Troponins  increase counts of myocardial infarction (MI) in clinical practice, but their impact on trends in admission rates for MI in National statistics is uncertain.Cases coded as MI or other cardiac diagnoses in the Hospital Morbidity Data Collection (MI-HMDC) in Western Australia in 1998 and 2003 were classified using revised criteria for MI developed by an International panel convened by the American Heart Association (AHA criteria) using information on symptoms, ECGs and cardiac biomarkers abstracted from samples of medical notes. Age-sex standardized rates of MI-HMDC were compared with rates of MI based on AHA criteria including troponins (MI-AHA) or traditional biomarkers only (MI-AHAck).ck declined by 18%.Between 1998 and 2003, rates of MI-HMDC decreased by 3.5% whereas rates of MI-AHA increased by 17%, a difference largely due to increased false-negative cases in the HMDC associated with marked increased use of troponin tests in cardiac admissions generally, and progressively lower test thresholds. In contrast, rates of MI-AHAck are consistent with long-standing trends in MI in Western Australia, suggesting that neither MI-HMDC nor MI-AHA reflect the true underlying population trends in MI.Increasing misclassification of MI-AHA by the HMDC may be due to reluctance by clinicians to diagnose MI based on relatively small increases in troponin levels. These influences are likely to continue. Monitoring MI using AHA criteria will require calibration of commercially available troponin tests and agreement on lower diagnostic thresholds for epidemiological studies. Declining rates of MI-AHA Coronary heart disease (CHD), despite declining mortality, remains a major health problem in developed countries -3. ReliaIn many jurisdictions, routinely collected mortality and hospital morbidity data are therefore the most commonly used alternative for monitoring trends in MI. While such data have major shortcomings, studies in Finland, Sweden and Western Australia have previously shown reasonable agreement between trends based on registers and administrative data -9. HowevIn recognition of the potential problems for epidemiological studies, a panel of international experts, meeting under the auspices of the American Heart Association (AHA), have developed new criteria for MI for use in epidemiological studies, referred to here as the 'AHA criteria', which emphasise the importance of troponin in the diagnosis of MI . So far,ck) including creatinine kinase (CK) or CK-MB in the classification algorithm.This study used the Hospital Morbidity Data Collection (HMDC), one of the core administrative datasets in the Western Australian Data Linkage System , to compth revision codes 401-429, 786.5; or ICD10 Australian Modification codes I10-I52, R07). Electronic records for the study population were extracted from the HMDC, identified by hierarchical discharge diagnosis as shown in Table The study population consisted of residents of the Perth Statistical Division (population 1.43 million in 2003) of Western Australia aged 35-79 years admitted to hospital in 1998 or 2003 for cardiac conditions or chest pain  9Non-fatal cases were defined as patients who were alive 28 days after admission. The episode records were then linked electronically to results of cardiac biomarkers provided by biochemistry departments. Cases were then selected for validation of discharge diagnosis codes against information abstracted from medical notes, using the sampling scheme outlined in Additional File Data were abstracted from medical notes directly into a Microsoft Access database by trained staff using a standardised data collection format. Data included: symptoms present on admission, results of biomarker tests (daily results for up to 5 days), reasons for possible false elevations of cardiac biomarkers , and photocopies of up to five electrocardiographs (ECG). Other data, including demographic details and dates, were added directly from the HMDC extract.A computer algorithm was developed to classify cases as Definite, Probable, Possible or Not MI according to AHA criteria based on the combination of symptoms, biomarker results and ECG abnormalities as defined in the International panel report  and illuAll analyses were carried out in SAS version 9.1.3 SP4 for Windows. Population estimates of the total number of cases in each sample group were calculated by inflating the sampled cases by their sampling fraction . To allow for the effect of population increase between 1998 and 2003, we estimated age-sex standardised rates of admission for non-fatal MI in age group 35-79 years using the direct method by 5-year age group and sex, with the Australian estimated population at 30 June 2001 as the standard.Counts of MI-HMDC were compared separately with counts of MI based on AHA criteria using all available biomarkers, including troponins (MI-AHA), or traditional biomarkers only (MI-AHADiagnostic thresholds for troponin tests were lower in 2003 than in 1998 as the sensitivity of the assays improved. We compared the distribution of ECG changes and biomarker results in cases of Positive MI coded in the HMDC as MI (true-positives) or Not MI  to examine the extent to which the lower thresholds were associated with false-negative cases.This study was approved by the Human Research Ethics Committees of the University of Western Australia, each of the eight hospitals from which data were collected, and by the Western Australian Confidentiality of Health Information Committee. The study was granted a waiver of informed consent.In 1998, there were 8939 28-day episodes of care for heart conditions or chest pain . In 1998, 49% of MI-HMDC had ECG evidence of MI (diagnostic or positive ECGs), compared with 31% in false-negative cases. In 2003, the corresponding percentages were 42% and 23%. Conversely, in both years 28% of MI-HMDC had normal/other ECGs compared with around 45% in false-negative cases. We also found that troponin levels were substantially lower in false-negative cases than in true-positive cases as illustrated in Figure Table ck (without troponin). When troponin is included in the AHA classification, MI-HMDC overestimated counts of Positive MI in 1998 but under-estimated the counts in 2003. This disparity between the clinical (coded) diagnosis in 2003 is in agreement with the finding by Roger and colleagues in their prospective study of the effects of troponin tests on counts of MI in Olmsted County, that found substantially lower counts of cases with a final coded diagnosis of MI in medical records compared with counts based on troponin tests [The introduction of troponins as highly sensitive and specific diagnostic tests for MI has revolutionised the clinical management of suspected myocardial infarction, but while their utility in clinical practice is unquestioned, problems in monitoring population trends in MI remain unresolved. We have made a three-way comparison of counts of MI-HMDC, MI-AHA (with troponin) and MI-AHAin tests . They atck (without troponin). The decrease in MI-AHAck is consistent with the decline in admission rates for MI in Perth in the 10 years prior to the introduction of troponin tests [ck is also consistent with a further study in Olmsted County that found that from 1987 to 2006, rates of MI declined by 20% when traditional biomarkers only were used in the diagnosis of MI, whereas rates of MI increased when based on troponin tests [In contrast, when troponin was excluded from the AHA classification, MI-HMDC overestimated counts of Positive MI which actually decreased between 1998 and 2003. There was an even greater difference between the increasing rates of MI-AHA (with troponin) and the decreasing rates of MI-AHAin tests . The marin tests .An essential requirement for monitoring MI is that classification should be based on objective criteria that remain constant over time. In the case of troponin-based criteria this is unlikely because of progressive lowering of diagnostic thresholds as the precision of tests improves, and because of variation within and between hospitals in the several commercial, non-standardised tests in use. This is likely to increase the number of false negative cases. A further likely reason for the increase in false-negative cases in 2003 was the marked general increase (from 33 to 66%) in use of troponin tests in all cardiac admissions other than MI or UAP. Thus, even though the prevalence of positive troponin tests in such cases was small, it had a relatively large negative impact on the sensitivity of MI-HMDC against MI-AHA.Western Australia is geographically isolated with relatively small population losses from emigration, and is thus ideal for epidemiological studies. It has comprehensive, linked health statistics systems spanning 30 years , and hasLimited resources forced us to adopt a sampling strategy to validate the coding of MI in the HMDC. If sampling was not strictly random, errors may have occurred in population estimates. Our strategy of linking biomarker test results to identify possible false-negative cases of MI in the HMDC may have identified some cases in which elevated troponins were not due to an acute ischaemic event  but which nevertheless met the 'AHA criteria' for MI. Lack of statistical power due to limited resources also prevented us from fully exploring possible differences in the impact of troponins on rates of MI by age and gender.Our study has identified a number of issues for further investigation before the predominately troponin-based 'AHA criteria' can be used with confidence in studies of trends in MI. The most urgent need is for calibration of the several commercial troponin tests in use, and for agreement on stratification of troponin results that would permit valid comparison of positive tests over time, despite changing diagnostic thresholds. While universal standardization of troponin tests is unlikely ever to be possible, calibration for studies of trends at regional level, particularly when only a few laboratories are involved, should be possible. This would not invalidate comparative studies within or between countries in which the primary focus is on trends rather than cross-sectional differences between rates, and providing that the methods of calibration are explicitly described.In view of the marked general increases in the use of troponin tests in acutely ill patients with both cardiac and non-cardiac conditions, further research is required to determine whether the search for false-negative cases should be restricted to cases coded within the ICD rubrics for ischaemic heart disease or chest pain.Finally, the International expert panel made no recommendations about the AHA categories of MI  that should be included in epidemiological studies. Definite plus Probable MI in the AHA classification appears to be comparable to "Definite MI" used as the main non-fatal event in the MONICA Project  and appe• Troponin tests increase counts of myocardial infarction (MI) in administrative data compared with counts based on traditional cardiac biomarkers.• Long-term trends in MI based on traditional biomarkers are declining, whereas those based on troponin tests are increasing.• Increased counts of MI associated with troponin tests using revised criteria for MI are only partly reflected in administrative data, possibly because physicians are reluctant to diagnose MI on the basis of relatively small increases in troponin levels.• This is the first population-wide study to explore the practical issues of implementing the revised criteria for myocardial infarction (MI) for use in epidemiological studies published by the American Heart Association (AHA) International panel in 2003. It shows that for trend analysis at least, the AHA criteria are flawed because they do not recognise variation in troponin assay thresholds in different laboratories or changes in diagnostic thresholds over time. This problem will increase with the development of even more sensitive troponin assays.• Studies of trends in MI (as exemplified by the WHO MONICA Project) have a basic requirement for unchanging objective criteria. Our study identifies further work that is required to standardise troponin testing for analysis of trends, particularly at the International level, if the AHA criteria are to have any utility.