The aim of this study was to describe treatment outcomes for multi-drug resistant tuberculosis (MDR-TB) outpatients on a standardized regimen in Nepal.Data on pulmonary MDR-TB patients enrolled for treatment in the Green Light Committee-approved National Programme between 15 September 2005 and 15 September 2006 were studied. Standardized regimen was used (8Z-Km-Ofx-Eto-Cs/16Z-Ofx-Eto-Cs) for a maximum of 32 months and follow-up was by smear and culture. Drug susceptibility testing (DST) results were not used to modify the treatment regimen. MDR-TB therapy was delivered in outpatient facilities for the whole course of treatment. Multivariable analysis was used to explain bacteriological cure as a function of sex, age, initial body weight, history of previous treatment and the region of report.In the first 12-months, 175 laboratory-confirmed MDR-TB cases  had outcomes reported. Most cases had failed a Category 2 first-line regimen (87%) or a Category 1 regimen (6%), 2% were previously untreated contacts of MDR-TB cases and 5% were unspecified. Cure was reported among 70% of patients (range 38%–93% by Region), 8% died, 5% failed treatment, and 17% defaulted. Unfavorable outcomes were not correlated to the number of resistant drugs at baseline DST. Cases who died had a lower mean body weight than those surviving . Default was significantly higher in two regions . At logistic regression, cure was inversely associated with body weight <36 kg  and treatment in the Eastern region .The implementation of an ambulatory-based treatment programme for MDR-TB based on a fully standardized regimen can yield high cure rates even in resource-limited settings. The determinants of unfavorable outcome should be investigated thoroughly to maximize likelihood of successful treatment. The emergence of strains of tuberculosis (TB) that resist drugs poses a potentially devastating threat to TB control globally Treatment of MDR-TB is resource intensive and lasts for 24 months or more, requiring a combination of second-line drugs that are more expensive, less effective and more toxic than those used in standard first-line treatment regimens In Nepal TB is a major public health issue. In 2007, there were an estimated 48,766 incident TB cases  In this article we describe the outcomes of patients with laboratory confirmed MDR-TB who received treatment during the first 12-months of the MDR-TB treatment programme.Mycobacterium tuberculosis we excluded those with no baseline drug susceptibility testing (DST) results available (6 cases), and those whose baseline DST results did not meet the definition of MDR-TB (confirmed susceptibility to either isoniazid and rifampicin) (13 cases).Between 15 September 2005 and 15 September 2006 a total of 224 patients were enrolled in the GLC-approved treatment programme in Nepal to receive standardized, second-line drug therapy for pulmonary MDR-TB. Of these, 175 patients with confirmed MDR-TB who began treatment in this period were retained for the study . ExclusiPatients were categorized according to the registration groups used by the Nepal NTP: Category 2 treatment failure, Category 1 treatment failure with DST confirmed MDR-TB, and previously untreated MDR-TB family contact. Cases for whom the registration group was not known were retained in the analysis.This analysis was carried out retrospectively and involved basic data collected on a routine basis within the NTP services to regularly monitor the performance of the MDR-TB treatment program. The data were analyzed anonymously. The implementation of such program activity, including the monitoring and evaluation system, is a routine and regular component of the NTP activities and approved by the Ministry of Health and Population of Nepal; therefore no ethical approval was required for this analysis.M. tuberculosis complex, the GenoType® Mycobacteria CM test was applied additionally to identify the NTM. In the study period DST was available for the following four first-line drugs: rifampicin, isoniazid, streptomycin, and ethambutol. DST for pyrazinamide and second-line drugs was not available in the country at the time of this study. In this article, DST was considered as baseline if the sample (sputum) was obtained from a patient within a period of 60 days prior to starting treatment until 40 days after start.Diagnosis of MDR-TB was performed at GENETUP TB-laboratory which functions as the National Reference Laboratory in the capital Kathmandu using the indirect proportion method on Löwenstein-Jensen medium. The external quality control for this laboratory is organized and performed by the Supranational Laboratory in Gauting, Germany since 1994 using proportion method. At least one positive culture per patient was also tested using the GenoType® MTBDR plus test of Hain Lifescience. All discrepant results were retested in both systems. If the identification of strains in the GenoType® MTBDR plus test revealed not a Laboratory follow-up to monitor treatment progress is conducted by sputum smear microscopy and culture conducted monthly during the first eight months of treatment (or 12 months if intensive phase was extended) and then every two months till the end of treatment.All patients enrolled received the same standardized treatment regimen for MDR-TB. Individual DST results were not used to modify the treatment regimen. The regimen was designed based on the results of DST to first- and second-line anti-TB drugs conducted within the context of a nationwide drug resistance survey, as described elsewhere The duration of the initial phase was also discussed with the GLC. In Nepal is was decided to prolong the initial phase to 8 months to allow the administration of kanamycin over a longer period. It was felt that to provide the full dosage of daily 6 months kanamycin over a slightly longer period (4 month daily and 4 months thrice weekly) - 8 months in total, might reduce the toxic effects that are usually seen between 4 to 6 months, and extend the time of impact on the overall regimen.The same drugs, excepting kanamycin, are used in the continuation phase, which usually lasts 16 months but may be extended by up to 8 months if the patient's culture converts to negative only between 12 and 18 months of treatment. Treatment is available in two dosages according to the weight of the patient at the beginning of treatment . The weight categories were carefully worked out using the spread of actual weights of a large sample of previously treated TB patients, and showed to work well in practice. Weight cut-off were similar to those used in another study in the same geographical area Treatment is delivered daily at the clinic. Support during treatment is guaranteed by the presence of a treatment support person nominated by the patient. A gastroprotective agent (ranitidine) is also taken daily during the course of treatment to decrease side effects. Treatment of minor adverse reactions with symptomatic drugs as well as admission to hospital for severe side effects are free of costs for the patients.An active defaulter tracing system is in place and implemented locally by the clinics with support and supervision from the District TB/Leprosy Officers.Patient data are kept peripherally on treatment cards, key outcome and treatment information transferred to district MDR-TB registers and from these reported in aggregated quarterly reports. Data from all the district registers is transferred to the national MDR-TB treatment register, which is kept and updated at the National Tuberculosis Centre (NTC) in Kathmandu. This includes demographic information, initial body weight, patient category based on previous treatment, treatment starting date, bacteriology and outcome (dated). Information on socioeconomic background, co-morbidities, or compliance with treatment was not available from this source. An electronic register based on Open Medical Record System (openmrs.org/wiki/Mdrtb_Module) was installed for use at the NTC in October-November 2008 and updated retrospectively with data from patients started in the national MDR-TB treatment programme since 2005.After cleaning the data were analyzed using the Stata SE/10.0  and the Statcalc function of EpiInfo version 6 (CDC). Chi-squared test was used to identify significant associations (p<0.05) between categorical variables and outcomes and to test linear trend. Strength of association is shown as crude odds ratios with their corresponding 95% confidence limits. Two sample t-test was used to explore associations between continuous variables (age and body weight) and cure. A logistic regression model was built to analyse cure as a function of sex, age, initial body weight, previous treatment history, and the region where the patient was treated.A total of 175 confirmed pulmonary MDR-TB patients were enrolled in the national MDR-TB treatment programme in the study period . More thA full description of the resistance patterns to first-line anti-TB drugs at baseline DST is given in Most patients have been cured , 14 patients died (8%), 29 patients defaulted (17%), and nine patients failed (5%) . No statThe median duration of bacteriological monitoring and of treatment of cured cases was 730 days . Among the 123 who were cured, 112 (91%) experienced a durable sputum-smear conversion to negative within the first six months after treatment start. Patients who failed to be cured through the programme were treated for a median duration of 721 days (range: 404–959 days). Of the 14 patients who died, one converted to negative at month three and one at month seven after treatment while 12 patients were culture positive at the time of death. Adverse events recorded during treatment of this cohort of patients are presented in Bivariate analysis showed a significant positive linear relationship between cure and initial body weight (p = 0.001). In the Eastern region, cure was significantly lower than in the Central  and Mid-western  regions. The same associations