Coeliac disease (CD), due to its protean clinical manifestation, is still very under diagnosed in adults and delays in diagnosis may take years and even decades. Simple tools to find cases in primary care may help to identify patients for further diagnostic tests. We have evaluated the usefulness of an on site rapid fingertip whole blood point-of-care test (POCT) for such a purpose.As CD is known to run within families, we tested 148 healthy relatives of 70 Romanian index cases with biopsy-proven CD  for the presence of circulating autoantibodies. In addition to performing the POCT (which measures blood erythrocyte self-TG2-autoantibody complexes) on site, blood was drawn for later evaluations of serum IgA-class endomysial antibodies (EMA). EMA-positive sera were further tested for transglutaminase 2 antibodies (TG2-IgA). All serological parameters were analyzed blindly in a centralized laboratory that had no knowledge of the on site POCT result. Endoscopic small intestinal biopsies was recommended for all POCT- or EMA-test positive subjects.In on site testing the POCT was positive in 12/148 first-degree relatives (8%) and all these subjects were also serum EMA-positive. A positive EMA test was found only in one other subject. All remaining 135 healthy first-degree relatives were negative for both POCT and EMA. Four subjects positive for both POCT and EMA were negative for TG2-IgA. Ten out of thirteen of the antibody-positive subjects agreed to undergo endoscopy. The POCT was found to be positive in 8/9 first-degree relatives having coeliac-type mucosal lesions of grade Marsh 2 (n = 3) or Marsh 3 (n = 6). The three POCT-positive subjects not agreeing to undergo endoscopy were also both EMA- and TG2-IgA-positive.The fingertip whole blood rapid POCT might fulfill the unmet need for a simple and cheap case-finding biomarker for early detection and presumptive diagnosis of CD. Confirmatory studies are warranted in adult case-finding in specialized outpatient clinics and in primary care. Coeliac disease (CD) is a systemic disorder triggered by the ingestion of gluten, a protein present in wheat, rye and barley. Gluten injures the small intestinal mucosal lining leading to villous atrophy and crypt hyperplasia . IndividIt is well known that CD runs within families, and first-degree relatives of patients are at risk of developing the disorder -15. By rActive case-finding in primary care using sensitive and specific biomarkers of CD might be a solution to shorten diagnostic delays. Centralized laboratories provide autoantibody tests such as endomysial (EMA) and antitransglutaminase 2 (TG2) IgA antibody tests ,26. Howe170 first-degree healthy relatives of 70 index cases (77 children and 15 adults) were invited over a five years period (2007–2012) to be evaluated for the presence of coeliac-autoantibodies. The inclusion criterion for the index cases was biopsy-proven CD . Of the 170 subjects, 148  agreed to participate. The remaining 22 could not been reached or declined. At the time of the evaluation, all participants confirmed that they were consuming a regular gluten-containing diet.The screening for the presence of CD autoantibodies in the healthy relatives took place on the same day as the diagnostic examinations of the index patients or in certain cases later, during regular follow-up visits. All centralized laboratory serologic determinations were made in blinded fashion without knowledge of the on-site fingertip whole blood POCT results.The Biocard Celiac Test, AniBiotech, Vantaa, Finland, was selected as the point-of-care biomarker test for CD case-finding. This rapid whole blood self TG2-based IgA-class fingertip test uses the patient’s own endogenous TG2 found in the erythrocytes of a whole blood sample. When the finger-tip blood is haemolyzed, the liberated TG2 forms a complex with circulating autoantibodies, if present in the same sample. Blood erythrocyte self-TG2-autoantibody complexes are captured from the haemolyzed sample by TG2-binding proteins onto a solid phase and the presence of autoantibodies is measured by labeled antihuman IgA . The resOn the same day when the POCT was performed, venous blood was drawn for laboratory serum autoantibody measurements. Serum IgA-class antiendomysial antibodies (EMA) were determined using an indirect immunofluorescence method  with cut-off for positivity at a serum dilution of 1:5. Further serum dilutions  were made for initial positive samples till the highest positive titer was obtained for each sample. In the present series, no cases of selective IgA deficiency were found.Serum samples showing positivity in the