• Future epidemiological studies using AHA criteria will need to recognise the large increase in false-negative cases of MI associated with the large general increase in the prevalence of troponin testing in cases with other cardiac conditions or chest pain.• Since the introductions of troponins, routinely collected hospital statistics no longer reflect true underlying trends in MI.ck: MI classified by the AHA criteria using traditional cardiac biomarkers only (excluding troponin tests); Positive MI: AHA Definite or Probable MI; Any MI: AHA Definite, Probable or Possible MI; UAP: unstable angina pectorisAHA: American Heart Association; HMDC: Hospital Morbidity Data Collection; MI: myocardial infarction; MI-HMDC: MI coded in the HMDC; MI-AHA: MI classified by the AHA criteria (including troponin tests); MI-AHAThe authors declare that they have no competing interests.All authors read and approved the final manuscript. MH, MK, JF, JR, PS, JH are chief investigators for this study. They designed the study, provided epidemiological , clinical  and statistical (MK) guidance, and assisted in preparation and review of the manuscript. MH was the lead author for sections on Background, Discussion and Conclusions. FS implemented the study, managed research staff and the collection of data, carried out the data mining and all of the statistical analyses, created the Endnote library of references, and prepared the manuscript. FS was the lead author for the Methods and Results sections of the manuscript, and reviewed the Background, Discussion and Conclusions. SR prepared the data files, merging and linking laboratory data with administrative data, assisted with identifying and sampling of the validation population and sample, and reviewed the manuscript. PB prepared the list of variables for data collection, trained the research staff in the data collection techniques from medical notes, and reviewed the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/11/35/prepubMethods: Additional Detail. Provides additional information on the selection of the validation sample, and the classification of biomarkers, ECGs and symptoms using AHA definitions. Includes two additional tables associated with methods and results sections of the main text, including Table 1 from the AHA 2003 classification [fication  showing Click here for file


The cerebrovasuclar artery disease as a common complication of type-2 diabetes mellitus (T2DM) caused huge economic burden and lives threatening to patients. We evaluated the prevalence and morphology of carotid and cerebrovascular atherosclerotic plaques in T2DM patients with transient ischemic attack (TIA) or stroke using multidetector CT (MDCT).64-MDCT and dual-source CT (DSCT) angiographies were performed in 195 T2DM patients with TIA or stroke  between January 2009 to August 2011. During the process, plaque type, its distribution, extensive and obstructive natures were determined for each segment derived from the patients.Atherosclerotic plaques were detected in 183 (93.8%) patients. A total of 1056 segments with plaque were identified, of which 450 (42.6%) were non-calcified, 192 (18.2%) were mixed and 414 (39.2%) calcified ones. Among them, 562 (53.2%) resulted in mild stenosis, 291 (27.6%) moderate stenosis, 170 (16.1%) severe stenosis and 33 (3.1%) occlusion. Non-calcified plaques contributed 91.8% to non-obstructive lumen narrowing, while mixed and calcified plaques contributed 89.0% and 65.0% respectively.MDCT angiography detected a high prevalence of plaques in T2DM patients with TIA or stroke. A relatively high proportion of plaques were non-calcified, as well as with non-obstructive stenosis. MDCT angiography might further enhance the detection and management of carotid and cerebrovascular atherosclerosis in T2DM patients with TIA and stroke Currently, over 200 million individuals are known to have diabetes mellitus (DM) worldwide and this number is expected to double by the year 2025. Type-2 Conventional digital subtraction angiography (DSA) is considered as the “gold standard” for the evaluation of the degree of carotid and cerebrovascular artery stenosis, but it is unable to make any predictions about plaque type, and this time-consuming method remains invasive and is still associated with catheter-related complications. Thus, sFrom January 2009 to August 2011, we retrospectively observed 195 consecutive T2DM patients  with neurologic symptoms of TIA or stroke at West China Hospital and Military General Hospital of Chengdu PLA. The presenting neurologic symptoms for all patients were classified as: (1) transient symptoms, i.e., TIA or amaurosis fugax; (2) prior stroke, i.e., any ischemic event with neurologic symptoms. The exclusion criteria for MDCT were known allergy for iodinated contrast agents, renal insufficiency (creatinine level ≥120 μmol/L), pregnancy, and lack of lab or clinical data. All the MDCT examinations were performed within 2 to 4 weeks of neurologic symptom onset. All subjects gave informed consent, and the Ethics Committees of West China Hospital and Military General Hospital of Chengdu PLA approved the study.Baseline demographics and DM-ralated complications were provided, such as age, gender, age at diabetes diagnosis, duration of DM, hypertension, duration of hypertension, daily smoking, diabetic retinopathy (DR), diabetic foot (DF), diabetic nephropathy (DN), atrial fibrillation (AF), myocardial infarction (MI), and CI. Laboratory tests included measurement of fasting plasma glucose (FPG), triglycerides (TG), total cholesterol (TC), glycosylated hemoglobin (HbAlc), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Hypertension was defined as systolic blood pressure of ≥140 mmHg or diastolic blood pressure of ≥90 mmHg. DN was present if the 24-h urinary albumin expression (24-h UAE UA1b) was ≥30 mg [Examinations were performed with 64-MDCT   at Military General Hospital of Chengdu PLA, DSCT   at West China Hospital. For the 64-MDCT group, examinations were performed with the following parameters: 64 row × 0.5 mm detector collimation, 0.5 s/gantry rotation, 120 kV, 350 mA, and 0.94 beam pitch. Unenhanced images were first obtained from lower neck level to mid skull, which was used to determine the scan range of MDCT angiography. Injection of 90 ml of iodinated contrast agent , immediately followed by 40 ml of saline chaser solution, was administered through an 18-gauge intravenous antecubital catheter at a flow rate of 6 ml/sec with a dual-head power injector . DSCT examination was obtained following standard protocols similar to conventional 64-MDCT.Image reconstructions were performed at 3D image analysis workstation  with the following parameters: a slice thickness of 0.75 mm, and increment of 0.4 mm. The MDCT angiography reader was permitted to utilize any or all of available postprocessing image reconstruction algorithms, including multiplanar reformat (MPR), curved planar reformat (CPR), maximal intensity projection (MIP) or volume rendered technique (VRT).All data sets were assessed by two independent observers unaware of the clinical history of the patients. In cases where disagreement occurred, agreement was reached in a joint reading.According to the criteria of North American symptomatic carotid endarterectomy trail (NASCET), carotidContinuous variables were expressed as means ± standard deviation (SD). The categorical variables were presented as numbers (percentages). All statistical analyses were per formed using SPSS16.0 . P < 0.05 was considered statistically significant.P < 0.05). Age ranged from 41 to 89 years, with a mean age of 65.7 ± 12.8, and the duration of diabetes was 6.2 ± 5.4 years. There was a high incidence of risk factors for atherosclerosis and associated diseases: 132 (67.7%) patients were presented with hypertension, 126(64.6%) with CI, and 67(34.4%) with a history of smoking. All clinical data and laboratory test results are shown in Table A carotid and cerebrovascular MDCT investigation was performed in 195 patients and a total of 7368 artery segments were included in the analysis, and 432 vascular segments were excluded because of deficiency or diameter <1.5 mm. There were significantly difference between men and women  and 12 (6.2%) patients with and without cerebrovascular disease. A total of 1056 segments with plaque were identified. With regard to plaque constitution, 450 (42.6%) of the plaques were classified as non-calcified plaques, 192 (18.2%) as mixed and 414 (39.2%) as calcified plaques , moderate (18.9%), severe stenosis (6.4%) and occlusion (1.8%), mixed plaques resulted in mild (60.4%), moderate (28.6%), severe stenosis (8.9%) and occlusion (2.1%), and calcified plaques resulted in mild (28.5%), moderate 36.5%), severe stenosis (29.9%) and occlusion (5.1%), respectively. As shown in Figure 6.5%, sevThe distributions of plaques were different, non-calcified plaques mainly located in intracranial arteries (80.0%), mixed plaques mainly in intracranial ICA 59.4%), and calcified plaques mainly in intracranial ICA (64.5%) and extracranial arteries (30.4%), as shown in Figure %, and caIn our study, only T2DM patients were enrolled, lack of control group of non-diabetic patients, and the main findings could be summarised as follows: Firstly, in T2DM patients with TIA or stroke, relatively high proportions of plaques were non-calcified and calcified, mainly resulted in non-obstructive stenosis. Secondly, the distributions of plaques were different, non-calcified plaques mainly located in intracranial arteries, both mixed and calcified plaques mainly in intracranial ICA. Finally, the distribution of plaques was extensive, and the most common site of plaque was the cavernous segment of ICA.In the present study, 42.6% of the plaques are classified as non-calcified plaques, 18.2% as mixed and 39.2% as calcified plaques. Several studies also shoSymptomatic patients with carotid stenosis will benefit from carotid endarterectomy or stenting as secondary prevention of ischemic stroke, thus prWe also find that the distributions of plaques are different: non-calcified plaques mainly located in intracranial arteries (80.0%), mixed plaques mainly in intracranial ICA (59.4%), and calcified plaques mainly in intracranial ICA (64.5%) and extracranial arteries (30.4%). Because non-calcified plaques was considered as vulnerable plaques, and maiAdditionally, when considering the distribution of plaques, we find that the most common site of plaque is the cavernous portion of ICA in T2DM patients with TIA or stroke. Fields et al. reportedOutlining the significance of our study, the prevalence and morphology of carotid and cerebrovascular atherosclerotic plaques in T2DM patients with TIA or stroke by 64-MDCT and DSCT angiography is reported comprehensively and systematically. Secondly, the uniqueness of our study lies in the scanner technology, which has better visualization of arteries with calcified plaque or vessels located next to the skull bone than early CT,25. In tLimitations are there. Firstly, only T2DM patients were studied, lack of a control group of non-T2DM patients, and data concerning the prevalence of atherosclerosis as determined by MDCT in larger populations are needed. Secondly, the capabilities of current generation CT in the evaluation of disease involving small and very small arteries with an average diameter of 1.5 mm or less have not been well characterized. Finally, MDCT is still associated with a risk of radiation dose, and administration of contrast media is also required.MDCT angiography detected a high prevalence of plaques in T2DM patients with TIA or stroke. A relatively high proportion of plaques were non-calcified, as well as with non-obstructive stenosis. MDCT angiography might further enhance the detection and management of carotid and cerebrovascular atherosclerosis in T2DM patients with TIA and stroke.The authors declare that they have no competing interests.CH made the main discovery, performed the statistical analysis, and drafted the manuscript. MG helped to draft and reviewed the manuscript. RJ, and JH contributed in execution, conceived of the study, and participated in its design. All authors read and approved the final manuscript.