with body weight and with region became stronger after adjustment in the multivariable model . No signIn this manuscript we describe the performance of an MDR-TB treatment program in a resource-limited setting designed around two main elements: a) integration of MDR-TB management in an existing ambulatory-based TB care delivery system; b) utilization of a fully standardized treatment regimen, i.e. DST results are not used to modify treatment regimens. To our knowledge the large majority of studies describing ambulatory- or community-based approaches to MDR-TB treatment adopt individualized treatment regimens, i.e. patients are offered an empiric treatment regimen which is tailored to the resistance patterns of the infecting strain as soon as DST results became available. Vast experience from Peru shows that community-based treatment for MDR-TB and even XDR-TB is feasible and can yield high cure rates In Peru the only cohort of MDR-TB patients treated with a fully standardized regimen on ambulatory basis gave discouraging results, with only 57% of the patients being cured An individualized treatment regimen for MDR-TB would likely not have worked in our setting. The lack of experienced health workers on MDR-TB management and the long distances between TB treatment centers would not have made possible the expansion of MDR-TB services had it not been for the utilization of a fully standardized regimen. The utilization of a standardized regimen makes training, drug forecasting, and management of adverse events much easier, in addition to reducing significantly the overall cost of treatment The large majority of patients enrolled in Nepal were Cat 2 failures (87%), and there was no recruiting among previously untreated TB patients (except family contacts). The strict application of the enrolment criteria has made possible to use a standardized treatment regimen throughout the country.Despite decentralization of the services in ambulatories in several regions overall default rates are quite high, but in line with previous publications from countries implementing similar programs As expected, patients with low body weight have significantly higher rates of death than those weighing more. In this cohort the higher the body weight the greater was the chance of being cured . These fAdverse events were managed by local health workers using a standardized protocol based on the WHO guidelines M.fortuitum were cured. The remaining 6 patients (5 infected with M.intracellulare and 1 with M.avium) defaulted. The proportion of patients with NTM that end up being treated with regimens for MDR-TB might not be negligible in resource-limited settings. International guidelines on how to treat these patients are urgently needed. For the moment guidance documents have been produced by some medical societies A total of 8 patients had disease caused by NTM strains  and endeOne limitation of this study is that, due to lack of access to routine DST for second-line drugs, XDR-TB cases could not be detected at the start of MDR-TB treatment. Undiagnosed XDR-TB may therefore be the cause of death of treatment failure in some of the patients in this cohort. XDR-TB is though to be present in around 7% of the patients enrolled in the MDR-TB treatment programme in Nepal. Recently DST for key second-line drugs has become more available and there are plans in Nepal to make it available to all patients with confirmed MDR-TB.The experience from the first year of the MDR-TB treatment programme in Nepal shows that fully standardized treatment for MDR-TB delivered through a network of outpatient facilities is not only feasible but can also yield high cure rates in resource-limited settings.


Thrombolysis with intravenous rt-PA is currently the only approved acute therapy for ischemic stroke. Re-occlusion after initial recanalization occurs in up to 34% in patients treated with rt-PA, probably caused by platelet activation. In acute myocardial infarction, the combination of thrombolysis and antiplatelet therapy leads to a greater reduction of mortality compared to thrombolysis alone. In patients with acute ischemic stroke, several studies showed that patients already on antiplatelet treatment prior to thrombolysis had an equal or even better outcome compared to patients without prior antiplatelet treatment, despite an increased risk of intracerebral bleeding. Based on the fear of intracerebral haemorrhage, current international guidelines recommend postponing antiplatelet therapy until 24 hours after thrombolysis. Remarkably, prior use of antiplatelet therapy is not a contra-indication for thrombolysis. We hypothesize that antiplatelet therapy in combination with rt-PA thrombolysis will improve outcome by enhancing fibrinolysis and preventing re-occlusion.ARTIS is a randomised multi-center controlled trial with blind endpoint assessment. Our objective is to investigate whether immediate addition of aspirin to rt-PA thrombolysis improves functional outcome in ischemic stroke. Patients with acute ischemic stroke eligible for rt-PA thrombolysis are randomised to receive 300 mg aspirin within 1.5 hours after start of thrombolysis or standard care, consisting of antiplatelet therapy after 24 hours. Primary outcome is poor functional health at 3 months follow-up .This is the first clinical trial investigating the combination of rt-PA and acute aspirin by means of a simple and cheap adjustment of current antiplatelet regimen. We expect the net benefit of improved functional outcome will overcome the possible slightly increased risk of intracerebral haemorrhage.The Netherlands National Trial Register NTR822. The condensed rationale of the ARTIS-Trial has already been published in Cerebrovascular Diseases. Stroke is an important cause of death and acquired disability in industrial world. In the large majority of ischemic strokes, cerebral arteries become occluded either by a cardiac embolus or by thrombus formation in atherosclerotic vessel walls. The process of thrombus formation is initiated by two separate but interacting mechanisms: fibrin formation and platelet activation. Current standard treatment in acute ischemic stroke with intravenous recombinant tissue plasminogen activator (rt-PA) aims at breaking down the fibrin cloth in order to restore recanalisation of the occluded artery. Rt-PA treatment results in an absolute 10% reduction of poor outcome compared to placebo , while tWithin this current time window, early recanalisation is strongly associated with improved functional outcomes and reduced mortality ,5. UnforRe-occlusion after initial recanalisation is probably initiated by increased platelet activation. Thrombolytic therapy strongly activates the coagulation cascade leading to thrombin formation, a potent platelet activator . This haAmong all antiplatelet drugs, aspirin  is the most widely used drug. Inhibition of platelet aggregation by aspirin is caused by the irreversible acetylation of cyclo-oxygenase 1 and inhibition of prostaglandin thromboxane A2. Aspirin has a rapid onset of action resulting in substantial elimination of activated platelets.In myocardial infarction, large clinical trials have shown that adding aspirin to thrombolysis prevents re-occlusion thereby improving outcome considerably. The results of the second International Study of Infarct Survival Trial (ISIS-2) showed that mortality was reduced by 42% if patients were treated with streptokinase in combination with aspirin while mortality was reduced by only 25% if patients were treated with streptokinase alone . AspirinIn acute ischemic stroke, the Multicenter Acute Stroke Trial - Italy (MAST-I) duplicated the design of the ISIS 2 and showed an absolute risk reduction of 14% for disability in patients treated with the combination of streptokinase and aspirin as compared streptokinase alone. This overall net benefit overcame an excess mortality rate which was observed in the combination group. Symptomatic intracerebral haemorrhages (SICH) largely contributed to this increased mortality rate . Meta-anThe addition of aspirin to rt-PA, which is a fibrin-selective thrombolytic agent, has never been investigated prospectively. In the protocol of the Neurological Institute of Neurological Disorders and Stroke (NINDS) Trial the use of antiplatelet agents was postponed for 24 hours after rt-PA treatment to prevent possible bleeding complications . HoweverSubgroup analysis of the NINDS-trial reveals that patients with prior aspirin use had a better outcome, with lower frequencies of clinical deterioration and the same SICH rate as compared to patients without previous aspirin use . RegardiThe Antiplatelet therapy in combination with Recombinant t-PA Thrombolys in Ischemic Stroke (ARTIS) Trial is a multi-center, prospective open, randomised controlled trial with blind endpoint assessment (PROBE-design). We compare direct addition of 300 mg aspirin to intravenous rt-PA thrombolysis for ischemic stroke versus standard thrombolysis care, in which antiplatelet drugs are usually delayed by 24 hours after rt-PA. All participating centres are experienced in thrombolytic treatment for acute stroke.The primary objective of the ARTIS-Trial is to investigate whether the addition of asprin to standard rt-PA thrombolysis reduces poor outcome in acute ischemic stroke. Poor outcome is defined as death or dependency assessed by the modified Rankin Scale  at 3 months follow-up.The study population are acute ischemic stroke patients who present at participating centres and are treated with intravenous (IV) thrombolysis with rt-PA. Patients aged 18 years or older can be enrolled. Patients will be asked for written informed consent. The trial itself has no other firm exclusion criteria than those established by the judgment of the individual treating physician using local protocols for IV rt-PA treatment. When the patient has a diminished decision-making capacity as result of the stroke (e.g. aphasia), informed consent will be obtained from a representative of the patient. Exclusion of these patients would lead to a selective patient sample. Patients are also excluded if they have:▪ known antiplatelet therapy in the previous 5 days (in case of uncertainty the patient may be included);▪ known thrombocytopenia or thrombocyte count < 100 * 10E9/l;▪ known contra-indications to acetylsalicylic acid treatment;▪ known anticogualant therapy in the previous 5 days;▪ known legal incompetence of the patient prior to this stroke.Randomisation will be performed per participating centre to ensure a equal distribution of patients between both group. The randomisation procedure will be computer- and web based, using permuted blocks. Randomisation will be stratified by centre, age , gender and the time between symptom onset and time of rt-PA bolus .