’ family practitioners of trial participation, we will ask the family practitioner to notify the trial co-ordinator if their patient sustains either an adverse or serious adverse event as a result of this trial.There are usually very few side-effects from corticosteroid injections, delivered either unguided (blind) or with the use of US-guidance. ,20,27-29A common adverse event from the exercise regime is expected to be symptoms of temporary, mild soreness of the shoulder joint or upper limb. Participants randomised to the physiotherapist-delivered exercise programme will be advised how to manage such symptoms by the treating physiotherapist. Participants randomised to receive the Advice and Exercise Leaflet will be able to find similar information in the leaflet.Outcome measures will be collected before randomisation and at 1 week, 6 weeks, 6 months and 12 months  and to rate their current shoulder pain intensity ). Non-responders to the initial text-message/telephone call will be contacted again 24 hours later. Those who do not respond to this reminder will be contacted by telephone a further 24 hours later. Data will be transposed by the research nurse onto a standard case report form.Outcome at 6 weeks, 6 months and 12 months will be collected by postal questionnaire  in shoulder pain and disability (SPADI) for the two main effects . To address whether the physiotherapy-led intervention consisting of individualised, supervised and progressed exercise provides better improvements in pain and function than a standardised advice and exercise leaflet, we will use the total SPADI score at the 6 month time-point, as the primary outcome measure. Since previous trials have shown that corticosteroid injections improve pain and function more rapidly than exercise ,20,21, wA total sample size of 250 participants is needed given 80% power; 5% two-tailed significance; and assuming 20% loss to follow up.The primary intention-to-treat (ITT) analysis will be double-analysed independently by two blinded statisticians. Between-group analysis will be undertaken according to randomised allocation with retention of all participants throughout follow up for the purposes of full evaluation. The primary evaluations will be mean differences in total SPADI scores at 6 weeks between those randomised to receive US-guided injection versus unguided injection, and mean differences in total SPADI scores at 6 months between those randomised to physiotherapist-led individualised, supervised and progressed exercise versus advice and exercise leaflet. Secondary analyses will include comparison of pain and disability subscales of the SPADI; long-term SPADI outcomes (i.e. at 12 months); and analysis of other outcome measures. Estimates of clinical effect of the two main comparisons will be shown as percentage differences (for dichotomous outcomes) and mean differences , with 95% confidence intervals. P-values will be given alongside statistical testing of the data. Multilevel longitudinal (mixed-) model regression methods  will be used to generate estimates of effects. By including the baseline values as outcome the model retains all available data (as opposed to complete-case data only) and upholds the full intention-to-treat analytic criterion. An assumption of this approach is that the missing data is missing at random (MAR) – being dependent on baseline factors utilised in the model. Covariates included in the regression models are as follows: age, gender, baseline SPADI scores, duration of pain and location of clinic. Sensitivity analyses will include any other variables which are unbalanced between the groups at baseline.Primary interest focuses on main effects (‘at the margins’) evaluation as our assumption is that the two interventions will act largely independently of each other. As a secondary analysis, we will estimate the interaction effect from a separate regression model (inclusive of interaction term) since any potential synergistic/antagonistic effect of the combination of treatments should be reported . DescripA per protocol analysis  will be carried out as a sensitivity analysis.Economic evaluation will estimate the cost-effectiveness of the trial interventions. A cost-consequence analysis will initially be reported, describing all the important results relating to costs and consequences in the intervention arms over the 12-month trial period. Subsequently, an incremental cost-utility analysis will be undertaken using patient responses to the EQ-5D-5L, to calculate the cost per additional Quality-Adjusted Life Year (QALY) gained over the same period. The analysis will adopt a broad perspective, considering NHS, patient and productivity costs. Data on health care resource use and time off work over 12 months follow-up will be obtained from the patient questionnaires. The resource use questions will capture details covering a broad range of health care resources, including prescribed and over-the-counter purchased medications, primary care and secondary care (inpatient and outpatient) attendances, treatments and investigations. Unit costs will be obtained from standard sources and health care providers, including NHS reference costs and the British National Formulary. Owing to the paucity of high-quality unit cost data for private health care consultations, these data will be costed as the NHS equivalent. Indirect costs, over 12 months follow-up, will be based on productivity losses due to work incapacity linked to presenteeism, and also work absenteeism. Costs for productivity loss will be computed through the product of lost productivity time (whether presenteeism and/or absenteeism) and the mean hourly wage of patients classified from data on wages corresponding to self-reported baseline occupation  QALYs will be calculated using the EQ-5D 5L index scores at baseline, 6 weeks, 6 months and 12 months, using the area-under-the-curve method.There is no consensus regarding how economic evaluations should be carried out alongside factorial trials and previously published economic evaluations alongside factorial trials have used two main methods: the ‘within the table’ approach  and the Missing cost and utility data will be imputed using multiple imputation through chained equations, with the number of imputation datasets set to reflect the proportion of missingness in the data as recommended by previous guidelines into the use of MI approaches . All anaThe SUPPORT trial will compare the clinical and cost-effectiveness of (1) US-guided corticosteroid injection to unguided (blind) corticosteroid injection and (2) a physiotherapist-delivered individualised, supervised and progressed exercise programme to provision of a standardised advice and exercise leaflet, for treatment of patients with SIS. In comparison to existing trials of injection and exercise interventions for SIS, the strengths of the SUPPORT trial are its size, long-term follow-up and inclusion of an individualised, supervised and progressed exercise programme which builds upon existing guidelines  to optimGP: General practitioner; GPwSI: General practitioner with a special interest; MSUS: Musculoskeletal ultrasound; NHS: National Health Service; NRS: Numeric rating scale; SIS: Subacromial impingement syndrome; US-guided: Ultrasound-guided.The authors declare that they have no competing interests.All authors participated in the design of the trial and the drafting of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/81/prepub