E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in  Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine important for the stimulation of proliferation, differentiation and survival of many hemopoietic cells E.coli. However, with many over-expression systems it was found that rhGM-CSF formed inclusion bodies E. coli and are an ongoing issue in biotechnology and biomedical research E. coli lysate, solubilize the inclusion bodies and then refold the protein of interest E. coli and isolate the inclusion bodies followed by the addition generally of chaotropic agents (e.g. GuHCl or Urea) to solubilize them. It is the refolding of the protein of interest that is often the limiting step with aggregation and improper disulphide bond formation being two major issues to overcome. During protein refolding, aggregation of partially folded intermediates can result in a significant decrease in final yields. L-arginine, a natural amino acid, is commonly used to enhance protein refolding by suppressing protein aggregation GM-CSF is a 127 amino acid compact glycoprotein composed of a two-stranded antiparallel β-sheet and a four α-helix bundle E. coli. The refolding protocol described was also successfully used to refold Fab fragments of antibodies and thus may be used as a general refolding strategy for proteins forming inclusion bodies in E. coli such as many cytokines We describe in detail here a straightforward method to refold and purify rhGM-CSF from inclusion bodies that generates milligram amounts of active protein from a single litre of AACATATGGCACCCGCCCGCTCG) and asGMCSF1 (5′TTCTCGAGCTCCTGGACTGGCTCC) were used to amplify the cDNA to initiate with the MAPARS protein sequence at the N-terminus and to remove the stop codon at the C-terminus so to allow incorporation of the C-terminal his-tag. The resultant construct contained the additional amino acids, LEHHHHHHHH, C-terminal to the GM-CSF sequence and was termed pET40-GM-CSF.The cDNA of rhGM-CSF, minus the signal sequence, was amplified and ligated into the NdeI and XhoI sites of the pET40b(+) vector (Novagen). The primers, sGMCSF1 (5′ E. coli. A 10 ml culture using LB containing 30 µg/ml of kanamycin was grown overnight shaking at 37°C and used to inoculate 1 L of LB containing kanamycin (in a 2.8 L Fernbach flask) the following morning. Through prior variation of induction times and lengths, it was found that addition of 1 mM isopropyl β-D-1-thiogalatopyranoside (IPTG) at a culture optical density (600 nm) of approximately 0.3 and subsequent culturing for 5 hrs at 37°C yielded the highest expression of rhGM-CSF. Cells were harvested by centrifugation at 5000×g  at 2–8°C for 10 minutes and stored at −20°C.The pET40-GM-CSF construct was transformed into the BL21(DE3) strain of Cells were resuspended in 25 ml of lysis buffer ). Mechanical disruption was used to lyse the cells (i.e. French Press or sonication) and the inclusion bodies isolated from the cell supernatant by centrifugation at 18000×g at 2–8°C for 20 minutes. The inclusion body pellet was solubilized in resuspension buffer , 10 mM DTT) by repeatedly passing the inclusion bodies through an 18g syringe. It is worth noting that any insoluble material can be centrifuged out at this time at 18000×g at 2–8°C for 20 minutes. The resuspended protein material was then diluted 50% in dialysis buffer #1  resulting in a 4 M GuHCl containing solution. The protein solution was then dialyzed overnight at 4°C in snakeskin dialysis tubing (Pierce) against 2 L of buffer #1. The following day the dialysis buffer was changed to 2 L of dialysis buffer #2 , 3 mM Reduced Glutathione, 0.9 mM Oxidized Glutathione) for overnight dialysis at 4°C. The following day the dialysis buffer was diluted 50% with water and dialysis continued overnight. Any insoluble material was centrifuged (18000×g at 2–8°C for 20 minutes) and the remaining protein solution dialyzed overnight at 4°C against 1 L of dialysis buffer #3  to remove the remaining GuHCl.280nm = 14 238) 0.1%) of the folded rhGM-CSF at 280 nm is 0.89 compared to 0.98 of rhGM-CSF minus the additional amino acids.The final dialyzed protein solution was clarified by centrifugation (18000×g at 2–8°C for 20 minutes) and the supernatant loaded onto a ProBond (Invitrogen) His-tag affinity column equilibrated in equilibration buffer . The column was washed with an excess of wash buffer  followed by a wash with equilibration buffer to remove the Triton X-100. The rhGM-CSF was eluted in 1 ml aliquots from the resin using elution buffer . To quickly visualize the eluted protein, 10 µl from individual fractions was pipetted on Whatmann filter paper, and stained with Coomassie Brilliant Blue for 2 min before destaining or analyzed by 15% reducing SDS PAGE. Fractions containing eluted protein were pooled and dialyzed against 2 L of 20 mM HEPES, pH 7.8, overnight at 4°C. Protein that precipitated out during the overnight low salt dialysis was removed by centrifugation before proceeding. The purified rhGM-CSF was quantified by UV spectroscopy using a calculated molar absorption coefficient corresponding to the non-reduced rhGM-CSF sequence . The peptide mixtures were separated on a PicoTip column  from New Objective  packed with reverse-phase C18 material . Solvent A (0.5% acetic acid) and solvent B (80% acetonitrile +0.5% acetic acid) were employed. A linear gradient of 6% to 80% solvent B in 30 min at a flow rate of 600 nl/min was applied via an Agilent 1100 nano HPLC pump. Peptide sequences were identified by searching spectra against the Swiss-Prot database using MASCOT The rhGM-CSF bioactivity was analyzed with a proliferation assay using TF-1 cells 2. Commercial rhGM-CSF was obtained from ImmunoTools , reconstituted, aliquoted and stored at −80°C. For the proliferation assay, TF-1 cells were washed 5 times in RPMI with no supplements and seeded 1000 cells per well in growth medium with no IL-3 or rhGM-CSF in 96 well tissue culture plates . rhGM-CSF was serially diluted two-fold in growth medium and added to the washed cells to give final concentrations ranging from 12.8 ng/mL to 1.56 pg/mL, and 100 µL total volume per well. Control wells containing cells but no rhGM-CSF (blank) and rhGM-CSF with no cells were performed. The plates were incubated in a 37°C humidified incubator with 5% CO2 for 4 days. Cell Proliferation Reagent WST-1 (Roche cat # 11 644 807 001) was added and incubation continued as above for 4 hrs. The absorbance values at wavelength 450 nm, with reference wavelength of 690 nm values subtracted, were determined using a plate reader (Molecular Devices). The absorbance values were directly proportional to the number of viable cells because the tetrazolium salts in the WST-1 reagent were cleaved to formazan by mitochondrial dehydrogenases in the cells. The blank values were subtracted from all wells on each plate and the values plotted. Unit values were defined as 50 U/mL being equivalent to the concentration of GM-CSF that supports 50% of maximal growth under the assay conditions used.The TF-1 cells (ATCC number CRL-2003) were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, sodium pyruvate, and penicillin/streptomycin  with 2% gibbon IL-3 added just prior to use. The cells were grown in a 37°C humidified incubator with 5% COE. coli  is not available, it is possible to proceed directly to the resuspension step. There will simply be more contaminating proteins in the dialysis tubing and more extensive washing of the purification column may be required.The rhGM-CSF protein was efficiently expressed using the pET40b(+) vector in E. coli . HoweverE. coli . Lysis oAfter centrifugation of the cell-lysate, the pelleted inclusion bodies were first solubilized in a buffer containing 6 M GuHCl and 10 mM DTT and then slowly dialyzed against buffers containing decreasing amounts of GuHCl, as well as L-arginine and the redox pair of oxidized and reduced glutathione. This process efficiently refolds the rhGM-CSF and any unfolded/insoluble material is removed by a centrifugation step. The solubilized rhGM-CSF was subsequently purified using a nickel-chelating resin via the 8×His tag engineered on its C-terminus . From a To confirm the identity of the purified rhGM-CSF, LC-MS/MS (FT-ICR) was performed on trypsin and protease V8-digested fragments. Trypsin cleaves C-terminal to