®) as lysine salt intravenous as bolus. Since there is a peak in platelet activation after 2 hours after initiation of rt-PA thrombolysis [In order to prevent delay of start of thrombolytic treatment, informed consent and randomisation procedures will be performed as soon as continuous infusion of rt-PA (0.9 mg/kg) has started after bolus administration 10%) Patients allocated to the active group will receive 300 mg aspirin . Thrombocyte aggregation is irreversibly reduced by this calcium-ureum-salt, causing longer coagulations times. Aspirin use may lead to gastro-intestinal reactions. However, due to the single use adverse reactions caused by the trial medication are expected to be limited.Aspirin intravenous is registered in the Netherlands as Aspégic® (Boehringer Ingelheim GmbH) is essential and important co-medication in the ARTIS-Trial. Interaction of Aspégic® with rt-PA is unknown, although rt-PA treatment might increase the risk of intracerebral bleeding in aspirin pre-treated stroke patients. The characteristics of rt-PA may therefore influence our results even though rt-PA itself is not under investigation.AlteplasePatients will receive rt-PA treatment in both groups according to local protocols at participating centres. Recommendations of rt-PA treatment concerning hypertension and thrombocyte count are based on standard international guidelines . Blood pAll medication used before the stroke may be continued, except anticoagulance. Standard secondary prophylaxis is recommended according to the following scheme:▪ carbasalate calcium 300 mg - once/daily - 24 hours after rt-PA for 14 days▪ carbasalate calcium 100 mg - once/daily - 14 days after rt-PA▪ simvastatine 40 mg - once/daily - 0-24 hours after rt-PA▪ dipyridamole 200 mg - twice/daily - 24 hours after rt-PAAdditional anti-diabetic or antihypertensive medication may be started as regarded appropriate by the treating physician.The primary endpoint is poor functional health at 3 months defined as dependency or death (mRS 3 - 6).The secondary objectives are:▪ complications within 48 hours after randomisation including the occurrence of SICH and serious systemic bleeding. SICH is defined as CT-documented haemorrhage and a clinical deterioration leading to 4 or more points increase on the National Institute of Health Stroke Scale (NIHSS) as compared to the best score on the NIHSS since admission. Serious systemic bleeding is defined as a potentially life threatening bleeding which requires immediate medical intervention;▪ neurological symptoms quantified by the NIHSS 7 - 10 days after randomisation or at discharge if the patient is discharged within 7 days;▪ survival at 3 months;▪ disability at 3 months assessed by the AMC Linear Disablility Scale;▪ functional health at 3 months non-dichotomized ;▪ causes of poor outcome.At baseline following patient characteristics are collected at each participating site: age, sex, ethnicity, medical history, pre-stroke medication, pre-stroke mRS, blood pressure, Glasgow Coma Scale (GCS), National Institutes of Health Stroke Scale (NIHSS), time of symptom onset, rt-PA bolus and (if applicable) trial medication, thrombocyte count and coagulation-International Normalized Ratio. Baseline CT-scans will be collected from participating centres and assessed blindly centrally at the coordinating centre for dens media sign, early ischemic changes and degree of leukoariosis by an independent blinded neuro-radiologist.At follow-up, neurological deficits are assessed by the NIHSS at 7-10 days or at discharge, if this is before 7 days. Clinical deterioration, defined as a 4 or more points increase on the NIHSS, will be followed by CT-scan and registration as (serious) adverse events including possible cause by each participating site. This CT-scan will be assessed at the coordinating centre as well.Primary outcome will be assessed by a blind research nurse from the clinical trial office of the coordinating centre, who will score the mRS by telephone using a structured interview. To increase the inter-observer reliability the number of research nurses will be limited to a maximum of three. Disability will be assessed by the same research nurse during the same telephone interview using the Amsterdam Linear Disability Scale . See AddIn patients with a poor outcome at three months, the Data Collection Committee composed of the investigators of the coordinating centre and the local investigator, judges whether this poor outcome is attributed to the initial ischemic stroke, reported adverse event or other causes.All adverse event reported by the subjects or observed by the treating physicians will be recorded. In case of serious adverse events (SAE), the principal investigator will be notified by email or telephone within 24 hours. The principal investigator subsequently reports SAE to the Data Safety Monitoring Board (DSMB). This is an independent committee of trial experts, who will focus on both safety monitoring and analysis of effectiveness on unblinded data. The DSMB will perform ongoing safety surveillances, especially with regard to the occurrence of serious adverse events in terms of SICH and serious systemic bleeding within 48 hours. The DSMB can recommend the Steering Committee of the ARTIS-Trial to terminate the trial when there is clear and substantial evidence of harm. All SAE will be reported to the central METC according to their requirements as well.Based on our own experience in the stroke unit cohort and the results of the rt-PA thrombolysis trials  and the 2 test with a 0,05 two-sided significance level will have 80% power to detect the difference between the control group proportion of 0,50 and an experimental group proportion of 0,40  when the sample size in each group is 400 . With this sample size, a two-sided 95% confidence interval for the difference between the proportions will extend 0,069 from the observed difference in proportions. With this sample size we are also able to statistically detect a minimal effect size (difference between mean scores of both treatment arms divided by the SD of the control group ) of d = 0,20 as benchmark for assessing the relative magnitude of score differences on the continuous AMC Linear Disability Scale (ALDS) which is a secondary outcome parameter.A two group Xt-test, the Mann-Whitney test, linear regression and ordinal logistic regression, when appropriate. The remaining secondary outcomes will be analyzed using simple 2 × 2 tables and logistic regression. In all analyses, statistical uncertainty will be quantified via 95% confidence intervals.Baseline characteristics will be summarized using descriptive statistics. The main analysis of this trial consists of a single comparison between the trial medication groups of the primary outcome after three months (dichotomized Rankin score). The analysis will be based on the intention-to-treat principle. The effect size will be expressed in a relative risk (RR) estimates and absolute risk reduction (ARR). Additionally the primary outcome will be analyzed using multivariate logistic regression, adjusting (if necessary) for clinically relevant baseline imbalances. The differences between NIHSS, ALDS scores and non-dichotomized mRS will be analyzed using the two-group Besides interim analyses on the safety data the DSMB will also perform an unblinded interim analysis on the primary outcome to assess the strength of the efficacy data when half of the patients are enrolled. The DSMB will also check the assumptions for sample size calculations. The analysis will be performed by an independent statistician of the Academic Medical Centre Clinical Research Unit, who is not involved in managing the trial. The DSMB can recommend the Steering Committee of the ARTIS-Trial to▪ adjust the sample size;▪ early terminate the study when there is clear and substantial evidence of benefit;▪ early terminate the study in case the data suggests no benefit or in case accrual rates are too low to provide adequate statistical power for identifying the primary endpoint.With respect to the primary outcome a predefined subgroup-analyses will be performed:▪ rt-PA treatment < 2 hours versus 2-3 hours versus > 3 hours from symptom onset Effectiveness of IV thrombolysis declines over time from symptom onset probably caused by an increase in clot stability. Regarding clot dissolution and reocclusion the beneficial effect of adding antiplatelet therapy might therefore be different over time. The risk of bleeding can also change over time [▪ trial medication within 1 hour versus between 1-1.5 hours from rt-PA bolus. Reocclusion occurs at a median time of 65 minutes after start of rt-PA treatment. Administration of Aspegic in the first hour after the start of rt-PA treatment is therefore expected to result in better outcome [▪ based on ethnicity differences: whites versus blacks, whites versus Hindu's, whites versus blacks and Hindu's, Hindu's versus the other ethnic groups. Previous studies on thrombolytic therapy in acute myocardial infarction suggest that racial differences do exist with an increased thrombolytic effect in blacks accompanied by an increased risk of bleeding