Superparamagnetic iron oxide nanoparticles (SPION) have emerged as an MRI contrast agent for tumor imaging due to their efficacy and safety. Their utility has been proven in clinical applications with a series of marketed SPION-based contrast agents. Extensive research has been performed to study various strategies that could improve SPION by tailoring the surface chemistry and by applying additional therapeutic functionality. Research into the dual-modal contrast uses of SPION has developed because these applications can save time and effort by reducing the number of imaging sessions. In addition to multimodal strategies, efforts have been made to develop multifunctional nanoparticles that carry both diagnostic and therapeutic cargos specifically for cancer. This review provides an overview of recent advances in multimodality imaging agents and focuses on iron oxide based nanoparticles and their theranostic applications for cancer. Furthermore, we discuss the physiochemical properties and compare different synthesis methods of SPION for the development of multimodal contrast agents. These techniques are powerful tools for visualizing the abnormal state of the body and for monitoring biological situations at the target site. Examples of non-invasive imaging techniques include the following: computed tomography (CT), magnetic resonance imaging (MRI), nuclear imaging of positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical (or fluorescence) imaging. These imaging techniques have provided scientists and clinicians with the ability to acquire in vivo images of anatomy and physiology in both animals and humans  (dithiocarbamate bisphosphonates) was stable in PBS and human serum for 48 h. Then, the [64Cu(dtcbp)2] complex was labeled with clinically available dextran-coated SPION by virtue of BP’s high affinity to iron oxide. In vivo studies were conducted in a 9.4 T NMR magnet and a NanoPET–CT scanner. The in vivo model for the study was the lymphatic system, which provided information regarding the early spread of cancer. A foot pad injection of [64Cu(dtcbp)2]–endorem resulted in a notable decrease in MR signal in popliteal lymph nodes after 3 h, which provides support for endorem accumulation. PET imaging of 64Cu-induced signal confirmed the uptake of nanoparticles in popliteal lymph nodes and iliac lymph nodes  . The cheph nodes .in vivo non-invasively in the pico-molar concentration range. However, a disadvantage of SPECT is that it offers limited anatomical details and spatial resolution. MRI is used in conjunction with SPECT to obtain quality anatomical images, thus offering both the structural and functional benefits of dynamic imaging. MRI/SPECT reduces discomfort associated with multiple sessions and scan times required for separate imaging procedures [There has also been increased interest in combining SPION with SPECT probes for MRI/SPECT dual-modality imaging . One advocedures .111In labeled anti-mesothelin monoclonal antibody (mAbMB) coated on iron oxide nanoparticles. A cell uptake study showed specific uptake of In-mAbMB-SPION by mesothelin-positive cells. An in vivo study of a mouse xenograft model of A431K5 tumors showed a signal drop post-24-h scan. The biodistribution study using SPECT imaging also showed relatively low uptake in the other normal organs  compared to the tumor. This result was well correlated with autoradiography images  [99mTc-SPION was comparable to the other similar sized nanocolloids. The MR image showed a non-homogenous uptake of 99mTc-SPION. This approach can be used for breast cancer and malignant melanoma imaging.In another example, Madru de (SLN) . The SLN111In was used to obtain autoradiography and biodistribution data. In the work of Madru et al., the radiolabeling efficiency of 99mTc-labeled PEG coated iron oxide reached 99%. The SLN uptake of nanoparticles was 100% ID/g, suggesting that the nanoparticle efficiency and SPECT/MRI detection in lymph node should encourage its usage in other cancers.The dual-modal probe developed by Misri and colleagues displayed better MR relaxivity compared to previously developed MRI/SPECT agents. The targeting efficiency with mesothelin-positive tumors was proven, and the SPECT detection motif The multi-functionality of nanoparticles further enables the integration of imaging and therapy . Theranostics refer to agents that target molecular biomarkers of a disease and are expected to contribute to personalized medicine. Among the currently available nano-vehicles, SPION have received great attention in the development of theranostic nanomedicines because they are not only used as contrast enhancement agents for MRI but can also deliver therapeutic agents, such as anticancer drugs and siRNA, to disease sites. In addition, SPION can continuously emit heat upon exposure to an alternating external magnetic field (AMF) by converting electromagnetic energy into heat –61. FiguChemotherapy is a common therapeutic approach to fighting cancer. However, it is non-selective and causes significant potential side effects to healthy tissues. To overcome this problem, magnetic nanoparticles loaded with the drug can serve as potential drug carriers in a new drug delivery strategy . The taret al. [et al., was modified in an adjunct study to include a DOX in the HSA (Human Serum Albumin) matrix to form DOX conjugated HSA–iron oxide nanoparticles [The multi-functionality of a SPION-based nanosystem was explored using drug and imaging modalities. With the development of biocompatible polymer-coated SPION loaded with DOX, Yu et al.  successfet al.  developearticles . The evaet al. [in vivo MRI and optical imaging  is a class of double-stranded RNA molecules that can inhibit any specific protein expression at the post-transcriptional level. The process is known as RNA interference (RNAi). Genes are integrated into SPION, which protect the nucleic acids against enzymatic degradation and facilitate cellular internalization and endosomal release . SPION aet al. using the CD44v6 single-chain variable fragment as a targeting agent for gastric cancer, and siRNA was loaded