EMA test were further evaluated for TG2-IgA  using an enzyme-linked immunosorbent assay (ELISA) with a positivity cut-off at 5 U/ml, as recommended by the product manufacturer.Upper gastrointestinal endoscopy with small-bowel mucosal biopsies was recommended for all subjects with positive POCT or EMA tests. During endoscopy, up to 5 duodenal mucosal specimens were taken. The biopsy specimens were fixed in formalin, embedded in paraffin, cut, stained with haematoxylin-eosin and scored by pathologists (AE and FV) according to the Marsh-Oberhuber classification  ,30.The study was approved by the Ethical Committee of the University and Pharmacy “Carol Davila” and Ethical Committee of the Institute for Mother and Child Care “Alfred Rusescu” Bucharest, Romania. The antibody-positive subjects or their parents gave written informed consent for upper gastrointestinal endoscopy.In total, 12 out of 148 (8%) first-degree healthy relatives showed positivity in the POCT. All POCT-positive subjects proved to be EMA-positive, giving 100% specificity for the POCT against the reference standard for serum CD autoantibody . Only onAltogether 10/13 antibody positive family members were adults (median age 38.5 years). Four subjects positive for both POCT and EMA were negative for TG2-IgA when using the manufacturer’s positivity cut-off of 5 U/ml. When again using 3 U/ml as cut-off, as suggested by the new ESPGHAN 2012 criteria , only onTen of the thirteen antibody-positive first-degree relatives agreed to undergo upper gastrointestinal endoscopy and duodenal biopsies. All but one showed coeliac-type mucosal lesions of grade Marsh 2 (n = 3) and Marsh 3 (n = 6), as summarized in Table The present study emphasizes the usefulness of a simple rapid fingertip whole blood POCT for a presumptive diagnosis of CD. The test was able to select correctly individuals, even clinically silent ones, for further confirmatory diagnostic studies such as duodenal biopsies. We show that the POCT tested on site worked hand in hand with the centralized laboratory golden standard CD autoantibody testing, the EMA test ; among 1To confirm or disprove our hypothesis that the used POCT biomarker is able to pick out CD also in adults, we chose to study first-degree family members of CD patients. In fact, 77% of the family members positive for the test were adults and our results are in agreement with those previously obtained in children ,27,28. TOn-site use of the POCT seems to provide a strong tool for the health care system in case-finding of CD. This offers immediate results and potentially shortens the diagnostic process in adults -19. MoreA limitation in the present study is the relatively small population studied. The study should be repeated in larger adult patient populations in primary care and specialized outpatient clinics dealing with patients with vague and various symptoms or signs where CD could be the underlying cause of ill health.The serologic prevalence of CD in our study in Romania was 8.7%, similar to that reported in other family studies -15. The In conclusion, case-finding in the primary care using a fingertip whole blood on site POCT could be the way forward in the early detection and diagnosis of celiac disease.CD: Coeliac disease; POCT: Point-of-care test; EMA: Endomysial antibodies; TG2-IgA: Transglutaminase 2 antibodies; HLA: Human leukocyte antigen; ELISA: Enzyme-linked immunosorbent assay.Ani Biotech, who donated the point of care test kits, did not take part in designing or executing the study, were not involved in reading the results or in writing the manuscript. MM is one inventor of the patent “Methods and Means for Detecting Gluten-Induced Diseases”, USA States Patent Number 7,361,480 - USA, Patent Granted 22.4.2008; European Patent No. 1390753, European Patent Office 22.10.2008. Finn Medi Oy Ltd, owned by the University of Tampere and Tampere University Hospital, Finland, has commercialized the innovation and licensed it to Ani Biotech, which used it to develop the Biocard Celiac Test. Finn Medi and Ani Biotech were not involved in the study. The other authors declare no competing interests.AP contributed to the conception and design, supervised all aspects of the evaluation, carried out the collection and analysis of data, wrote the initial version of the manuscript and approved the final manuscript as submitted. MJ provided supervision in all aspects of the evaluations, including design, clinical examination, endoscopic biopsies critically revised the manuscript and approved the final version as submitted. CJ contributed to the literature search and analysis of data, clinical