complications. The beneficial or detrimental effect of the addition of antiplatelet therapy to IV rt-Pa might therefore differ among different ethnic groups -26.Subgroup analyses consist will consist of a simple comparison of these different groups on primary and secondary outcome measures.The ARTIS study will be conducted according to the principles of the Declaration of Helsinki (version of 2004) and in accordance with the Medical Research Involving Human Subjects Act (WMO) and other guidelines, regulations and Acts. The Medical Ethics Committee of the Academic Medical Centre approved the protocol before start of the trial. Data management, monitoring and reporting of the study will be performed in accordance with the ICH GCP guidelines. Approval by the local medical ethical review board is required for each participating centre before start of inclusion.The AMC Medical Research BV has insurance, which is in accordance with the legal requirements in The Netherlands . This insurance provides cover for damage to research subjects through injury or death caused by the trial.The trial results will be published by the coordinating investigator on behalf of the ARTIS-study group. Members of the ARTIS-study group will then be listed at the end of the article.We present the protocol of a randomised controlled clinical trial to investigate the efficacy of direct addition of 300 mg aspirin to rt-PA thrombolysis in acute ischemic stroke. In accordance with thrombolysis in myocardial infarction, in which the combination of acute aspirin and thrombolysis improves outcome considerably , we hypowithin 24 hours after rt-PA thrombolysis in the first European Cooperative Acute Stroke Study (ECASS-I) showed a slight trend towards increased mortality from all causes (including SICH). This risk is now explained by the higher rt-PA dose (1.1 mg/kg) used in this trial since there was no increased risk in ECASS-II where the currently standard dosage of 0.9 mg/kg rt-PA was used [A major safety concern in this trial refers to the occurrence of symptomatic intracranial haemorrhage (SICH). Subgroup-analysis of patients receiving APT -PA dose .1 mg/kg Several cohort studies could not find a significant association between pre-treatment with antiplatelet agents and SICH -30. OtheARTIS is a randomised controlled trial investigating the efficacy of the acute addition of aspirin to intravenous rt-PA thrombolysis in patients with acute ischemic stroke. ARTIS will answer a highly relevant question in acute stroke care by means of a simple adjustment of current antiplatelet regimen with regard to rt-PA thrombolysis. A condensed version of the protocol has been published in Cerebrovascular Diseases .th 2010, 361 of the 800 patients have been included so far. This trial is set up in the Netherlands. However, other centres - also from foreign countries - experienced in thrombolysis are invited to participate as well. The principle investigator can be contacted by e-mail.The ARTIS-Trial has started at the end of 2008. Thirty-seven centres are actively randomizing patients. As of May 10APT: antiplatelet therapy; ASA: acetylsalicylic acid; ICH: intracranial haemorrhage; mRS: modified Rankin Scale; NIHSS: National Institute of Health Stroke Scale; rt-PA: recombinant tissue plasminogen activator, SICH: symptomatic intracranial haemorrhage.The authors declare that they have no competing interests.SMZ wrote all drafts and final manuscript, is concerned with patient recruitment and data management. YBR is the principal investigator and conceived the study, designed the protocol, applied for financial support and commented on all drafts and final manuscript. RV, JS and RJH helped conceive the study and design the protocol and commented on final manuscript.All authors read and approved the final manuscript.Data collection forms.Click here for file


In a case-control study in northeastern Italy hepatitis C virus infection seemed to increase by about 50-fold the risk of non-Hodgkin's lymphoma involving the liver and major salivary glands (i.e. larger than that for hepatocellular carcinoma) and by about fourfold the risk of lymphomas at other sites.


A micro-cytotoxicity assay was adapted for the detection of cell-dependent antibodies (CDA). Using normal rat spleens as the source of effector cells such CDA activity was readily demonstrable in allo-immune sera tested on cultured sarcoma cells. The same technique was then used to examine for tumour specific antibodies in the sera of Hooded rats bearing a “non-immunogenic” syngeneic metastasizing sarcoma. During the early stages of tumour growth, at Days 7 and 14, tumour specific CDA cytotoxicity was detectable at high titres. By Day 21, however, this activity had completely disappeared from the serum. This cell-dependent cytotoxicity was tumour specific in that it did not kill cells from an unrelated syngeneic sarcoma, and the activity was probably confined to immunoglobulin G as detected by molecular weight separation techniques. Following tumour amputation at Day 21, this type of specific antibody activity rapidly re-appeared in the serum. The presence of tumour specific CDA showed an inverse correlation with the presence of specific inhibitors of cell-mediated immunity in the same sera. At no stage in tumour growth could complement-dependent cytotoxicity be detected in tumour bearing rat sera.It is concluded that cell-dependent cytotoxic activity is not associated with conventional complement dependence, that this CDA type of assay is exquisitely sensitive and is suitable for the detection of anti-TSTA antibodies in tumour bearing rats.The possible significance of CDA activity in syngeneic tumour immunity is discussed briefly. The results suggest that the role of humoral immune mechanisms in host resistance to tumour growth needs re-appraisal.


To evaluate the use of unbiased computer-assisted lateralization of temporal lobe epilepsy (TLE) by z-score parametric PET imaging (ZPET).38 patients with histologically proven unilateral TLE due to pure hippocampal sclerosis, referred for pre-surgical PET evaluation of intractable seizure over a 5-year period, were included. The F-18 FDG images were oriented along temporal long axis and then transformed into ZPET images on a voxel by voxel basis. Multiple regions of interests  were placed on cortical, subcortical and cerebellar structures on twenty-eight out of 38 patients with totally seizure-free (class I) outcome. Paired t-tests with Bonferroni correction were used to determine the location of the most asymmetric regions as variables for subsequent discriminant analysis of the entire group of the patients.The computer program identified the anterior half of the temporal lobe (p < 0.0005) and thalami (p = 0.021) as the most asymmetric regions in TLE patients with Class I outcome. Discriminant analysis using z-scores from a total of 8 ROIs (in 4 pairs) on these structures correctly lateralized thirty-seven out of 38 (97%) patients . The only false localization came from a patient with equivocal z-scores on the temporal lobes and this patient turned out to have poor outcome.The computer-assisted lateralization of TLE using ZPET provides an accurate, fast and objective way of seizure evaluation. The interictal state of temporal lobe epilepsy (TLE) is characterized by decreased uptake due to hypometabolism on F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) images. Spanaki et al. suggested that seizures might have a reversible effect on brain areas connected with, but remote from, the epileptogenic cortex . Choi etRoutine interpretation of PET images to evaluate hypometabolism involves visual assessment of images normalized to the region of maximal uptake. Color scales or continuous gray scales are used to determine qualitative differences in metabolism. Unfortunately, besides human observer variability, factors such as medications and external stimuli yield variability in glucose metabolism, which affects the maximal pixel count between patients and temporally within the same patient. Traditional analysis also involves comparison of homologous regions, which implicitly assumes a relatively normal contralateral side. Wong et al  proposedThe average intra-subject coefficient of correlation between the individual normal subjects' right and left hemispheric metabolism has been shown to be 80% . The aveThe purpose of this study is to retrospectively evaluate the use of totally unbiased automatic computerized determination of the lateralization and the extent of epileptic foci utilizing the z-map parametric statistical imaging.Interictal PET images from 38 patients with surgically proven unilateral temporal lobe epilepsy due to pure hippocampal sclerosis referred for pre-surgical PET evaluation of intractable seizure over a 5-year period at mean age of 31 were analyzed. All patients had extensive clinical, EEG, neuro-psychologic and MRI evaluation. In 18 patients, the seizure focus was found on the left temporal lobe. The other 20 patients had right side temporal focus. Twenty-eight of the 38 patients had seizure free (Engel Class I ) outcome-1. The images were re-oriented along the temporal axis and zoomed to a final pixel size and slice thickness of 3.4 mm.All patients received an average dosage of 10 mCi (370 MBq) of F-18 FDG in a fasting state. EEG monitoring was performed shortly before the tracer injection and during the uptake period of the F-18 FDG. PET image acquisition was begun about 45 minutes after injection. The PET camera was a Posicam 6.5  and data were acquired in a 256 × 256 matrix with a pixel size of 1.7 mm, using a uniform attenuation coefficient of 0.096 cmA voxel by voxel transformation of the PET image into a z-score parametric PET image was performed using PV-WAVE ADVANTAGE  using the methods described below. The mean and standard deviation of pixels whose counts were greater than 50% of the maximal count were calculated. Then the whole volume of brain image was transformed