for the therapy [SPION functionalized with PEG grafted polyethylenimine (PEG-g-PEI) was synthesized by Chen  therapy . Compareet al., in which the fabricated core material was manganese-doped magnetism-engineered iron oxide (MnMEIO) nanoparticles coated with BSA [N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) treatment. Thus, activated MnMEIO nanoparticles were treated and functionalized with thiolated poly(ethylene glycol) , cyclic Arg-Gly-Asp (RGD) peptide and Cy5-dye labeled thiolated siRNA (HS–siGFP–Cy5). The effect of MnMEIO–siGFP–Cy5/PEG–RGD nanoparticles was studied in cell lines expressing GFP (MDA-MB-435-GFP and A549-GFP cells), and the result showed dose-dependent decreases in GFP expression, which validate the effect of synthesized nanoparticles.An all-in-one nanoparticle was developed with multimodal imaging and siRNA delivery capability by Lee with BSA . This naHyperthermia is a form of treatment in which the temperature of body tissue is raised to 42 °C. This treatment drastically affects the functionality of cellular structures, proteins, cell membrane, nucleic acid repair enzymes, and induces cell death. Tumor cells can also be killed due to their lower tolerance of a sudden variation in temperature using magnetic hyperthermia (MHT). SPION possess the inherent property of generating heat by ferromagnetic resonance. However, the heating process is generated by Néel and Brownian relaxations during MHT treatment . Therefoet al. [3O4-PEI-Au-PEG nanoparticles with improved biocompatibility. To study the hyperthermia effect, the nanoparticles were suspended in agar to mimic in vivo conditions. Applying laser irradiation at a 532-nm wavelength for 90 s to a nanoparticle concentration of 50 μg/mL induced changes in temperature of approximately 32 °C. The MRI contrast effect was found to be comparable to that of Feridex®. PEI acts as a cushion between the core and shell and helps to maintain their physical properties. The magnetic properties of SPION had also been retained after the gold coating was applied. This study raises the possibility of using SPION-GOLD hybrid nanoparticles for multimodal contrast imaging and cancer treatment by hyperthermia.The possibility of using SPION in both multimodal and nanoheater applications has been studied by Hoskins et al. . SPION wMultimodal and multifunctional probes are cutting-edge technologies in which the advantages of nanoparticles are maximized. The use of MRI has prompted the development of a variety of multimodal probes based on T2 contrast agents. SPION is considered an ideal vehicle for multimodal and multifunctional applications. The theranostic agents based on SPION are crucial in cancer detection and the delivery of therapeutic payloads, such as chemotherapeutic drugs and genes. New research is dedicated to developing multifunctional and multimodal theranostic agents, which form the ultimate weapon against cancer. However, there are several challenges facing multimodal and multifunctional techniques, including the standardization of therapy response and the stability of complex nanoparticles under certain biological conditions. Future research must focus on rectifying the current flaws in multimodal and multifunctional imaging probes by introducing new biocompatible polymers. Finally, multimodal and multifunctional techniques may enhance clinical theranostics in the near future.


ZFN technology is a powerful research tool and has been used for genome editing in cells lines, animals and plants. The generation of functional ZFNs for particular targets in mammalian genome is still challenging for an average research group. The modular-assembly method is relatively fast, easy-to-practice but has a high failure rate. Some recent studies suggested that a ZFP with low binding activity might be able to form a working ZFN pair with another binding active half-ZFP. In order to unveil the potential ZFP candidates among those with low binding activities, this paper established a highly sensitive mammalian cell-based transcriptional reporter system to assess the DNA binding activities of ZFPs by inserting multiple copies of ZFN target sequence fragment (TSF) of an interested gene . Our results showed that this system increased the screening sensitivity up to 50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each hPGRN or hVEGF ZFN pair was in proportion with the combination of the binding activities of the ZFL (Left zinc finger) and ZFR (Right zinc finger). A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Lastly, site-specific genome editing by hPGRN ZFNs generated by this system was confirmed by sequencing, and the PGRN knock-out cell line showed significantly decreased cell growth compared with the control. Our system will provide a valuable tool for further optimizing the nucleases with regard to specificity and cytotoxicity. The PGRd by PCR .in vivo activities in vitro cutting assays can rapidly identify whether one or both halves of the nuclease are working. When both halves fail, choose another target site. When only one half of the nuclease fails, screen for variants with a mini-library by different methods including bacterial one-hybrid In this paper we established a highly sensitive mammalian cell-based transcriptional reporter system to unveil the potential ZFP candidates among those with low binding activities. Our results showed that this system increased the screening sensitivity up to 50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each PGRN ZFN pair was in proportion with the combination of the binding activities of the ZFL and ZFR. A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Lastly, site-specific genome editing by hPGRN ZFNs generated by this system was confirmed by sequencing, and the PGRN knock-out cell line showed significantly decreased cell growth compared with the control. Compared with the selection methods, which are highly labor-intensive and time-consuming, modular assembly is faster and easier to engineer ZFPs. Simply by using a computer-generated archive the researchers can build ZFNs from one- or two-finger modules In this paper, ZFPs assembled based on the publicly available resources for single zinc finger modules were screened for binding activities by using a mammalian cell-based reporter system, followed by analysis of genome editing activities. The results suggested that ZFNs with good genome editing activities did not necessarily have high binding abilities on both right and left ZFPs. For example, a right hPGRN ZFP with low binding ability was able to form a biological active hPGRN ZFN if combined with a left hPGRN ZFP with relatively high binding ability. The targeted chromosomal gene repair efficiency of each hPGRN or hVEGF2 ZFN pair was in proportion with the combination of the