arginine and lysine residues whereas protease V8 cleaves C-terminal to glutamic and aspartic acids. The mass spectral analysis confirmed the identity of GM-CSF with almost complete sequence coverage .9 U/mg) with the commercial rhGM-CSF having comparable activity at 11.4 pg/ml (4.4×109 U/mg).To confirm the bioactivity of the refolded, purified rhGM-CSF, a human leukemia cell, TF-1, that was dependent on GM-CSF for its survival and proliferation, was washed extensively and 1000 cells were placed into 96 wells with a titration of refolded rhGM-CSF in triplicate. Four days later the metabolic activity of the surviving TF-1 cells was assessed by a WST assay according to the manufacturer’s instructions. In parallel, a titration of a standard commercial preparation of rhGM-CSF obtained from ImmunoTools was performed. The titrations of purified rhGM-CSF or the commercial source started with concentration of 1.28 ng/ml of each. As can be seen in E. coli, we adapted our refolding protocol for its purification. Our protocol is based on a combination of strategies for refolding of inclusion body proteins. For a review of isolation and purification of proteins from inclusion bodies see Vallejo and Rinas E. coli cell lysate, the first step in the rhGM-CSF refolding procedure is the solubilization of the inclusion bodies.  The conditions we used to resuspend the rhGM-CSF were harsh, however they were effective at solubilizing the protein and reducing non-native, inter- and intramolecular disulfide bonds. As well, although expensive, we found that use of a high grade of GuHCl was necessary to achieve efficient refolding.We had previously developed a protocol that efficiently allowed for the refolding of Fab fragments of antibodies from inclusion bodies Once solubilized, the protein was dialyzed against decreasing amounts of GuHCl. It is common to use dilution to reduce the amount of GuHCl during protein refolding, however this results in large volumes of dilute protein which is difficult to scale-up. Dialysis is more compatible with higher concentrations of protein and we found that it was effective for refolding both Fabs and rhGM-CSF. To aid in the rhGM-CSF refolding, we first reduced the disulfide bonds using DTT. hGM-CSF contains 4 cysteine residues forming 2 disulphide bonds that likely formed non-native, inter- and intramolecular disulfide bonds during inclusion body formation 9 U/mg and very similar to that of a commercial source of rhGM-CSF.The rhGM-CSF was purified using a nickel-chelating resin via the 8×His tag engineered on its C-terminus. Immediately after elution from the nickel column, we dialyzed the rhGM-CSF against a low salt containing buffer. In doing so, any partially folded protein precipitated and was removed during a subsequent centrifugation step. In cloning the rhGM-CSF into the expression vector, the additional amino acids, LE +8×His tag, were added to the C-terminus of the protein. Crystallographic analysis of rhGM-CSF E. coli). Likely it was the lack of addition of L-arginine and reduced and oxidized thiols that may have lowered the final yields of rhGM-CSF in these refolding protocols. Indeed, it has been demonstrated that enabling proper disulphide bond formation through the use of reduced and oxidized thiols and the addition of L-arginine to prevent aggregation are key to improving yields of purified, active protein E. coli have been successful, although the final yields can be significantly lower (∼500 µg/ml) rhGM-CSF has been refolded from inclusion bodies for many years. Early attempts similarly used chaotropic and reducing agents to solubilize the rhGM-CSF from inclusion bodies with dialysis into neutral Tris buffer E. coli, solubilization from inclusion bodies, refolding by dialysis, and purification on a nickel-chelating resin via a C-terminal His-tag. This protocol does not require extensive experience in protein purification nor elaborate chromatography equipment. Using this protocol we routinely generate approximately 7 mg of bioactive rhGM-CSF per litre of cell culture.Once inclusion bodies are formed, it can be difficult to refold the protein of interest into an active form. Here we present an easy, straightforward and efficient protocol for the purification of rhGM-CSF from inclusion bodies that was also successfully used in the refolding and purification of antibody Fab fragments. It involves the expression of the protein of interest in


GM-CSF is mostly known for its capacity to promote bone marrow progenitor differentiation, to mobilize and mature myeloid cells as well as to enhance host immune responses. However the molecular actions of GM-CSF are still poorly characterized. Here we describe a new surprising facet of this “old” growth factor as a key regulator involved in IL-1βsecretion. We found that IL-1β release, a pivotal component of the triggered innate system, is heavily dependent on the signaling induced by GM-CSF in such an extent that in its absence IL-1β is only weakly secreted. GM-CSF synergizes with LPS for IL-1β secretion mainly at the level of pro-IL-1β production via strengthening the NF-κB signaling. In addition, we show that expression of Rab39a, a GTPase required for caspase-1 dependent IL-1β secretion is greatly augmented by LPS and GM-CSF co-stimulation suggesting a potential GM-CSF contribution in enhancing IL-1β exocytosis. The role of GM-CSF in regulating IL-1β secretion is extended also in vivo, since GM-CSF R−/− mice are more resistant to LPS-mediated septic shock. These results identify GM-CSF as a key regulator of IL-1β production and indicate GM-CSF as a previously underestimated target for therapeutic intervention. Dendritic cells (DCs) and macrophages (MØ), the major sentinels of the innate immune system, are probing and recognising microbes and danger signals via specific pattern-recognition receptors. The resultant intracellular signalling cascades lead to the maturation and to secretion of several pro-inflammatory cytokines, such as IL–1 (IL-1α and β), TNF-α, IL-6. All these cytokines are ultimately involved in the pathogen elimination by promoting and sustaining an effective adaptive immune response IL-1β is a particularly potent pyrogenic cytokine and a key modulator involved in the regulation of immune responses against a variety of microbes as well as in several acute and chronic inflammatory disorders and septic shock The tightly controlled production and secretion of IL-1β typically requires two separate “external” signals. The first signal, provided by several pathogen-associated molecular patterns (PAMPs), promotes transcription, production and intracellular accumulation of the cytokine pro-form. The second signal, sometimes called “danger” signal is mediated by ATP, uric acid and aluminium salt (Alum) and many other factors, is needed for NLRP3 and caspase-1 activation and subsequent release of the biological active form of the cytokine. To avoid uncontrolled NLRP3 priming, its activation is strictly dependent on the PAMP-mediated NF-κB activation which regulates also the expression of NLRP3 This multi-level control ensures that the highly pyrogenic and inflammatory IL-1β is secreted only under circumstances when an inflammatory response is required. The loss of this stringent control can lead to autoinflammatory disorders, such as familial Mediterranean fever, familial cold autoinflammatory syndrome and Muckle-Wells syndrome. All these inflammatory diseases respond to specific IL-1β or caspase-1 rather than TNF-α blocking therapies Although GM-CSF was originally characterized as a haematopoietic growth factor responsible for the differentiation of bone marrow (BM) progenitor cells To test the effects of GM-CSF on the cytokine production in “danger situation” we stimulated FLTL3- derived DC in the presence of ATP with LPS alone or in combination with GM-CSF. Cultures were then measured for their contents of several pro-inflammatory cytokines, such as of IL-1α, IL-1β, TNF-α, and IL-6. GM-CSF alone did not induce any cytokine release but to our surprise combination with LPS dramatically enhanced the secretion of IL-1s with all tested concentrations of LPS about ten fold . InteresWe then extended our analysis to other APCs, such as GM-CSF derived BM DCs, M-CSF-derived BM MØ as well as L929 conditioned supernatant -derived BM MØ, focusing on