into z-scores on a pixel by pixel basis using the formula :where μ and σ are the respective mean and standard deviation of the pixel counts. The 50% of maximum count was chosen to exclude ventricles and white matter from being involved in the calculations. Since at least 20,000 pixels are used in the calculations, the pixel distribution is essentially Gaussian and a small percent of outlier values due to seizure focus will not significantly affect the results .Multiple regions of interest (ROIs) were then placed on the z-map images guided by an automatic sectorial gird generated by computer . A totalThe asymmetry in FDG metabolism was evaluated by comparing differences between homologous regions in the subset of patients  with totally seizure free (class I) outcome. Paired t-tests with Bonferroni correction were performed to determine the location and hence the extent of the most asymmetric regions on metabolism Figs.  to 5. DiAll ZPET images analyzed were truly interictal based on the EEG and clinical monitoring of seizure activity during the uptake phase of F-18 FDG before PET imaging. The computer program identified the anterior half of the temporal lobe (p < 0.0005) and thalami p = 0.021) as the most asymmetric regions in TLE patients with Class I outcome. Discriminant analysis using z-scores from the most asymmetric regions correctly lateralized thirty-seven out of the 38 (97%) of patients . The only false localization came from a patient with equivocal z-scores on the temporal lobes and this patient had poor outcome. The results suggested also extension of functional abnormality outside the temporal lobe into the ipsilateral thalamic, frontal and parietal areas  and clinical and neuropsychological evaluation converged to the same conclusion before surgery was performed. Thus the numbers with poor surgical outcome might be small or the analysis might be biased towards good prognostic implications. Further studies on both surgical and non-surgical candidates using long term follow up are required to further address the true prognostic implications of the location, extent and severity of hypometabolism measured by PET.It was interesting to note the computer findings in current study about the involvement of ipsilateral thalamus in TLE. It might be due to sensory deafferentation from the severity or chronic nature of TLE. As Choi et al  had poinStatistical parametric mapping removes inter-observer variations as shown by Vinton et al  and WongAs the effects of epileptic focus are usually contiguous  and the main focus of the study is to evaluate to the extent of functional abnormality from pure temporal lobe epileptic focus, the traditional ROI sampling guided by automatic individualized grid used in the current study will be a sufficient sampling method for detecting the extent of the abnormality. The more precise method of statistical probabilistic anatomical mapping (SPAM) would be useful if one want to know the exact focus of activation or abnormality, which was first used in MRI  and receThe results show significant metabolic differences between homologous brain regions determined by computer assisted z-score lateralization that accurately identify regions and the extent of hypometabolism associated with temporal lobe seizures. The computer-assisted lateralization of TLE using ZPET provides an accurate, fast and objective way of seizure focus and extent evaluation.EEG, Electroencephalography; FDG, F-18 fluorodeoxyglucose; MRI, Magnetic resonance imaging; PET, Positron emission tomography; ROI, Region of interest;TLE, Temporal lobe epilepsy; SPAM, Statistical probabilistic anatomical mapping;SPECT, Single photon emission computed tomography; ZPET, Z-score parametric PET imaging.The author(s) declare that they have no competing interests.CYOW conceived of the study, participated in its design and coordination, performed computer programming and statistical analysis and involved in manuscript writing. JG and JB carried out data collection and literature search and involved in manuscript writing. COW helped in data collection, performed literature search and prepared illustrations. GBS participated in data analysis, study coordination and manuscript writing. All authors read and approached the final manuscript.The pre-publication history for this paper can be accessed here:


The oxidation of fatty acids in mitochondria plays an important role in energy metabolism and genetic disorders of this pathway may cause metabolic diseases. Enzyme deficiencies can block the metabolism at defined reactions in the mitochondrion and lead to accumulation of specific substrates causing severe clinical manifestations. Ten of the disorders directly affecting mitochondrial fatty acid oxidation have been well-defined, implicating episodic hypoketotic hypoglycemia provoked by catabolic stress, multiple organ failure, muscle weakness, or hypertrophic cardiomyopathy. Additionally, syndromes of severe maternal illness (HELLP syndrome and AFLP) have been associated with pregnancies carrying a fetus affected by fatty acid oxidation deficiencies. However, little is known about fatty acids kinetics, especially during fasting or exercise when the demand for fatty acid oxidation is increased (catabolic stress).A computational kinetic network of 64 reactions with 91 compounds and 301 parameters was constructed to study dynamic properties of mitochondrial fatty acid β-oxidation. Various deficiencies of acyl-CoA dehydrogenase were simulated and verified with measured concentrations of indicative metabolites of screened newborns in Middle Europe and South Australia. The simulated accumulation of specific acyl-CoAs according to the investigated enzyme deficiencies are in agreement with experimental data and findings in literature. Investigation of the dynamic properties of the fatty acid β-oxidation reveals that the formation of acetyl-CoA – substrate for energy production – is highly impaired within the first hours of fasting corresponding to the rapid progress to coma within 1–2 hours. LCAD deficiency exhibits the highest accumulation of fatty acids along with marked increase of these substrates during catabolic stress and the lowest production rate of acetyl-CoA. These findings might confirm gestational loss to be the explanation that no human cases of LCAD deficiency have been described.In summary, this work provides a detailed kinetic model of mitochondrial metabolism with specific focus on fatty acid β-oxidation to simulate and predict the dynamic response of that metabolic network in the context of human disease. Our findings offer insight into the disease process (e.g. rapid progress to coma) and might confirm new explanations (no human cases of LCAD deficiency), which can hardly be obtained from experimental data alone. The primary sources of fatty acids for oxidation are dietary and mobilization of triacylglycerols mainly stored in adipocytes of adipose tissue. The release of metabolic energy, in the form of fatty acids, is controlled by a complex series of interrelated cascades that result in the activation of hormone-sensitive lipase, which hydrolyzes fatty acids from triacylglycerols and diacylglycerols. The final fatty acid is released from monoacylglycerols through the action of monoacylglycerol lipase, an enzyme active in the absence of hormonal stimulation. Once released, these fatty acids travel through the blood to other tissues such as muscle where they are oxidized to provide energy through the mitochondrial β-oxidation pathway. The β-oxidation spiral of fatty acid metabolism involves these four steps: oxidation, hydration, a second oxidation, and finally thiolysis. These occur in repeating cycles through the sequential removal of 2 carbons and production of acetyl-CoA, which then enters the Krebs cycle for oxidation and ATP production Figure . AnotherMitochondrial fatty acid oxidation deficiencies are due to genetic defects in enzymes of fatty acid β-oxidation and transport proteins . Genetic defects have been identified in most of the genes where nearly all types of sequence variations (mutation types) have been associated with disease . In partTen of the disorders directly affecting mitochondrial fatty acid oxidation have been well-defined, implicating episodic hypoketotic hypoglycemia provoked by catabolic stress, multiple organ failure, muscle weakness, or hypertrophic cardiomyopathy. Additionally, syndromes of severe maternal illness (HELLP syndrome and AFLP) have been associated with pregnancies carrying a fetus affected by fatty acid oxidation deficiencies. The incidence of one of these disorders, MCAD deficiency (MIM 201450), is 1:14 600 in almost 8.2 million newborns worldwide .The introduction of tandem mass spectrometry (MS/MS) for the analysis of plasma acylcarnitines has greatly facilitated the identification of patients with a defect in fatty acid β-oxidation and has unquestionably been the most striking recent advance in newborn screening. Pre-symptomatic diagnosis is important to prevent morbidity as most of the diagnosed defects are treatable and the prognosis is generally favorable -12.Besides statistical model building and data mining based approaches -15, compTo date, little is known about fatty acids kinetics, especially during catabolic stress or exercise when the demand for fatty acid oxidation is increased. Here we introduce a detailed kinetic model of mitochondrial metabolism with specific focus on fatty acid β-oxidation to simulate and predict the dynamic response of that metabolic network toward distinct enzyme deficiencies. The simulation results are compared and validated using experimental data. Finally, the dynamic response to changes in the input to the system representing catabolic stress is simulated and results are interpreted in a biological and clinical context, followed by a discussion on limitations of the model.The major objectives of this study were to construct a dynamic simulation environment allowing