binding activities of the ZFL and ZFR. Consistent with our observation, an analysis of off-target cutting also suggested that a shortage of binding energy from a half-site can be energetically compensated by excess zinc finger: DNA binding energy in the other half-site Most of ZFNs generated in this study were shown to have site-specific genome editing abilities in mammalian genome. The overall high success rate of our system was partially due to the highly sensitive screening for ZFP candidates with binding activities by our mammalian cell-based luciferase reporter system. Our results showed that with insertion of multiple copies of ZFN TSF of an interested gene, this system increased the screening sensitivity up to 50-foldand markedly amplified the differences in the binding activities between different ZFPs, which allowed us to unveil the potential ZFP candidates among those with low binding activities. In addition to high sensitivity, this system is also simple and easy to utilize. However, there were still rare ZFPs with relatively high binding ability but low targeted genome editing activity. The underlying mechanism may be related to chromatin accessibility or the 3-D structure of ZFPs. There is a need for further study.Previous studies suggested that ZFN-associated cytotoxicity was due to the creation of off-target DSBs Human kidney cell line, HEK 293 and human hepatocyte cell line, HepG2 were purchased from ATCC, cultured in 10% DMEM (high glucose)  containing 10% newborn calf serum. Cell lines with a site specific integration of exogenous genes in the genome were positively or negatively screened in the presence of selection medium containing G418 (200 µg/ml) or GCV (0.5–1.0 mg/ml). Selection medium was changed every two days.Zinc finger arrays were selected using the OPEN method essentially as previously described Left zinc finger nuclease (LZFN) containing zinc finger protein (ZFP) targeted human VEGF and mutated Fok1 Donor DNA vector was constructed by a sequential cloning of up and down homologous sequence targeting human PGRN amplified from human genomic DNA respectively, into Ad5 E1 shuttle. The cassette expressing eGFP-T2A-neo under the control of CMV promoter was inserted into donor DNA vector between up and down homologous sequence, and the cassette expressing TK-T2A-mcherry under the control of PGK promoter was inserted downstream of the down homologous sequence. The resultant vector was called pAd5-E1-hPGRN-CMV-eGFP-T2A-neo-up/down-PGK-TK-T2A-mcherry, which could be used for positive and negative selection (All primer sequences used for the vector construction will be provided upon request).Donor vector pAd5-E1-hPGRN-CMV-eGFP-T2A-neo-up/down-PGK-TK-T2A-mcherry was co-transfected with pAd5-E1-hPGRN-L/R-ZFN into HepG2, the cells were then cultured in the presence of neomycin 48 hour post-transfection, followed by the screening of GCV. The cell line with site specific integration in the genome of HepG2 was used for extracting genomic DNA. Site specific integration was confirmed by the sequence of amplified fragment using indicated primers based on the template of genomic DNA extracted from cells above. (All primer sequences will be provided upon request).ZFPs targeting AAVS1 were synthesized based on previously published data ZFP-p65 AD expressing plasmid and renilla luciferase expressing plasmid as an internal control were co-transfected with reporter vector containing a different copy number of TSF into HEK 293 cells. Renilla luciferase expressing plasmid and reporter vector were co-transfected into HEK 293 cells as negative control. 48 hours post-transfection, the cell lysates were subject to luciferase activity assay using Dual Luciferase Assay kit  according to the manufacture manual using Thermo Scientific Varioskan Flash .ZFNs expressing plasmid was transfected into HEK 293 cells, 48 hours post-transfection, the cell lysates were subject to SDS-PAGE, then transferred on N.C membrane, followed by the incubation with McAb against HA or Flag. The film was developed using ECL kit (Thermo) according to manufacture manual.Figure S1Scatter plot of binding abilities (Y) against gene repair activities (X) of hPGRN ZFNs. The gene repair ability of each hPGRN ZFN pair was in proportion with the combination of the binding activities of hPGRN ZFL1 and each paired ZFR  except small deviations in pairs involving ZFR4 and ZFR5 (see arrows).(TIF)Click here for additional data file.Table S1Raw data of luciferase correction assay. The biological activities of hPGRN ZFNs were expressed as firefly luciferase values normalized to renilla luciferase (FL/RL).(DOC)Click here for additional data file.Table S2Full DNA and protein sequences of the best ZFN (hPGRN ZFL1 and ZFR2) for human PGRN.(DOC)Click here for additional data file.


Many public health problems in modern society affect the gastrointestinal area. Knowledge of the disease occurrence in populations is better understood if viewed in a psychosocial context including indicators of the social environment where people spend their lives. The general aim of this study was to estimate the occurrence in the population and between sexes of common gastrointestinal conditions in two neighborhood cities representing two different social environments defined as a "white-collar" and a "blue-collar" city.We conducted a retrospective register study using data of diagnosed gastrointestinal disorders (cumulative incidence rates) derived from an administrative health care register based on medical records assigned by the physicians at hospitals and primary care.Functional gastrointestinal diseases and peptic ulcers were more frequent in the white-collar city, while diagnoses in the gallbladder area were significantly more frequent in the blue-collar city. Functional dyspepsia, irritable bowel syndrome, and unspecified functional bowel diseases, and celiac disease, were more frequent among women while esophageal reflux, peptic ulcers, gastric and rectal cancers were more frequent among men regardless of social environment.Knowledge of the occurrence of gastrointestinal problems in populations is better understood if viewed in a context were the social environment is included. Indicators of the social environment should therefore also be considered in future studies of the occurrence of gastrointestinal problems. Many public health problems in modern society affect the gastrointestinal area. Knowledge of the disease occurrence in populations is better understood if viewed in a psychosocial context including indicators of the social environment where people spend their lives. The etiology of functional gastrointestinal diseases (FGD) such as irritable bowel syndrome (IBS), functional dyspepsia (FD) and gastroesophageal reflux (GERD) is complex and in many