the IL-1β secretion profile. In this study, we did not analyse the secretion profile of neutrophils, known to be one of the major IL-1β producers, since they secrete Il-1β independently from caspase-1 + DCs were co-incubated with GM-CSF  on DCs treated with LPS alone or the LPS/GM-CSF combination. The secretion of IL-1β was clearly dose-dependently decreased by both inhibitors pointing to a crucial role of GM-CSF in sustaining NF-κB activation . The decWe then analysed the proteolytic processing of pro-caspase-1 triggered by nigericin in WT and in GM-CSF R−/− MØ upon LPS stimulation in presence or absence of GM-CSF. The cleavage of the active form of the enzyme, p20 was clearly detectable in both WT and GM-CSF R−/− cells independently whether GM-CSF was co-applied . HoweverIt is commonly accepted, that excessive production of pro-inflammatory cytokines such as TNF-α and in particular IL-1, is one of the major causes of septic shock induced by endotoxin. To determine whether GM-CSF contributes to septic shock, we injected 50 µg/g LPS i.p. and both mouse survival and serum cytokine levels were monitored in WT and GM-CSF R−/− mice. At this high concentration, LPS induces a caspase-1 dependent endotoxin shock caused by elevated levels of pro-inflammatory cytokines like TNF-α and IL-1β It was recently shown that over expression of Rab39a, a member of the RabGTPase family, leads to an enhancement of IL-1β secretion and that its expression is regulated by TLR agonists, like MALP2, LPS and Pam3Cys Although GM-CSF was originally characterized as a haematopoietic growth factor responsible for the differentiation of BM progenitor cells and mobilization of myeloid cells, in the last years GM-CSF has been recognized as a key pro-inflammatory cytokine during inflammation or in response to infection Here we describe a novel and unexpected finding that GM-CSF can act as a strong synergistic enhancer of the inflammasome-dependent IL-1β secretion in response to NF-κB activating agonists, such as LPS, TNF-α and Dectin-1 ligands. In fact, unlike TNF-α, IL-6 and IL-12 whose secretion can be maximally increased only about 2-fold by the LPS/GM-CSF co-stimulation, microbial-induced IL-1β secretion can be synergistically augmented by GM-CSF up to 10-fold. Our data strongly suggest that GM-CSF acts as enhancer of the synthesis of the pro IL-1β form. The enhanced pro-IL-1β levels could be explained by the GM-CSF ability to regulate the IL-1β gene expression at transcriptional as well as post-translational levels as shown by Fernandez et al in human polymorphonuclear leukocytes In addition, we could rule out its role in the cleavage process required for the formation of the IL-1β active form which is actually stimualted by many so called “danger signals” such as extracellular ATP, Alum as well as uric acid. Interestingly, GM-CSF revealed to be the most potent modulator of the IL-1β secretion when compared to other pro-inflammatory cytokines, such as TNF-α, IL-6, INF-γ and M-CSF, which hardly primed its release. Furthermore, the observed synergistic enhancement of pro-IL-1β synthesis upon co-stimulation with LPS/GM-CSF is not a consequence of an augmented GM-CSF-mediated cell survival since no difference in number of viable cells between the two stimulated groups (LPS versus LPS/GM-CSF) was observed during the short window of stimulation (3 h to max 16 h) (unpublished data).Although we have related the “GM-CSF phenomenon” to increased synthesis of pro-IL-1β it is possible, maybe likely, that GM-CSF signalling could also influence molecular pathways controlling the still mysterious and unconventional secretion of IL-1β. In fact, it is reported that GM-CSF triggers granule exocytosis in human neutrophils GM-CSF/LPS combination also increased greatly the expression of NLRP3, caspase-1 and caspase-11 mRNAs in several experiments  suggesting that in some unknown situations the protein levels of the most inflammasome components can be further boosted.c  and C57BL/6 mice were obtained from The Jackson Laboratories , bred and maintained in the animal facility of the Nanyang Technological University under specific pathogen-free conditions.B6.129S1-TLRgrade™ LPS was purchased from Alexis , Bay 11-7082, Wedelolactone, Alum, Pam3CysSK4 and Curdlan from Sigma , MSU, ATP, Nigericin from InvivoGen . Following antibodies were used for western blotting: anti-tubulin, anti-caspase-1 p20, anti-c-Rel and anti-USF-2 , anti-IL-1β antibody (R&D), anti-NLRP3 (Alexis), anti-caspase-11 . Recombinant GM-CSF, TNF-α, IL-6 and IFN-γ were obtained from Biolegend, M-CSF from Milteny Biotech .+ cells were purified by magnetic separation (MiltenyiBiotec). Two types of MØ were generated from BM cells using L929 cellconditioned medium (30%) (L929-derived BM Mφ) or 20 ng/ml M-CSF (M-CSF-derived BM Mφ), respectively.GM-CSF and FLT3L-derived BM DCs were generated by incubating freshly prepared BM cells for 8 days in IMDM medium supplemented with 20 ng/ml GM-CSF or 100 ng/ml FLT3L, respectively. In the case of the FLT3L generated DCs, CD11bCells were stimulated overnight with LPS with or without GM-CSF and subsequently treated for 1 h or 6 h with different danger signals  and cell supernatant was analysed for IL-6, TNF-α, IL-1α and -β by ELISA following manufactures instructions (Biolegend).+ fraction of FLT3L generated BM DCs and L929-derived BM MØ were stimulated for 24 hours with 100 ng/ml LPS in presence or absence of 5 ng/ml GM-CSF. Total RNA was extracted using the Pure Link RNA Isolation Kit (Invitrogen) as per manufacturer's instructions and cDNA was prepared. SYBR green I-based quantitative real-time PCR (QPCR) was performed following manufacture's protocol . Rab39a and Rab39b expression was calculated using a comparative method for relative quantitation upon normalization to HPRT gene expression.CD11bFor detection of the pro IL-1β, caspase-1, caspase-11, NLRP3 and tubulin, as an internal control, lysates of L929-derived MØ and FLT3L-derived DCs were resolved by SDS-PAGE and equal amounts transferred onto nitrocellulose membranes. The blots were developed by chemiluminescence according the manufactures instructions .For detection of the caspase-1 cleaved p20 subunit as well as of the bioactive IL-1β p17 form, L929-derived BM MØ were incubated overnight untreated or with 5 ng/ml GM-CSF, 100 ng/ml LPS or the combination of 5 ng/ml GM-CSF and 100 ng/ml LPS, respectively. After 1 h stimulation with 1 µM nigericin, supernatants were collected and equal amounts of concentrated fractions were separated by SDS-PAGE.+ DCs were kept untreated or stimulated for 20 h with 5 ng/ml GM-CSF, 100 ng/ml LPS or the combination of 5 ng/ml GM-CSF and 100 ng/ml LPS, respectively. NF-κB DNA binding activity of p65, p50, p52 was determined using a DNA-binding ELISA kit  using equal amounts of nuclear extracts. c-Rel and USF-2 as an internal standard were monitored by western blotting.To measure nuclear NF-κB subunits p65, p50, p52 and c-Rel levels, FLT3L-derived CD11bWT and GM-CSFR−/− mice (6–8 weeks old) were i.p. injected with 50 µg/g of LPS from E.coli  The mice were monitored for sign of septic shock and for lethality over a period of 7 days. For serum cytokine analysis, mice were bled 3 h after treatment.Calculations, statistical analysis and graphs were performed on Graphpad Prism 4.0 .This study was carried out in strict accordance with the recommendations of the NACLAR  guidelines under the Animal & Birds  Rules of Singapore. The protocol was approved by the by the Institutional Animal Care and Use Committee (IACUC) of the Nanyang Technological University of Singapore . All efforts were made to minimize the suffering.Figure S1+ FLT3L generated DCs (3×105/well) were primed for 24 h with 100 ng/ml LPS in absence (white bars) or presence (back bars) of 5 ng/ml GM-CSF, 50 ng/ml M-CSF, 50 ng/ml TNF-α, 50 ng/ml IL-6, 50 ng/ml IFN-γ.CD11b 5 mM ATP was added for the last 1 h as a danger signal. Released IL-1β was measured in the culture supernatants by standard ELISA and each value represents the mean of triplicates +/− SD.(TIFF)Click here for additional data file.Figure S2Effect of Bay 11-7082 and Wedelolactone on DC viability. FLT3L derived DCs were pre-treated for 30 min with a different concentration range of inhibitors, stimulated as described in (TIF)Click here for additional data file.