the exploration of complex biochemical processes involved in fatty acid β-oxidation, the validation of the model with experimental data, and finally the application of these pathway models to the analysis of metabolic diseases. We therefore built a deterministic model describing the biochemical reactions and pathway in the form of kinetic rate equations, and investigated the dynamic response of the system to specific perturbations of enzyme activities. Based on a publicly available computational model for mitochondrial metabolism (see the methods section) and previously described enzymatic activity distributions Figure , we wereThe reduced activity of acyl-CoA dehydrogenases leads to a deviation of the total activity as compared to healthy controls Figure  and, subIsotope-dilution MS/MS on plasma or whole blood facilitates the measurements of acylcarnitines and the diagnosis of newborns with a defect in fatty acid β-oxidation ,26. The In order to compare the data from the screening programs to accumulating fatty acyl-CoA concentrations for the simulated enzyme deficiencies, we calculated relative concentrations with respect to the simulated control group (Controls) and healthy control from the screening programs, respectively. We did not directly compare acyl-CoA in the mitochondria with measured acylcarnitine concentrations outside the mitochondria. Based on diagnostic findings and conclusions in newborn screening programs we assumed similar relative behavior of acylcarnitines and acyl-CoAs. Simulation revealed that LCAD deficiency showed the largest effect of acyl-CoA accumulation (179-fold), followed by MCAD deficiency (53-fold) corresponding well with experimentally derived MCAD deficiency data (51-fold) of the Middle Europe data set Figure . A less We additionally provide acylcarnitine ratios with respect to C4 concentration on experimentally derived data of newborn screening programs in Middle Europe and South Australia as well as simulation data with different C16 input  in Table Clinical manifestation of MCAD deficiency usually starts after significant catabolic stress. When carbohydrate stores are depleted, the organism switches to energy production from stored triacylglycerols, which results in lipolysis and release of fatty acids. In MCAD deficiency a dramatic rise of plasma levels of specific free fatty acids is observable, indicating impaired β-oxidation of respective chain-length acyl-CoA. Additionally, ketones remain inappropriately low, reflecting the defect in hepatic fatty acid oxidation. Hypoglycemia develops shortly thereafter, probably because of excessive glucose utilization due to the inability to switch to fat as a fuel . Thus, wNext we studied the repeating cycles of the β-oxidation spiral of fatty acid metabolism that sequentially removes 2 carbons. After the palmitoyl-CoA (C16) concentration was increased by about 20%, acyl-CoA concentrations reached a steady-state after 6 hours in healthy controls Figure . The depAcetyl-CoA – the final product of the β-oxidation spiral – is required for the production of energy and ketone bodies, especially during periods of fasting. Deficiencies of acyl-CoA dehydrogenases resulted in reduced production of acetyl-CoA. Following a 20% increase of palmitoyl-CoA (C16) concentration, the highest shortage of 30 days acetyl-CoA production was found in LCAD deficiency. The acetyl-CoA generated by the β-oxidation within 30 days was reduced by 6.3% compared to acetyl-CoA generated in the healthy situation. Figure The present computer simulation attempts to contribute to a better understanding (explanation) of pathophysiological aspects of a group of hereditary disorders impairing mitochondrial β-oxidation. Several enzymes are involved in mitochondrial fatty acid oxidation. For all of them, genetic defects impairing their function have been described. The availability of modern analytical methods has facilitated newborn screening for these disorders. For the identification of patients with a defect in β-oxidation, acylcarnitines in blood were evaluated showing a characteristic profile depending on the affected enzyme even in asymptomatic stage. We simulated the steady-state concentrations of acyl-CoAs in acyl-CoA dehydrogenase deficiencies and compared the results to acylcarnitine data from screening programs in Middle Europe and South Australia. Results indicate that the overall characteristics of the simulated accumulation of acyl-CoA show good agreement with experimental data and findings in the literature Figure .Differences in simulated C16 acyl-CoA and measured C16 acylcarnitine might be caused by the special biological role of C16 acylcarnitine. From a biochemical perspective it is not clearly evident why a decrease of acyl-CoA dehydrogenase activity Figure  would noDifferences in simulated acyl-CoA ratios and measured acylcarnitine ratios shown in Table The clinical phenotypes of most of the disorders are very similar. As MCAD deficiency is the most prevalent defect among them we focused our discussion and biochemical interpretation on this particular defect. Nevertheless, the aspects are relevant to the pathogenesis of all fatty acid oxidation defects. Patients with MCAD deficiency are without clinical manifestations until a prolonged fasting period sometimes in combination with infection or fever. As a physiological response to this catabolic stress triacylglycerols from adipose tissue are released and energy production switches from carbohydrate to lipid utilization. In healthy individuals, subsequently ketone body formation by the liver is increased to provide this metabolic fuel for brain and muscles.The response to catabolic stress in patients with MCAD deficiency shows a marked increase in plasma fatty acids, mitochondrial acylcarnitines and acyl-CoAs. Severe symptoms of lethargy and nausea develop as a consequence of encephalopathy, and patients can become dangerously ill, sometimes before plasma glucose falls to hypoglycemic levels. The progression to severe sickness proceeds within a few hours. Patients often die in the course of the first episode or at least suffer from persistent brain damage. The underlying pathogenetic mechanisms have been poorly understood until now. To simulate the response to fasting we assumed a 20% increase of palmitoyl CoA (C16) and observed significant differences of the calculated metabolic changes in acyl-CoA deficiencies with respect to the healthy controls Figure . One majThe simulated accumulation of specific acyl-CoAs according to the investigated enzyme deficiencies are in agreement with the accumulation of plasma free fatty acid intermediates, which enter the central nervous system and exert toxic effects, which may explain the observed encephalopathy and cerebral edema. In vitro experiments on cerebral cortex of rats indicate that inhibition of energy metabolism and oxidative stress induction by the accumulating fatty acids may contribute to the pathophysiology of encephalopathy ,31.Although several patients have been found to have VLCAD deficiency, none have been documented with LCAD deficiency . This coOur model can be extended to comprehensively test and study deficiencies of mitochondrial trifunctional protein and β-hydroxy-acyl-CoA dehydrogenase, or other diseases of fatty acid oxidation such as carnitine cycle, electron transfer and ketone synthesis defects. Furthermore, differences in the expression level of the enzymes in different cells and tissues and their consequences on the dynamical behavior of the β-oxidation can be investigated. Future work will incorporate the enzymatic steps for unsaturated fatty acids.In summary, this work provides a stimulating example for Systems Biology in the context of human disease revealing insights into dynamic properties of complex biochemical networks under the constraints of various disease conditions. As analytical technologies for global and targeted measurements mature, especially with regards to metabolites, new findings and hypothesis can be verified utilizing quantitative data. Furthermore, while mitochondrial deficiencies are often treated with metabolites to stimulate the enzyme activities, models will allow evaluation of the influences of metabolite treatments at the mitochondrial level, visualization of the dynamic behavior of the pathway and exploration of a hypothetical rationale of the treatment. In this respect, computational biology proves to be able to uncover insights, which can hardly be obtained from experimental data alone.. This work offered a perfect starting point to construct a kinetic model for fatty acid metabolism with particular focus on the β-oxidation cycle and the objective to enhance our understanding of various deficiencies of acyl-CoA dehydrogenases.The computational model for mitochondrial β-oxidation was based on a publicly available E-Cell2  simulatiIn order to strike a balance between simplicity and complexity of our simulation model, we extracted the β-oxidation part of the mitochondrial model (reducing complexity), modified and completed it with respect to simulating mitochondrial fatty acid oxidation deficiencies. The β-oxidation cycle together with the metabolite transporting system originally comprised 8 different enzymatic reactions with 5 different reaction mechanisms. The modifications of the original model comprise three central parts that are essential for simulating mitochondrial fatty acid oxidation deficiencies: (i) extension to the oxidation of stearoyl-CoA (C18), arachidonoyl-CoA (C20) and behenoyl-CoA (C22) (ii) input of short, medium and long chain fatty acids entering the β-oxidation cycle (iii) modeling of acyl-CoA dehydrogenase using four enzymes classified by their fatty acid chain length specificity.There is no evidence that the first three steps of the β-oxidation cycle of stearoyl-CoA (C18), arachidonoyl-CoA (C20) and behenoyl-CoA (C22) are different from the steps of the other acyl-CoAs. We used the same parameters for