ways still unclear and there is still a lack of understanding of the pathology ,2. The dKnowledge of the disease prevalence rates is crucial for describing the population burden of diseases and assessing the associated health care utilization and health care cost . AvailabThe role and importance of social and physical environments for health, the effect of residence and the question of whether we should focus on context rather than people is still an open question in health sciences -17. A geThe general aim of this study was to estimate the occurrence in the population and between sexes of common gastrointestinal conditions in different social environments defined as a "white-collar" and a "blue-collar" city.We focused on the population in two equally sized cities (the twin cities) located 40 km (25 miles) apart in the same county in the south-east of Sweden, namely, the city of Norrköping (N) and the city of Linköping (L). The two cities are quite equal in size and there are neither any age nor sex distribution differences between them . Both ciThere is a long tradition in the Scandinavian countries of documenting diseases in registers and the integrated health care delivery systems are obligated to serve all inhabitants, and each inhabitant is assigned a unique personal code number based on birth date and sex . The heaWe conducted a retrospective register study using a health care register to identify diagnosed gastrointestinal problems in medical records. The Health Care Register (HCR) used is based on computerized data files linked by a unique personal code to birth date and sex of all inhabitants in the county. The same personal code is used for all visits and diagnoses in the HCR. The international classification of diseases (ICD) 10-code was used. The selected GI disorders in this study were: Irritable bowel syndrome-constipation (IBS-C) and Irritable bowel syndrome-diarrhoea (IBS-D), respectively , esophageal reflux , functional dyspepsia , functional bowel disease unspecified , peptic ulcer disease , inflammatory bowel disease , coeliac disease (ICD-K90) diseases in the gallbladder area (ICD-K80-K83.9), cancers in the GI tract  and alcohol-related liver diseases . The attending physician assigned all diagnostic codes. The diagnoses were extracted from the HCR using a case-finding algorithm that retrospectively searched the register from 1/1/2002 to 31/12/2007.All data were stored in a common database and statistically analysed using the SPSS version 17.0 program . The algorithm captured the cases (one case = one patient) regardless of whether the disorders of interest constituted the main or secondary diagnoses, and it also specified the health care level at which the patient was diagnosed, that is, primary care, outpatient hospital care, and/or inpatients hospital care. The following case definition was applied: the first contact with health care services with a GI diagnosis during the period studied (2002-2007) was regarded as a case. Cumulative incidence rates were calculated and expressed as number of cases per 1,000 inhabitants. The mean population in different age groups for the period 2002-2007 was used as the denominator. We initially estimated the occurrence of gastrointestinal diagnoses for all ages up to the age of 80 in a gender comparison. However, the comparison between the blue-collar and the white-collar cities were only focusing on the population between 25 and 79 years, i.e. the working ages and retirement ages. The numerator for the prevalence calculation was the number of first diagnosed cases identified during the six-year study period. The denominator was the number of inhabitants calculated as the mean number in each age group of the population for the six-year study period. The prevalence rates are presented as numbers per 1,000 inhabitants. We also calculated the Relative Rates (RR) and 95% confidence intervals (CI) and differences between the cities were also calculated with Pearson's chi square.The Research Ethics Committee of the Faculty of Health Sciences, Linköping University, Sweden, approved the study (2009).The comparison between the twin cities revealed significant differences for functional GI-disorders and peptic ulcer disease (PUD) (ICD-K25 to ICD-K26) that were more frequent in the "white-collar" city than in the "blue-collar city. On the other hand, the diagnoses in the gallbladder area (ICD-K80.0 to ICD-K83.9) were significantly more frequent in the blue-collar city for both sexes and in all age groups. Also alcohol-related liver disease (ARLD) (ICD-K70) among 45-64 year old patients of both sexes was more frequent in the "blue-collar" city; see Table Significantly differences between sexes in the total cumulative incidence rates of different gastrointestinal disorders were also seen, the same pattern concerning sex differences were seen in both cities, the total distribution of these differences are illustrated in Figure Coeliac diagnoses were significantly, RR = 1.57 (95% CI 1.37-1.79), more frequent among women compared to males as well as diagnoses of the gallbladder area, RR = 1.80 (95% CI 1.71-1.91). Even ARLD showed a sex difference, more frequent among men, RR = 2.69 (95% CI 2.05-3.53). Gastric (ICD-C16) and rectal cancers (ICD-C20) were totally more frequent among men, RR = 1.73 (95% CI 1.34-2.21) and RR = 1.27 (95% CI 1.08-1.48), respectively, as illustrated in Figure This population-based study revealed differences in the occurrence of GI diagnoses between the studied social environments and also between sexes regardless of the social environment. The comparisons showed that GI-diagnoses that are predominantly related to stressful life events like GERD, IBS, and PUD were more common in the population of the "white-collar" city. Possibly this difference in type of gastrointestinal occurrence between these social environments reflects differences in perceived and actual exposure to stressful life conditions, more predominant in the "white-collar" city. Recent studies of functional gastrointestinal disorders such as IBS, showed that e.g. lack of influence on planning work, anxiety and sleep disruptions were associated with an IBS diagnosis in women and lack of influence on working pace among men ,23. AlthHowever, certain GI diagnoses like alcohol-related liver diseases and gall-tract disorders were more prevalent in the "blue-collar" city. This may suggest a less healthy life style in its population leading to these problems. This is supported by our previous observations showing that diagnoses predominantly related to life-style problems like coronary heart diseases and chronic obstructive pulmonary disease were more common in the studied blue-collar city .The observed differences in the occurrence of GI diagnoses between the two social