Highly sensitised children have markedly reduced chances of receiving a successful deceased donor renal transplant, increased risk of rejection, and decreased graft survival. There is limited experience with the long-term followup of children who have undergone desensitization. Following 2 failed transplants, our patient was highly sensitised. She had some immunological response to intravenous immunoglobulin (IVIg) but this was not sustained. We developed a protocol involving sequential therapies with rituximab, IVIg, and plasma exchange. Immunosuppressant therapy at transplantation consisted of basiliximab, tacrolimus, mycophenolate mofetil, and steroids. At the time of transplantation, historical crossmatch was ignored. Current CDC crossmatch was negative, but T and B cell flow crossmatch was positive, due to donor-specific HLA Class I antibodies. Further plasma exchange and immunoglobulin therapy were given pre- and postoperatively. Our patient received a deceased donor-kidney-bearing HLA antigens to which she originally had antibodies, which would have precluded transplant. The graft kidney continues to function well 8 years posttransplant. Kidney transplantation remains the optimal treatment for end-stage renal failure (ESRF). However, highly sensitised children continue to have markedly reduced chances of receiving a successful kidney transplant and, if transplanted, they have a greater number of rejection episodes with decreased graft survival . Over thSeveral protocols have been suggested for desensitization of patients who have a positive cross-match against a potential donor, utilizing a combination of IVIg –5, plasmOur patient was a 12-year-old Caucasian girl with ESRF secondary to congenital nephrotic syndrome of the Finnish variety . She hadShe was then maintained on hemodialysis for 8 years without a suitable kidney becoming available. Her antibody levels remained high with a panel reactive antibody (PRA) determined by Complement Dependent Lymphocytotoxic (CDC) of 95% against peripheral blood lymphocytes and 100% against chronic lymphocytic leukaemia (CLL) cells. No suitable living donor was available. After 60 months on the waiting list for a deceased donor transplant, the decision was made to attempt desensitization. After failure of IVIg on its own to desensitize her, a novel regime was developed as outlined below.2), four infusions administered weekly.Rituximab (375 mg/mSingle volume plasma exchange with fresh frozen plasma (FFP) replacement monthly for 3 months.Infusion of IVIg (flebogamma 2 g/kg over 2 days) monthly for 3 months after each plasma exchange.Plasma exchange with FFP replacement.Infusion of IVIg (flebogamma 500 mg/kg).Plasma exchange with 4.5% human albumin replacement.Infusion of IVIg (500 mg/kg) daily for 4 days on day 3, 4, 5, and 6.2).Infusion of rituximab (375 mg/mBasiliximab 10 mg IV 2 hours pretransplant.2 IV.Prednisolone 600 mg/mTacrolimus 0.15 mg/kg IV.Mycophenolate mofitil 250 mg twice daily (14 mg/kg/day).Prednisolone tapered to 10 mg/day by day 7 posttransplant.Basiliximab 10 mg IV day 4 posttransplant.Tacrolimus 0.3 mg/kg/day post transplant adjusted with serum drug levels (aim 12–15 in first 3 months).Our patient received a blood group identical, HLA 1,1,2 mismatch kidney from an unrelated, standard criteria, deceased donor. CDC crossmatch was negative, but flow cytometry crossmatch was significantly positive against both T and B cells. Subsequent investigation of the day of transplant serum showed donor-specific antibodies against A31 and borderline reactivity to DQ2. The A31 and DQ2 antibodies were presumed to be due to transfusion, while the historic reactivity to DR53 was related to previous mismatches.Donor-specific antibodies were monitored by flow cytometry using donor cells or ELISA thrice weekly for 3 weeks, weekly for 6 weeks, and monthly thereafter. Donor-specific antibodies were noted to be mildly raised, day 3 posttransplant, and this was treated with plasma exchange and further doses of IVIg  on day 3, 4, 5, and 6 as outlined above. Donor-specific antibodies were no longer detectable by ELISA. Retrospective reanalysis of stored sera using Luminex single antigen technology has shown persisting donor-specific weak positivity against HLA A31 and DQ2, however, this has remained stable over the last 6.5 years.2 with a serum creatinine of 104 μmol/L.She is now 8 years post transplant and has had no episodes of rejection. Her most recent estimated glomerular filtration rate (eGFR) is 71 mL/minute/1.73 mSensitizing events include previous transplantation, blood transfusions, and pregnancy . As manyAt the time of transplantation, 10% of patients on the renal transplant deceased donor waiting list have >60% panel reactive antibody (PRA) levels to HLA . A high The cautious and cost-effective approach is to firstly maximise the chance of their receiving negative crossmatch grafts. This can be achieved through acceptable mismatch programs  and liviP = 0.02). 3-year graft survival rate was not significantly affected.IVIg is known to have immunomodulatory effects , 4, whenex vivo model of xenograft hyperacute rejection [Plasmapheresis has been used with success in an ejection , in patiejection , 35, in ejection , and as ejection –40. It iejection .The increased understanding of the role played by B-cells in rejection led to the use of agents which specifically target this facet of the immune response . One suc2 with a serum creatinine of 104 μmol/L.We developed a regime which involved sequential therapies with rituximab, IVIg, and plasma exchange. This resulted in a marked sustained reduction in antibody levels, allowing transplantation. Our patient remains well, 102 months posttransplant. She has been successfully transitioned to adult services. Immunosuppression is maintained on MMF and tacrolimus. Her most recent estimated glomerular filtration rate (eGFR) is 71 mL/minute/1.73 m


There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are being used in clinical practice. Anti-aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica (NMO), but we could expect novel CSF biomarkers that help define prognosis and response to treatment for this disease. One of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers. Collaboration between investigators is needed to establish large biobanks of well-defined samples. A key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by pre-analytical factors. Here, consensus guidelines for CSF collection and biobanking are presented, based on the guidelines that have been published by the BioMS-eu network for CSF biomarker research. We focussed on CSF collection procedures, pre-analytical factors and high quality clinical and paraclinical information. Importantly, the biobanking protocols are applicable for CSF biobanks for research targeting any neurological disease. NMO can be diagnosed based on a blood-derived biomarker, that is antibodies against aquaporin-4, a channel protein present on astrocytes, extensively discussed in other contributions in this special issue. The presence of antibodies against aquaporin-4 has been proven as one of the most successful results of biomarker studies, and is supportive for the idea that central nervous system (CNS) abnormalities are reflected in changes in body fluids. It also proofs the autoimmune component of this disorder and of pathologies that are related to the NMO spectrum disorders, such as longitudinally extensive transverse myelitis. Determination of serum anti-aquaporin-4 antibody levels is a mainstay in the diagnosis of NMO, but the discovery of such disease-specific antibodies is relatively recent , and, thhttp://www.bioms.eu/). The aim of this collaboration is to obtain well-proven, high-quality biomarkers, which will be achieved by sharing patient samples, standardization, and improvement of procedures important in the research area. One of the most urgent prerequisites for collaboration was felt to be standardization of biobanking protocols. Therefore, a consensus-meeting was organised and the result was collection and biobanking guidelines, which the network developed and published in 2009 [http://www.bioms.eu/) is a major achievement in the CNS biomarker field [One important flaw in several previously performed biomarker studies in CNS diseases has been the lack of large cohorts to sufficiently power the study. This is especially an issue for such a rare disease as NMO, where a single center will not be able to collect a large cohort within a reasonable time frame. The need for collaboration was the reason for biomarker researchers in Multiple Sclerosis to start a network  centers is allowed. Furthermore, to bring a biomarker to clinical practice, one may need patents and the involvement of industrial partners could be needed, who have the infrastructure for large-scale production, quality control procedures, and to reach as many laboratories as possible. For large-scale validation studies, patient samples are of course also needed, and it will be wise to consider this possibility at the start of biobank formation and, if ethical laws permit and the option is perceived to be important, indicate the possibility for industrial cooperation in the patient information and consent.Lastly, researchers should be willing to share their samples and information for the benefit of the whole, that is, obtaining reliable biomarkers that can be used for patient care and cure.The CSF volume taken can influence the concentration of biomarkers. Most molecules and cell numbers have a rostrocaudal concentration gradient , 7. If aUsually, diagnostic CSF is obtained by lumbar puncture. Because of the increasing gradient in protein concentration from ventricular to lumbar CSF , the sitμL should not be used for biomarker studies.A traumatic tap causing blood contamination of CSF occurs in about 14–20% of standard lumbar punctures . For bioThere is no evidence that the type of lumbar puncture needle influences biomarker concentrations. However, atraumatic needles are best tolerated by patients, and are associated with a lower risk for postlumbar puncture headache, that is about 12% for a needle size of 20–22 G compared to about 70% for a needle size of 16–19 G , 15.β peptides [There are several reports showing that the type of collection tube influences biomarker outcomes, for example, total tau proteins and amyloid peptides . TherefoFor biomarkers that are influenced by circadian rhythm, time of withdrawal is important . Since iIt is important to collect matched serum and/or plasma samples for evaluation of CSF biomarkers because the concentration of the marker in blood often influences that in CSF . FurtherLastly, DNA collection expands the possibilities for studying the phenotypes and genotypes within individuals. A protocol for storage and handling of DNA can be found in the supplementary files (E-Appendix  1).For CSF, there are no data available yet that support a preference for leaving the samples at room temperature or at 4°C until processing. For serum/plasma preprocessing temperature is more crucial. To avoid platelet activation , serum/pWe propose to adhere to a standardized spinning protocol of 400 g for 10 minutes at room temperature when fragile cells need to be preserved for RNA of cell isolation, and otherwise at 2,000 g. For serum/plasma, we propose to spin at 2,000 g for 10 min at room temperature. Standardization of spinning temperature and speed may be important for some biomarkers, although no studies have addressed these specific preanalytical variables for CSF. For plasma and serum, temperature of processing is known to be critical for specific biomarkers . After cStudies of the effects of preanalytical variables by MALDI-TOF/MS proteomics (proteins/peptides <20 kD) have shown that the time between sampling and storage is more crucial for specific serum proteins or peptides than for CSF, , 13, 23.Due to the same rationale as for CSF withdrawal (item 5), we recommend that polypropylene tubes should be used for aliquoting and storage. Furthermore, vials with screw caps should be used for a secure sealing. The proposed tube size is 0.25, 0.5, and 1 mL.