stearoyl-CoA (C18), arachidonoyl-CoA (C20) and behenoyl-CoA (C22) as well as for the other acyl-CoAs, and explored only the parameters of the oxoacyl-CoA thiolase – the fourth step of the β-oxidation cycle – in dependence on the carbon chain length. However, simulations revealed that the variation of these parameters with regard to carbon chain length had no impact on acyl-CoA concentrations (data are not shown). Therefore, we set the parameters to the same values as for palmitoyl-CoA (C16).Short and medium chain fatty acids (C4 – C12) were assumed to enter the β-oxidation cycle directly, whereas long and very-long chain fatty acids (C14–C22) were entering the cycle via the carnitine transporting system. The input of short, medium and long chain fatty acids entering the β-oxidation cycle depends on the concentration of the fatty acids in the inter-membrane space. A higher input results in a higher output – the production of acetyl-CoA – as can be seen in Figure The modeling of acyl-CoA dehydrogenase using four enzymes classified by their fatty acid chain length specificity is based on experimental findings shown by the distribution of the enzyme activity in Figure We treated the mitochondrial matrix and intermembrane-space free carnitines as well as the intermembrane-space free CoA and acylCoA as fixed parameters in order to guarantee constant input of acylCoA to the beta-oxidation cycle. We did not get any enlarged acylcarnitine pools in all our calculations. If we set the mitochondrial matrix and intermembrane-space free carnitines variable, very small changes of mitochondrial matrix and intermembrane-space free carnitines were found resulting in a little bit smaller flux of acylCoA entering the beta-oxidation cycle. These very small changes did not impact on our results (data not shown). The level of matrix CoA was set high and variation of multiple magnitudes of that level did not impact on our results (data not shown)., and modify this model of β-oxidation at different disease states.The final model consisted of 64 reactions with 91 compounds  and 301 parameters. The entire model at different simulation states in SBML format can be found in the Additional files. The users can directly open the SBML files in COPASI  or use oStarting with initial concentrations, steady-state values were calculated under acyl-CoA dehydrogenase activity conditions of healthy controls. Subsequently, that steady-state of the model was the starting point to calculate steady-state values under mitochondrial fatty acid oxidation deficiencies conditions. These calculations are summarized in simulation A. Results of simulation A were compared and validated with experimental data. Finally, the models obtained in simulation A were used in simulation B where dynamic responses with respect to catabolic stress were calculated.The initial metabolite concentrations  in the inter-membrane space by 20%, 30% and 40%. The dynamical behavior of a 20% increase for the control group as well as for the different acyl-CoA dehydrogenase deficiencies are shown in Figure  in the iThe three central modifications of the original model are based on findings from literature or from performed simulations.Overall validation was given by relating the calculated variables of the model (the metabolites of the β-oxidation cycle) to experimental available data from two new-born screening programs in Europe and Australia (simulation A). The overall characteristics of the simulated accumulation of acyl-CoA show good agreement with experimental data and findings in the literature Figure .A limitation of our work relates to the direct comparison of calculated and measured data. It has to be considered that experimental data of the (isolated) mitochondria are not available and that the available measured data are not only influenced by the respective subsystem (the beta-oxidation cycle), but also may reflect additional effects caused by other subsystems and disease conditions.Further limitations comprise that only saturated fatty acids were considered and that the β-oxidation of unsaturated fatty acids and odd-numbered chains of carbon were not modeled. Furthermore, we did not include carbon chain length dependencies of the activity of enoyl-CoA hydratase, β-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase.RMO participated in the design of the study, carried out the computational calculations and drafted the manuscript. IO participated in the design of the study and helped to draft the manuscript with respect to biochemical and medical issues. BT conceived of the study, and participated in its coordination. GS helped to draft the manuscript. KMW performed newborn screening data analysis and helped to draft the manuscript with respect to biochemical issues. AG participated in the design of the study, performed newborn screening data analysis and helped to draft the manuscript. All authors read and approved the final manuscript.Initial state healthy controls. Computational model of healthy controls at initial stateClick here for fileSteady state healthy controls. Computational model of healthy controls at steady stateClick here for fileSteady state SCAD deficiency. model of SCAD deficiency at steady stateClick here for fileSteady state MCAD deficiency. Computational model of MCAD deficiency at steady stateClick here for fileSteady state LCAD deficiency. Computational model of LCAD deficiency at steady stateClick here for fileSteady state VLCAD deficiency. Computational model of VLCAD deficiency at steady stateClick here for file


We find, through statistical analysis based on a novelstochastic population-dynamic T-cell model, that the carrying capacitycorresponding to TCR-specific resources is approximately 1000-fold larger thanthat of TCR-nonspecific resources, implying that the size of the peripheralT-cell pool at steady state is determined almost entirely by TCR-nonspecificmechanisms. Nevertheless, the diversity of the TCR repertoire depends cruciallyon TCR-specific regulation. The estimated strength of this TCR-specificregulation is sufficient to ensure rapid establishment of TCR repertoirediversity in the early phase of T cell population growth, and to maintain TCRrepertoire diversity in the face of substantial clonal expansion-inducedperturbation from the steady state.T cell populations are regulated both by signals specific to the T-cell receptor(TCR) and by signals and resources, such as cytokines and space, that actindependently of TCR specificity. Although it has been demonstrated thatdisruption of either of these pathways has a profound effect on T-celldevelopment, we do not yet have an understanding of the dynamical interactionsof these pathways in their joint shaping of the T cell repertoire. CompleteDiGeorge Anomaly is a developmental abnormality that results in the failure ofthe thymus to develop, absence of T cells, and profound immune deficiency. Afterreceiving thymic tissue grafts, patients suffering from DiGeorge anomaly developT cells derived from their own precursors but matured in the donor tissue. Wefollowed three DiGeorge patients after thymus transplantation to utilize theremarkable opportunity these subjects provide to elucidate human T-celldevelopmental regulation. Our goal is the determination of the respective rolesof TCR-specific vs. TCR-nonspecific regulatory signals in the growth of theseemerging T-cell populations. During the course of the study, we measuredperipheral blood T-cell concentrations, TCR de novo development of human T cell populations.We have developed a mathematical model to aid in the statistical analysis of theprecious data from these patients. In addition to helping elucidate the means bywhich the size and diversity of T cell populations are jointly regulated, theinsights gained from this study hold promise for the development of therapies topromote immune recovery after transplantation.Protective adaptive immunity depends crucially on the enormous diversity of theT-cell receptor repertoire, the antigen receptors expressed collectively onT-cell populations. T cells develop from T-cell precursors that originate in thebone marrow and migrate to the thymus, where their T cell receptors areconstructed stochastically, and tested for autoreactivity against a host of selfantigens. Complete DiGeorge anomaly is a rare congenital disease in which thethymus fails to develop, blocking all T cell development and causing profoundimmunodeficiency. Thymus transplantation, performed in the first two post-natalyears, allows the patient's own T cell precursors to develop in theengrafted thymus tissue into normal, functioning T cells. In addition to savingpatients' lives, this procedure provides an extraordinary opportunityto study the  An essential characteristic of T lymphocytes is their ability, as a population, torecognize an enormous number of peptide antigens. This capability is essential tothe function of the adaptive immune system and is attributable to the diversity ofthe T-cell receptors (TCR) they express. This diversity in turn comes about throughthe stochastic assembly of TCR from genomic “libraries” of genesegments for the TCR alpha and beta chains, the two polypeptides that together makeup the most common form of the TCR The peripheral T-cell population is maintained at a constant size in spite ofsubstantial, continual turnover, ongoing thymic production, and clonal expansion inresponse to immunological challenges Diverse lines of investigation imply that signals delivered, and resources provided,through both TCR-specific and TCR-nonspecific channels are essential for theestablishment and maintenance of the size and diversity of T cell populations Our intent in this paper is to elucidate the mechanisms that lead to expansion anddiversification of the TCR repertoire in a recovering T-cell-deficient host: in ourcase, a complete DiGeorge subject that has received an unrelated, unmatched thymustransplant. After transplantation, host T-cell precursors migrate from the bonemarrow to the donor thymus where they develop via thymopoiesis into host T cellsthat then emigrate into the peripheral blood. Here, T cells have the capacity toundergo spontaneous clonal expansion, thus leading to restoration of the peripheralT-cell pool. The expansion continues until a steady state is reached. Throughoutthis process, selective pressure for survival emerges among and within the clonesthrough competition for stimulatory signals.The steady state T-cell population size arises in the balance among severalphenomena, including the rapid and extensive expansion of rare clones throughactivation-induced peripheral division and their subsequent contraction  and the relatively slowturnover of a diverse pool of naive cells through continuous thymic emigrationand cell death. The specific memory T cells that arise as the result of clonalexpansion appear to be regulated largely independently of the naive cells Both the size and diversity of peripheral T cell populations are controlledthrough competition for limiting resources.  