environments are not likely to be explained by chance or by differences in access to health care services. However, it could not be ruled out that health care seeking patterns might differ in the two cities. Both the inpatient and outpatient care is delivered by public health services based on population needs in both cities. So there are no significant differences in the supply of health care facilities like primary care in the two cities. On the marginal, the "white-collar" city population might benefit from being more often referred for investigations requiring technology available only at a university hospital, since these facilities is situated in the "white-collar" city. There might also be variations between the cities in the diffusion of diagnostic skills of doctors within the field of GI disorders. This might impact the assignment of GI complaints in patients seeking advice in the primary care. Anyhow, we believe that the differences seen between the population in the two cities is related to differences in health behaviors, educational levels, psychosocial factors, nutritional habits, life styles in general and specifically alcohol consumption and smoking behaviors .It is well known that FGD diseases such as IBS and FD and FGD-UNS are more common among women than men, but surprisingly, IBS and FGD-UNS were more frequent among the youngest group of men (data not shown). The explanations of this gender phenomenon must be hypothetic and can differ. It might be randomly due to an unknown factor or due to the fact that more women suffer from IBS -26 and tThe total number of gall diseases was significantly more frequent among women compared to men except in the oldest age group. The causes of gallstone formation are multifactorial, although some risk factors are permanent, such as being women, increasing age and ethnicity and previous family history of the disease. Other recognized risk factors are a high caloric intake and obesity -30. TherPeptic ulcer diseases in this study occur more frequently among men in all age groups. These diseases are often induced by Helicobacter pylori (HP) or non-steroid anti-inflammatory drugs (NSAID), two independent risk factors, PUD is often decreasing with age, although a peak often occur at retirement ,43. HoweThis study also showed that gastric and rectal cancers occurred more frequently among men of all ages compared to women. Colon and small intestinal cancers were more equally distributed among both sexes, although colon cancer was slightly more frequent among men, but not significantly so. Colorectal cancer is the third most common form of cancer in Sweden . On the Possible limitation in this study is that we do not have any measures of individual socio-economic conditions. Living in a certain social environment like a "blue-collar" or a "white-collar" city could only be a proxy for real socioeconomic status. Further limitations in the study are that the data used was not initially collected for research purposes. The diagnoses registered, although they all derive from medical records, could in some cases be regarded as approximate. The strength of this study is that it is based on medical records in computerized data files (HCR) for all inhabitants linked by birth date and sex. The same personal code is used for all visits and diagnoses in HCR. An individual can thus be followed retrospectively or prospectively through the health care system using this personal code. The health care institution where the patient was diagnosed represents all health care levels - primary care, outpatient hospital care, and/or inpatient hospital care which gives a more complete panorama of the health care. Other strengths of this study are that our inclusion method gains reliability through being very general and covering a span of several years and that the number of included patients is high tending to level out possible misclassification within the groups defined as patients. Studies have also shown that general practitioners rarely misdiagnose for instance FGD -55 in faTo conclude, this population-based study revealed differences in the occurrence of gastrointestinal diagnoses between the populations in a "white-collar" and a "blue-collar" city. Gastrointestinal diagnoses that are predominantly related to stressful life events like GERD, IBS and peptic ulcer disease was more common in the population of the "white-collar" city. This might reflect differences in lifestyles, living conditions and factors in working life like differences in heavy, strenuous or even monotonous work of the populations in the two studied cities that constitutes two close but different social environments. Lifestyle-related gastrointestinal diseases like alcohol-related liver diseases and gall-tract disorders were more frequent in the "blue-collar" city. Significant differences in the cumulative incidence rates of the most common gastrointestinal conditions between sexes were also seen. Women tended to have more functional gastrointestinal diagnoses and gall diagnoses, while men had more peptic ulcer diseases and alcohol-related liver diseases. Many of these conditions also tend to increase with age. A forthcoming research project will investigate the validity of diagnose-issue and whether differences in working life conditions between individuals with functional gastrointestinal problems compared to healthy controls in the "white-collar" and the "blue-collar" city might contribute to explain the differences seen in these twin cities.Knowledge of the occurrence of gastrointestinal problems in populations is better understood if viewed in a context were the social environment is included. Indicators of the social environment should therefore also be considered in future studies of the occurrence of gastrointestinal problems.ARLD: Alcohol-related liver disease; ASA: Acetylsalicylic acid; CI: Confidence interval; FD: Functional dyspepsia; FGD: Functional gastrointestinal diseases; GERD: Gastroesophageal reflux; GI: Gastrointestinal; GPRD: The general practice research database; HCR: Population based health care register; HP: Helicobacter pylori; IBD: Inflammatory bowel disease; IBS: Irritable bowel syndrome; IBS: C Irritable bowel syndrome constipation; IBS: D Irritable bowel syndrome diarrhoea; ICD: International classification of diseases; L: Linköping; N: Norrkoping; NASID: Non-steroid anti-inflammatory drugs; PHC: Primary health care; PUD: Peptic ulcer disease; RR: Relative risks; UK: United Kingdom; UNS: Unspecific.The authors declare that they have no competing interests.EG, ÅF, TF participated in the study design and coordination and completed the data collection. EG, ÅF, TF, CH and EB drafted the manuscript as well as analysis and interpretation of data, read and approved the final manuscript.