β(1-42) which is decreased a further 20% after three more thawing cycles [Freeze/thaw cycles can influence biomarker concentrations . For exag cycles , 27. By g cycles .In principle, repeated freeze/thawing of samples should be avoided, as data addressing this topic are available for only a few biomarkers and the response to freeze/thaw cycles of new biomarkers is not known. Thus, splitting the pooled sample in multiple small aliquots is optimal, and possible freeze/thaw cycles should be recorded.Small aliquot volumes are optimal to avoid freeze/thawing and to avoid waste of CSF. Tubes should be filled up to 75% to prevent freeze-drying within the tube, which will affect the concentration of biomarkers, although it may only be a problem if the seal of the cryogenic tubes are not airtight. This issue has not been formally studied and is not referred to in related standard operating procedures .Unique codes are necessary to track samples and pair with clinical data. Ideally barcodes should be used to facilitate searching, to aid in blinding the analysis, and to protect the privacy of patients. It is important to have center-unique codes, to track data retrospectively. Labels must be water and frost (−80°C) resistant.Proteins may not be stable at −20°C for years. In one study, the effect of storing CSF at −20°C and −80°C on cystatin C, an abundant CSF protein, was investigated. Cleavage of this protein occurred in all samples stored at −20°C but not in samples stored at −80°C . Apart fTaken together, we recommend that samples are stored at −80°C to ensure long-term stability of biomarkers.To enable easy tracking and fast relocation of samples, storage information should include freezer location, freezer identification, and sample location within freezer.Freezers should be alarm controlled and a sample rescue plan established and documented. All freezers must be registered in a freezer log file. Ideally, daily temperature logs should be available for all freezers. Aliquots of samples should be distributed among different freezers, although not absolutely needed if good surveillance is in place. An empty, an empty back-up freezer should be available.Transport of frozen samples should always be performed on dry-ice, and the volume should be sufficient for transport for minimal 3 days. Preferably, transports are initiated on Monday for the samples to arrive within the same week. Once the samples have arrived and are ready for experiments, excessive thawing temperatures (such as 37°C) are to be avoided to prevent protein degradation. Furthermore, inadequate agitation can cause salt and protein gradients to form in thawed samples.Information on the age at sampling is needed to allow comparability to age-matched reference values, since many proteins show age-dependent changes, for example, albumin or IgG . IdeallyReference ranges of biomarkers can be influenced by the genetic status . For exaIt is well known that commonly used drugs for treatment of MS, including immunomodulatory agents and use of methylprednisolone for treatment or prevention of relapses, have an influence on expression of biomarkers , 35. OthOther relevant physiological variables that can influence CSF and blood analyte levels should be recorded including fasting versus non fasting, pregnancy, and underlying nonneurological conditions such as infections .To enable stratification of patients according to their CSF findings and to evaluate suitability of samples for further analysis, results of basic CSF analysis should be recorded. Primarily, the CSF profile serves for exclusion of other diseases. In addition, quantitative changes of immunological markers are likely to occur depending on disease stage, relapse activity, and medication. Inflammatory processes may influence the blood-CSF barrier function and thereby biomarker concentrations .The presence of Oligoclonal IgG bands (OGB) in NMO is quite distinct from that in MS in that OGBs in MS are persistent, while they are transient in NMO , 37. ForThe mask on the database screen could be in the local language, but the underlying files will need to be in English. It is strongly suggested to use a commercially available program, if not a common database for networks like BioMS-eu. The database should also adhere to standardized international units.The lists provided in


Allogeneic hematopoietic stem cell transplantation  is an effective and sometimes the only curative therapy for patients with certain hematological diseases. Allo-HSCT has been practiced in China for approximately 30 years, and great improvements have been made within the past decade, particularly in fields such as the haploidentical HSCT system, strategies to overcome relapse and GVHD, and modified HSCT for elderly patients. This review will describe the current situation and provide a prospective of these unique aspects of Allo-HSCT in China. It has been more than thirty years since the first allogeneic bone marrow transplantation was successfully performed in China , and durCurrently, there are 104 transplant units certified by China's Ministry of Health and the China Marrow Donor Program. Of these centers, 60% are active and routinely perform HSCT. According to data collected from 50 active centers by the Chinese Hematopoietic Stem Cell Transplantation Registry Group 2007-2011), 5 centers performed more than 100 HSCT cases annually, and approximately 30 centers performed 20-100 cases per year. The total number of HSCT cases in all 50 active centers increased steadily from 1093 cases in 2007 to 1633 cases in 2010 [007-2011,The types of donor sources for Allo-HSCT in China are related identical 47.3%), related mismatched/haploidentical (30.8%), unrelated matched 12.1%), unrelated mismatched (7.7%), umbilical cord blood  and Allo-HSCT accounts for 91% of the total HSCT cases.  annual meeting. Also, HSCT in China is not just imitation, there are some characteristic aspects which may contribute to other HSCT programs around the world.These unique characters of Chinese HSCT system can be divided into four categories: ① Haplo-HSCT system, ② strategies to overcome relapse and ③ GVHD, and ④ modified HSCT for elderly patients. The initiation of multi-center clinical trials and advances in translational research are important for promoting progress in HSCT methods in China.Haplo-HSCT is one of the acceptable alternatives in the absence of HLA-matched siblings or unrelated donors ,6. Therein vitro T cell depletion within the past 10 years (the GIAC protocol). This protocol includes the following steps: treating donors with granulocyte colony-stimulating factor (G-CSF) to induce donor immune tolerance, intensified immunological suppression to both promote engraftment and prevent GVHD, antithymocyte globulin (ATG) administration for the prophylaxis of GVHD and graft rejection, and combination of G-CSF-primed bone marrow harvest (G-BM) and G-CSF-mobilized peripheral blood stem cell harvest (G-PB) as the source of stem cell grafts [Due to the "One Child Policy", there has been a shortage of HLA-identical siblings for Chinese patients who need HSCT; what's more, the donor pool of Chinese Marrow Donor Program and cord blood banks are still relatively small in view of the massive Chinese population , Therefol grafts -21. A sil grafts ,21. The l grafts . Also, ml grafts ,23. Besil grafts . Based oThough the HLA polymorphism of China is more heterogeneous than that of Japan and Korea, as there are 56 different nationalities in China and the main nationality "Han" also varies in HLA alleles across the vast mainland -28; Haplin vitro and in vivo studies: ①G-CSF leads to T cell hyporesponsiveness and modulates the balance between Th1 and Th2 immune responses [+ and CD8 + T cells in bone marrow grafts. It was also found easier to polarize T cells from Th1 to Th2 in G-BM compared with G-PB [Nevertheless, there are still several problems confronting the future development of haplo-HSCT. First, although the underlying mechanism of crossing the HLA barrier has been partially explained by a series of esponses ; ②G-CSF ith G-PB ; ③T cellith G-PB . ④Depletith G-PB , etc. Hoith G-PB , our finith G-PB ; additioith G-PB , which nRelapsing hematologic malignancy after HSCT remains a major cause of death for patients, and finding methods to trigger a more potent graft-versus-tumor effect without increasing the risk of GVHD is still the ultimate goal of HSCT. Many Chinese researchers have attempted to address this challenge.In a recent study, Huang and his colleagues provide the first report of multiple mutations of CEBPA that contribute to the transformation of donor cells to the leukemic phenotype and provide clues of relapse from donor origin. Huang found that after the first genetic mutation involved in leukemogenesis occurs in the donor, the susceptible donor cells can acquire second or third mutations in different genes or recessive mutations in a single gene after transplantation to the recipient's particular microenvironment. This acquisition of mutations can lead to overt leukemic transformation and cause relapse. This mechanism provides a unique viewpoint that aids our understanding of the process of relapse due to the "multiple genetic-hits mechanism" and suggests that the targeting of mutated genes may be a possible strategy to prevent relapse . MeanwhiThere is a large gap between laboratory research and clinical application; however, by integrating minimal residual disease (MRD) monitoring and modified donor lymphocyte infusion (mDLI), researchers at Peking University have improved the management of relapse following HSCT in the clinic . TraditiGVHD is the main complication post-HSCT, and Chinese doctors must address this condition due to the high proportion of haplo-HSCT cases. Thus, several novel strategies to prevent GVHD have been devised.The application of low-dose MTX has been demonstrated as a novel regimen for the treatment of GVHD by Peking University research group in different studies. Data from a recent report suggested that MTX in combination with low-dose methylpredinisone was well-tolerated and effective when used as the first-line treatment for aGVHD . It has Lai et al. recently described the efficacy of a new GVHD prophylaxis regimen that combines CSA and MTX with a short 30-day course of low-dose (500 mg/d) mycophenolate mofetil. After allo-HSCT with identical sibling donors, the incidence of acute GVHD was 16% (9.5% for grades 2-4 GVHD and 1% for grades 3 and 4 GVHD). The cumulative incidence of chronic GVHD was 53%, and 28% of these patients experienced the extensive type of disease . Xia et Nevertheless, most of the studies mentioned above are retrospective, and thus, further prospective and multi-center, randomized, controlled studies are needed.In addition to drugs and antibodies, immunomodulatory cell transfusion may be a more specific method to protect patients from GVHD. Chen et al. reported that heterogenetic bone marrow mesenchymal stem cells (MSCs) could suppress T cell activation. MSC pretreatment was useful in the prevention of GVHD in HLA-mismatched bone marrow transplantation . Xiang eBasic and Pre-clinical researches on GVHD have also been highly regarded in China. Zhao showed blockade of osteopontin(OPN) could reduce alloreactive CD8+ T cell-mediated GVHD in animal model: OPN promoted the migration and infiltration of naive and alloreactive CD8(+) T cells into host organs; it also facilitated activation and viability of donor-derived CD8(+) T cells via synergizing with CD3 signaling. This findings could validate OPN as a potential target in GVHD prevention . As mentThere are also many promising pre-clinical studies