Our current understanding of lymphopenia-induced proliferation is due to studiesin mice demonstrating that T cells divide rapidly after transfer into Tcell-deficient  haveshown a reduction in T-cell capacity to proliferate upon stimulation, leading toa six- to seven-fold reduction in the frequency of clonogenic T cells comparedwith T cells from IL7R-sufficient mice (IL7R+/+),as well as a 50% reduction in the average clone size of singleIL7R−/− T cells compared with theIL7R+/+ T cells TCR-nonspecific signals include cytokines such as the cytokine interleukin-7(IL7), which is necessary for the survival of nave T cells To assess the contributions of thymic emigration rate on the steady-state T-cellpopulation size, Berzins et al Complete DiGeorge Anomaly (cDGA) is a congenital condition, the hallmark of whichis profound immunodeficiency arising from abnormal development of the third andfourth pharyngeal pouches resulting in thymic aplasia (complete absence of thethymus). This developmental irregularity may also cause various other anatomicalabnormalities including heart defects, hypoparathyroidism, and craniofacialmalformations Thymus transplantation is emerging as a valuable treatment for athymia generally.There is therefore substantial medical interest in elucidating the immunologicalrecovery of thymus transplant patients quite apart from the basic immunologythese patients may reveal.To illustrate the connection between these alternative homeostatic mechanisms andthe observations one might make on the DiGeorge subjects, consider the followingtwo scenarios as a thought experiment. First, suppose that TCR-specificresources such as spMHC are not limiting, either because they are produced ingreat excess or because they are rendered unnecessary. Under these conditions,homeostasis will be due exclusively to competition for TCR-nonspecificresources. The first T cells leaving the thymus will expand rapidly, consumingthe TCR-nonspecific resources required for growth of their own growth and forthe growth of all other clones. Subsequent T cells leaving the thymus willencounter a more impoverished environment and will grow more slowly, leading toearly dominance of one or a small number of early clones and therefore a limitedTCR repertoire .ConversReality will lie somewhere between these two limiting cases: TCR repertoirediversity will be shaped by both TCR-specific and TCR-nonspecific resources. Ourgoal is to explore, quantitatively, how these two sets of signals interactdynamically to shape the mature functional T cell population.The quantitative contributions of different signals in the regulation of T-cellnumber and the diversity of the TCR repertoire are very difficult to determineexperimentally in the context of an intact system. To examine the interplayamong the mechanisms responsible for the heterogeneity of the T-cell repertoire,we have developed a mathematical model for the effective interactions amongT-cell clones. We model the temporal evolution of T-cell clones and theirdynamics under the combined effect of TCR-specific and TCR-nonspecific signals.In particular, we consider competition within clones for spMHC complexespresented by antigen-presenting cells as well as competition among cells in allclones for cytokines, space and other TCR-non-specific resources.Such mathematical models have been used extensively to study the dynamicalinteractions between different branches of the immune system ix, We use a population-dynamic model for the analysis of patient data. The state of themodel at any time is given by the variables γ. Wemodel intraclonal competition for specific spMHC within the γ(1−ρ)T/κ.κ is the carrying capacity (the maximum population sizesustainable in the absence of immigration) and ith effective clone. In other words, the emigration of cellsfrom the thymus is modeled as a stochastic point-process, with the times betweenemigration events for specificity The maximum growth rate for model cells is denoted Eq.(1) provides the basis for the numerical data fitting we perform, but analysis ofthe model itself is facilitated by replacing the the Poisson process in Eq.(1) byits time-dependent mean value, ie, neglecting the fluctuations due to thediscreteness of the emigration process. This approximation is valid near steadystate, where each of the clones is substantially filled. In this approximation, thedynamic equations for, eg, the clone proportions Using this same approximation, the dynamics of the total T-cell population size isgiven byi, At the single stable steady state, which is unique and stable, all clones have thesame size, independent of tion of  .When Because the thymic graft is likely to become functional only gradually over time, wemodel the thymic emigration rate as a Hill function,This parameterization is atypical, but proves to be practical for performing thefitting, as will be described in the http://www.codeproject.com/KB/recipes/Random.aspx). The continuouscomponents are integrated numerically using a fourth-order Runge-Kutta scheme withadaptive step size. Thymic emigration is implemented as follows. For each timeinterval Sample paths under the model are generated using software written in C# under VisualStudio 2008, and the Troschuetz pseudorandom number generator classes .Between 13 and 17 blood samples per subject were collected over a period of2–3 years following thymus transplantation in three infants withcomplete DiGeorge anomaly. T cell receptor diversity in CD4 T cells wasassessed by obtaining frequencies of TCRBV family usage by flow cytometry,and of complementarity determining region 3 (CDR3) length distributionswithin TCRBV families by spectratyping and using these frequencies toestimate the diversity as described below.Standard flow cytometry on whole blood samples was performed as previouslydescribed Spectratype analysis provided information about CDR3 length diversity withineach functional TCRBV family. Briefly, CD4 T cells were isolated from theperipheral blood of subjects and controls. RNA was prepared and used forcomplementary DNA (cDNA) synthesis. The cDNA was used as a template for 23TCRBV-specific primer pairs covering 21 TCRBV families to amplify thecomplete CDR3 region by PCR The Kullback-Leibler divergence  to the patient T cell concentration and TCR diversitydata simultaneously using Markov Chain Monte Carlo Click here for additional data file.


This case report describes an enchondroma of the distal phalanx of the right little finger in a 37-year-old woman with Camurati-Engelmann disease. Curettage of the tumor and artificial bone grafting were performed in May 2004. Surgical treatment resulted in a good clinical outcome with no evidence of recurrence at 5-year follow-up. The genetic relationships between Camurati-Engelmann disease and benign chondroid tumors are discussed. HoweverIn 1990, a 23-year-old Japanese woman initially presented to our hospital with upper and lower limb pain. Radiographs on admission showed diffuse, bilateral sclerosis of the medullary cavity of her femur and tibia along with endosteal and subperiosteal cortical thickening affecting the diaphysis with epiphyseal sparing . The patIn 2004, this patient was admitted to our institute at age 37 with a complaint of pain in the distal phalanx of her right little finger. Physical examination showed swelling and tenderness of the right little finger, despite which she had full range of motion in all fingers. Radiographs showed an osteolytic lesion in the distal phalanx of the little finger with distension and thinning of the surrounding cortex . The cliSurgery was performed in May 2004. A longitudinal skin incision was made on the dorsal ulnar aspect of the distal phalanx of the right little finger. A cortical hole was easily made in the thinned cortex through which the intramedullary canal was curetted and filled with artificial hydroxyapatite . HistoloProgressive diaphyseal dysplasia (PDD), or Camurati-Engelmann disease (CED), is a rare, autosomal dominant genetic disorder characterized by sclerosing bone dysplasia . The revEnchondroma is a benign hyaline cartilage tumor that constitutes close to 20% of all cartilaginous tumors. Enchondromas most commonly present in the short tubular bones, the proximal femur, and the humerus . AlthougGenetic studies of CED have discovered that CED results from a mutation in the transforming growth factor-β1 (TGF-β1) gene on chromosome 19q13.1 ,9. TGF-βInterestingly, chromosome 19 loss has been reported as a common occurrence in both chondrosarcoma and in benign cartilaginous tumors, including enchondroma . GunawanIn conclusion, we presented here the case of an enchondroma in a 37-year-old female with Camurati-Engelmann disease. Since this is the first such report, we cannot determine whether this case was a coincidence or whether there is a genuine relationship between enchondroma and this extremely rare genetic skeletal disorder.