Plasmodium chabaudi (AS) by a virulent clone (AJ) in immuno-deficient and competent mice. We test whether competitive suppression is caused by clone-specific differences in one or more of the following processes: adaptive immune clearance of merozoites and parasitised red blood cells (RBCs), background loss of merozoites and parasitised RBCs, RBC age preference, RBC infection rate, burst size, and within-RBC interference. These processes were parameterised in dynamical mathematical models and fitted to experimental data. We found that just one parameter Malarial infections are often genetically diverse, leading to competitive interactions between parasites. A quantitative understanding of the competition between strains is essential to understand a wide range of issues, including the evolution of virulence and drug resistance. In this study, we use dynamical-model based Bayesian inference to investigate the cause of competitive suppression of an avirulent clone of  Malaria infections often consist of more than one strain of the same parasitic species. Understanding the within-host competition between these various strains is essential to understanding the evolution and epidemiology of drug resistance in malarial infections. The infection process and the competition between strains involve complicated biological processes that are explained by various hypotheses. Mathematical models tested against experimental data provide quantitative measures to compare these hypotheses and enable us to discern the actual biological processes that contribute to the observed dynamics. We use a group of models against experimental data on rodent malaria to test various hypotheses. Such quantitative measures, in understanding rodent malaria, can be considered as a step towards understanding within-host parasite dynamics. Our work presented here demonstrates how confronting mathematical models with data allows the discovery of subtle and novel interactions between hosts and parasites that would be impractical to do in an experiment and allows the rejection of hypotheses that are incorrect. It is our contention that understanding the forces controlling within-host parasite dynamics in well-defined experimental model is a necessary step towards understanding these features in natural infections. Malarial infections often consist of more than one strain of the same parasitic species P. chabaudi clones have demonstrated competitive suppression of less virulent clones by virulent clones P. chabaudi clones, by Taylor et al., provides evidence for competitive suppression of one of the clones irrespective of initial dose P. chabaudi with different ratios of initial parasite numbers. The competitive suppression of avirulent clone at the later stages of infection in all three treatment groups were attributed to clone-specific and cross-immunity of the host induced by the parasite strains. However, the exact role of host immune response on the suppression of CR could not be explored.Several experimental studies of mixed infections of P. chabaudi, differing in virulence, were tested against an unrelated, and more virulent strain of P. chabaudiIn another experimental study, 7 genetically closely related strains of Another study showed that a virulent clone obtained a competitive advantage due to larger parasite and gametocyte densities, compared to an avirulent clone, during mixed infections P. chabaudi were inoculated into mice either simultaneously or 3 or 11 days apart and their population sizes were tracked using immunofluorescence or quantitative polymerase chain reaction Recently a series of experiments were designed to study the effects of parasite genotype, residency and time of infection on within-host parasite densities during mixed infections. In these experiments two pairs of distinct clones of P. chabaudi (AS and AJ) were co-infected into mice. The AS clone is less virulent than the AJ clone, being associated with a lower peak parasitaemia, less RBC loss and less weight loss In a recent paper examining competition between malaria clones we found direct experimental evidence of immune-mediated competition Our aim in this paper is to re-examine this dataset using a dynamical model-based Bayesian inference approach in order to determine the nature of these competitive interactions, immune mediated or otherwise. Parameterised dynamical (process) mathematical models are fitted to the experimental data. Mechanism can then be inferred from the estimated parameters – i.e., a parameter for a mechanism (such as immune-mediated clearance rates of parasites) that is different across treatments suggests possible causes of competitive interactions We briefly describe the experiment here. See nu/nu mice ; and (iii) nude mice reconstituted with T cells taken from wildtype mice. The mutation nu is a recessive mutation that blocks the development of the thymus and hence these mice have no mature T-cells which impairs their immune systems Three different phenotypes of 12–14 week old, female BALB/c mice were used: (i) wildtype mice; (ii) female Mice of each phenotype  were inoculated intraperitoneally with We extend the model of malaria parasite bloodstream asexual replication developed in P. chabaudi, parasitised RBCs (pRBCs) rupture synchronously every 24 hours In https://code.google.com/p/bayesian-model-based-inference/We use a discrete-time formulation to model the dynamics, where each time step corresponds to a single day. The start of day We assume that the processes determining RBC and parasite densities occur on two non-overlapping timescales. The first corresponds to the short infection phase during which merozoites infect RBCs, which occurs within a few minutes following schizogony. The second and subsequent timescale (the remainder of the day) corresponds to the RBC turnover phase: the parasites replicate within pRBCs, and new unparasitised RBCs (uRBCs) migrate from the bone marrow and spleen into the bloodstream We model separate, time-dependent, adaptive immune responses against merozoites in the infection phase P. chabaudi1, considers this possibility . Our fifth hypothesis H5 considers competition caused by a combination of two processes: differential background loss rates of merozoites 6 and H7 we consider clone-specific adaptive immunity against merozoites and pRBCs respectively.Biologically, competition between clones can be mediated by several processes as listed in sibility . In our 6 and H7), so we might observe weaker adaptive immune clearance of the AJ clone compared to the AS clone.Competition is incorporated into the model via clone-specific parameters . We woul1 to H7 are allowed to be different between the two strains. This so called “all-cause” model is fit to the data. Each single-cause model is obtained by keeping the parameters clone-specific for the cause of interest and making parameters clone-non-specific for all other causes. Each single-cause model is fit to the data. If none of the single-cause models adequately predict the data, we would then examine dual-cause models, and so on. This was not necessary however. The all-cause model acts as a reference because it has the highest maximum likelihood. Any single-cause model that has a maximum likelihood similar to  the all-cause model fits the data as well as the all-cause model.We test the causes of competition as follows. The full model, described above and in more detail in the Supplementary Material, includes all possible causes of competition. That is, all parameters included in hypotheses Hin vivo and RBC invasion rates may vary between-mice due to the multi-factorial nature of such processes which involve the interaction of many host and parasite proteins. We therefore make no assumption about which parameters are invariant across mice and estimate each parameter separately for each mouse.There is considerable variability in parasite and RBC dynamics of the mice both between and within the treatment groups. This suggests that there is variability in the underlying processes that govern the dynamics and thus in the parameters. Furthermore, the immune responses are significant sources of variability 10-parasite densities are normally distributed with standard deviations In the experiment, measurements were taken at approximately 08:00 hrs, roughly We use maximum and marginal likelihoods to compare our competing hypotheses about the causes of