focused on the potential application of immune cells in controlling GVHD, such as umbilical cord blood (CB) derived stromal cells , ex vivoIn short, the progression of these basic and pre-clinical studies would hopefully ameliorate the clinic practices of controlling GVHD in near future.The outcome of transplantation in patients older than 60 years of age remains unsatisfactory with a low complete remission (CR) rate and poor overall survival (OS) due to prolonged pancytopenia and an elevated chemotherapy-related mortality, etc. . AllogenMouse models infused with high doses of G-CSF-mobilized allogeneic spleen cells after cytarabine chemotherapy without immunosuppressive pretreatment exhibited rapid hematopoietic recovery and persistent microchimerism without GVHD. Ai and colleagues designed a clinical control study to show the superiority of "mini-HSCT" over chemotherapy for elderly patients suffering from AML. This HSCT protocol mainly consisted of induction chemotherapy plus the infusion of HLA-mismatched G-PBSCs. Patients who received this treatment had a higher complete remission rate and disease-free survival over 2 years with no observable GVHD. These results indicate that G-PBSCs in combination with conventional chemotherapy may provide a promising treatment method for AML in elderly patients. This finding warrants further multi-center studies to confirm the results and determine if the same method can be applied in the treatment of ALL or other hematopoietic malignancies . BesidesAs mentioned above, multi-center prospective studies and researches connecting "bench to bed" are essential to promote the development of HSCT in China. Actually, there have been successful multi-center trials conducted in general hematology and molecular diagnostics, such as the prospective study "Molecular Diagnosis and Individualized Treatment of Acute Leukemia" supported by the National High Technology Research and Development Program (Program 863). Nevertheless, many challenges remain in the HSCT field: (1) Compared with many well developed international registries, for example, the European Group for Blood and Marrow Transplantation (EBMT), the Chinese Hematopoietic Stem Cell Transplantation Association must make considerable progress in establishing itself as a leader in clinical trials in addition to its role in data management . (2) StaNext, two strategies can be employed to promote the connection between basic research and clinical practice in the field of Chinese HSCT. Firstly, Chinese researchers must make significant progress in identifying the underlying mechanisms of well-developed clinical models, such as haplo-HSCT. This unique diagnosis and treatment system in turn provides a perfect opportunity to analyze and compare the differences among clinical and animal models. Secondly, translational researchers must pursue promising cellular or animal experiments. For example, researchers from Jilin University, China, found that IFN-γ promotes graft-versus-leukemia effects without directly interacting with leukemia cells in mice after HSCT . TherefoIn summary, it would be very meaningful for Chinese scholars to participate in translational researches of clinical significance, such as immune tolerance in HLA-mismatched HSCT, the distinction between GVHD and GVL, and the association between infection and chronic immunologic imbalance.China has made substantial progress in the field of allo-HSCT during the past three decades, particularly in the clinical applications and mechanism researches of haplo-HSCT system, strategies to overcome relapse and GVHD, and modified HSCT for elderly patients. With the development of multi-center clinical trials and advances in translational research, these unique fields of allo-HSCT in China will have a promising future and contribute more to the world HSCT system.HSCT: Hematopoietic stem cell transplantation; BMT: bone marrow transplantation; GVHD: Graft versus host disease; CIBMTR: Center for International Blood and Marrow Transplant Research; Allo: Allogeneic; ATG: Antithymocyte globulin; G-BM: G-CSF-primed bone marrow harvest; G-PB: G-CSF-mobilized peripheral blood stem cell harvest; MRD: Minimal residual disease; mDLI: Modified donor lymphocyte infusion; MSCs: Mesenchymal stem cells; CR: Complete remission; OS: Overall survivalThe authors declare that they have no competing interests.ML collected data and wrote the manuscript, XJH designed and revised the manuscript. All authors read and approved the final manuscript.


The cerebrospinal fluid (CSF) levels of the proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR) are increased in Alzheimer’s disease (AD), but whether CSF PKR concentrations are associated with cognitive decline in AD patients remain unknown. In this study, 41 consecutive patients with AD and 11 patients with amnestic mild cognitive impairment (aMCI) from our Memory Clinic were included. A lumbar puncture was performed during the following month of the clinical diagnosis and Mini-Mental State Examination (MMSE) evaluations were repeated every 6 months during a mean follow-up of 2 years. In AD patients, linear mixed models adjusted for age and sex were used to assess the cross-sectional and longitudinal associations between MMSE scores and baseline CSF levels of Aβ peptide (Aβ 1-42), Tau, phosphorylated Tau (p-Tau 181), PKR and pPKR. The mean (SD) MMSE at baseline was 20.5 (6.1) and MMSE scores declined over the follow-up . A lower MMSE at baseline was associated with lower levels of CSF Aβ 1–42 and p-Tau 181/Tau ratio. pPKR level was associated with longitudinal MMSE changes over the follow-up, higher pPKR levels being related with an exacerbated cognitive deterioration. Other CSF biomarkers were not associated with MMSE changes over time. In aMCI patients, mean CSF biomarker levels were not different in patients who converted to AD from those who did not convert.These results suggest that at the time of AD diagnosis, a higher level of CSF pPKR can predict a faster rate of cognitive decline. Alzheimer’s disease (AD) is classically marked by the progressive occurrence of memory disturbances followed by aphasia, apraxia and agnosia associated with behavioral symptoms The goal of the present study was to determine in a longitudinal cohort of AD and aMCI patients the possible links between the rate of cognitive decline and the initial levels of CSF biomarkers including PKR and pPKR. Our results show that CSF pPKR concentration can predict the future cognitive decline in AD patients.41 consecutive patients with a diagnosis of AD have been recruited from our outpatient Memory Clinic between January 2010 and January 2011, as previously described This work has been approved by the Ethics Committee of Paris University Hospital  and all patients or caregivers gave their written informed consent for this study. Next of kin, care takers or guardians consented on the behalf of participants whose capacity to consent was compromised. Usually, patients with mild AD forms signed the consent, for moderate AD forms patients and care givers signed the consent and in severe AD forms care takers signed the consent.Lumbar punctures were performed in fasting patients in the following month after the clinical diagnosis. CSF was collected in 12 mL polypropylene tubes. Within 2 hours, CSF samples were centrifuged at 1800 g for 10 minutes at 4°C. An appropriate part of CSF was used for routine analysis, including total cell count, bacteriologic exam, and total protein and glucose levels. CSF was aliquoted in polypropylene tubes of 500 µL and stored at −80°C until further analysis. CSF Aβ 1-42, Tau, and p-Tau 181 were measured with Innotest sandwich enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s procedures . Positivity criteria for biomarkers were defined by anomalies of Aß and T-tau or p181tau levels, according to the cutoffs of our center . PKR and pPKR CSF levels were analyzed by western blots procedures as previously reported (13). Results of PKR and pPKR are expressed in optical density units (ODU). All biological analyses were done in a single hospital laboratory. The biological team involved in the CSF analysis was unaware of the clinical diagnosis. The quality of CSF evaluations was validated by a European Consortium .Linear mixed models were used to study the relationship between baseline levels of CSF biomarkers and repeated measurements of MMSE scores. The intercept and the slope (time) of the models were treated as random effects, allowing them to vary between individuals, with an unstructured covariance matrix. Time in months since baseline was included as a continuous linear term after verification that a quadratic term did not provide a better fit. We used z-scores to estimate standardized regression coefficients β that allow comparing the strength of the relations between MMSE and the different biomarkers. Models included one of the CSF biomarkers, age, sex, time and the interaction between time and the different parameters.Results were also shown using tertiles of patients according to CSF pPKR at baseline. In a complementary analysis, 11 patients with amnestic MCI, coming from our previously reported cohort P-values were two-tailed and values ≤0.05 considered as statistically significant. Analyses were performed using SAS 9.2 .Baseline characteristics of the study cohort are depicted in The Our results show that the level of CSF pPKR is associated with a more pronounced cognitive decline, as measured with the MMSE, in a cohort of patients newly diagnosed with AD. While CSF Aβ 1-42 levels and p-Tau 181/Tau ratio were cross-sectionally associated with MMSE score at the time of the diagnosis, pPKR was the only biomarker to be linked to the cognitive decline over the follow-up survey.Several previous studies have reported an association between CSF biomarkers and cognitive change in AD, and results remain debated. In a previous study, one cluster of AD patients with extreme levels of CSF biomarkers were characterized by a faster progression of their cognitive deficit, no response to cholinesterase inhibitor treatment, and a higher mortality In a previous study we have shown that CSF PKR and pPKR levels were increased in AD patients as compared to neurological disease controls The second question that can be posed is to know if analyzing CSF pPKR levels could be useful at the onset of the clinical disease. Although classical biomarkers are very useful to predict the clinical outcome in patients with MCI There are some limitations in our study. Firstly, the number of patients included in our study remains limited. The lack of association between CSF biomarkers levels and conversion from MCI to AD we observed may be probably linked to the small number of MCI patients in our sample. A larger multicentric study including other types of dementia as well as MCI patients is needed to confirm the link between pPKR and the cognitive decline. Secondly, other indicators such as volumetric MRI or PET scan DFG glucose could also be compared with pPKR in future studies. Thirdly, the patients have been followed up during a mean period of time of 26 months. This is obviously limited and longer surveys are needed in order to detect exacerbated cognitive decline. We did not include the neurological controls of our initial discovery cohort of patients because those patients were suffering from an heterogenous group of diseases with various clinical evolutions. In addition, apolipoprotein E genotyping should be performed in future studies to determine a possible influence of ApoE4 genotype on pPKR and other biomarker CSF levels In conclusion, we report that pPKR is a biological indicator of the cognitive decline in AD patients that 1- could be utilized to complement the current CSF biomarkers, 2- could be used in future clinical trials, 3 - could represent a possible pharmacological target for future therapeutic approaches