competitive suppression. Marginal likelihoods naturally penalise models that over-fit data with too many parameters. Marginal likelihoods are computed for each mouse When comparing two hypotheses the ratio of their marginal likelihoods, their Bayes factor, is a convenient statistic. Bayes factors quantify how much more likely one hypothesis is over another given the observed data The experimental data on nude and reconstituted mice are discussed in The average parasite densities for the three mouse phenotypes for single (solid lines) and mixed (dashed lines) infections of the AJ (left panel) and AS (right panel) clones are shown in The all-cause competition model was fit to the single and mixed infection data from We first tested the fits for the three functional forms of the adaptive immune responses. The sigmoidal response gave the best fits in terms of maximum and marginal likelihoods see , the pieThe standardised residuals of the all-cause model for each mouse phenotype are given in the Supplementary Material  and S5. It is clear from The fits to individual mice data of the single-cause model with clone-specific In single clone infections there is no significant difference between In contrast to single infections, in mixed infections In mixed infections, the difference between In nude and reconstituted mice In wildtype mice, the opposite is the case: We discuss the significance of these results next.P. chabaudi clones, the AJ clone competitively suppresses the AS clone In mixed infections of virulent AJ and avirulent AS We tested seven possible mechanisms that could cause competitive suppression . Our ressimulate parasite and RBC dynamics in mixed infections. These simulations gave qualitatively similar dynamics to data from mixed infections thus suggesting that RBC age preference can cause competitive suppression. We went a step further in this study by fitting our model to the mixed infection data as well as the single infection data. This allowed us to quantitatively compare this mechanism with many others and demonstrate that, although it can explain competitive suppression, it does less well than clone differences in In fact, all of the mechanisms of competition we tested could explain competitive suppression (results not shown). However, these mechanisms did not predict the data as well as a clone-specific  see . This doBefore we discuss the biological interpretation of refer to . In singIn addition, in nude and reconstituted mice we found that Our definition of in vivo measurements of the loss of invasive ability of free merozoites The parameter Our finding that in vivo experiments show that parasite growth rate in the exponential phase increases at low parasite dose and saturates at high parasite dose This leads us to suggest that our definition of sed mice , ,  agaiAlthough clone-specific differences in the data , we cannpecific  . This ispecific  , hence tOur results leave us with two unanswered questions: Why should the clearance rate of parasites by the innate immune response change between single and mixed infections? And why is the change in clearance rates positive for the AS clone in nude and reconstituted mice and negative for the AJ clone in wildtype mice ? We beliP. chabaudi is most likely mediated through innate clearance of merozoites acting throughout an acute infection.In conclusion, our dynamical model-based inference approach can be used to compare multiple hypotheses about biological processes underlying infection dynamics data. Using this approach we have shown that competitive suppression of an avirulent clone of Figure S1Schematic of model showing the 24 hr cycle. The model includes the erythropoiesis cycle where new uninfected reticulocytes are produced that mature into normocytes, and the erythrocytic phase of the parasites which includes the infection phase, RBC turn over phase and schizogony.(TIF)Click here for additional data file.Figure S2Assessment of convergence of Markov chains. Gelman-Rubin statistics for each parameter sorted by mouse (top panel) and by parameter (bottom panel). A statistic below 1.1 suggests excellent convergence of the Markov chains (TIF)Click here for additional data file.Figure S3Standardised residuals of reconstituted mice. Assessment of the all-cause model fits to the data by standardised residuals for reconstituted mice; AS parasite density (top panel); AJ parasite density (middle panel); RBC density (bottom panel). Each cross represents the standardised residual of a time point for an individual mouse. The solid red line joins the means of the standardised residuals at each time point. The dashed lines represent the 95% interval for the expected mean for the same number of residuals as the data (see (TIF)Click here for additional data file.Figure S4Standardised residuals of nude mice. Assessment of the all-cause model fits to the data by standardised residuals for nude mice. See caption in Figure 8 for details.(TIF)Click here for additional data file.Figure S5Standardised residuals of wildtype mice. Assessment of the all-cause model fits to the data by standardised residuals for wildtype mice. See caption in Figure 8 for details.(TIF)Click here for additional data file.Figure S6Q-Q plots for reconstituted mice. The standardised residuals are approximately normally distributed suggesting adequate fits to the data. AS parasite density quantiles (top panel); AJ parasite density quantiles (middle panel); RBC density quantiles (bottom panel).(TIF)Click here for additional data file.Figure S7Q-Q plots for nude mice. The standardised residuals are approximately normally distributed suggesting adequate fits to the data. AS parasite density quantiles (top panel); AJ parasite density quantiles (middle panel); RBC density quantiles (bottom panel).(TIF)Click here for additional data file.Figure S8Q-Q plots for wildtype mice. The standardised residuals are approximately normally distributed suggesting adequate fits to the data. AS parasite density quantiles (top panel); AJ parasite density quantiles (middle panel); RBC density quantiles (bottom panel).(TIF)Click here for additional data file.Figure S9Statistical comparison of possible causes of competition for reconstituted mice. Marginal against maximum likelihoods on a 5).(TIF)Click here for additional data file.Figure S10Statistical comparison of possible causes of competition for nude mice. See (TIF)Click here for additional data file.Figure S11Statistical comparison of possible causes of competition for wildtype mice. See (TIF)Click here for additional data file.Table S1Model variables. Dependence on day (PDF)Click here for additional data file.Table S2Model parameters.1Dependence on AS and AJ removed for brevity. 2 NT is a Normal distribution truncated at 0.(PDF)Click here for additional data file.Text S1Models and methods. A detail description of the various models in the paper and the methods used to compare them.(PDF)Click here for additional data file.


Sepsis is a serious clinical condition with a considerable morbidity and mortality. Procalcitonin (PCT) is a good biomarker for early diagnosis and infection monitoring. A semi-quantitative PCT assay can be performed at the bedside and has good diagnostic value ,2. The pThe study design was a randomized diagnostic trial, which was also a pragmatic trial. Septic patients more than 18 years old with and without signs of organ hypoperfusion or dysfunction who were admitted to Cipto Mangunkusomo Hospital emergency department in the internal medicine unit were eligible. Subjects were randomly assigned to either a semi-quantitative PCT-examined group (study group) or a control group. Semi-quantitative PCT test results will be informed to the physicians taking care of the patients. The primary outcome was 14-day mortality. Secondary outcomes were the time of initiation and appropriateness of empirical antibiotics. A Tropical Infection Consultant will assess the appropriateness of empirical antibiotics based on Pedoman Umum Penggunaan Antibiotik Departemen Kesehatan Republik Indonesia.Two hundred and five patients met the inclusion criteria. Ninety-five of 100 subjects from the study group and 102 of 105 subjects from the control group were included in the analysis (Figure Semi-quantitative PCT examination affects the empirical antibiotic initiation time and mortality in septic patients, but not the appropriateness of empirical antibiotics.