management of adult participants, critically revised the manuscript and approved the final version as submitted. VB made a substantial contribution to data collection and analysis, assisted with the initial manuscript preparation and approved the final version as submitted. CB made a substantial contribution to all aspects of evaluation, including clinical examination and management of pediatric participants, acquisition and analysis of data, revised the manuscript and approved the final text as submitted. AE and FV made a contribution on the interpretation of data, provided histological interpretation of the mucosal biopsies, critically revised the manuscript and approved the final text as submitted. KL contributed substantially to the data results, revised the manuscript and approved the final version as submitted. IA contributed substantially to the design and interpretation of data, critically revised the manuscript for important intellectual content and approved the final manuscript as submitted. MM is the senior author and expert in the field; he was responsible for the conception, design and the interpretation of data, contributed to obtaining of funding, made a substantial contribution to the manuscript preparation, including review, critique and advice on each version of the manuscript, directed the literature search and approved the final version of the manuscript as submitted.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/13/115/prepub


Although selective or whole body cooling combined with optimal intensive care improves outcomes following neonatal encephalopathy in high-income countries, the safety and efficacy of cooling in low-and middle-income countries is not known.We performed a systematic review and meta-analysis of all published randomised or quasi-randomised controlled trials of cooling therapy for neonatal encephalopathy in low-and middle-income countries.Seven trials, comprising a total of 567 infants were included in the meta-analysis. Most study infants had mild (15%) or moderate encephalopathy (48%) and did not receive invasive ventilation (88%). Cooling devices included water-circulating cooling caps, frozen gel packs, ice, water bottles, and phase-changing material. No statistically significant reduction in neonatal mortality was seen with cooling . Data on other neonatal morbidities and long-term neurological outcomes were insufficient.Cooling therapy was not associated with a statistically significant reduction in neonatal mortality in low-and middle-income countries although the confidence intervals were wide and not incompatible with results seen in high-income countries. The apparent lack of treatment effect may be due to the heterogeneity and poor quality of the included studies, inefficiency of the low technology cooling devices, lack of optimal neonatal intensive care, sedation and ventilatory support, overuse of oxygen, or may be due to the intrinsic difference in the population, for example higher rates of perinatal infection, obstructed labor, intrauterine growth retardation and maternal malnutrition. Evaluation of the safety and efficacy of cooling in adequately powered randomised controlled trials is required before cooling is offered in routine clinical practice in low-and middle-income countries. Neonatal encephalopathy occurs in 1 to 3 per 1000 live births in high-income countries, and in up to 20 per 1000 live births in low and middle-income countries. Recent meta-analysis of therapeutic cooling trials conducted in high-income countries show that whole-body, or selective, head cooling reduces mortality (risk ratio (RR) 0.78;  0.66 to 0.93), and improves survival with normal neurological outcome after neonatal encephalopathy The burden of neonatal encephalopathy in low-and middle-income countries is far higher than in high-income countries, and it accounts for approximately one million deaths annually We performed a systematic review, and meta-analysis of the published literature on the safety and efficacy of cooling therapy in low-and middle-income countries.We included all randomised, or quasi-randomised controlled trials comparing either selective head or whole body cooling , with standard care, in term or near term infants with neonatal encephalopathy consequential to perinatal asphyxia.Asphyxia was considered if at least one of the following criteria was met: Apgar score ≤5 at 5 min, cord or arterial blood pH ≤7.1, base deficit ≥12 mmol/L within the first hour of life, or ongoing resuscitation or mechanical ventilation at five minutes of life. Neonatal encephalopathy was defined by detailed neurological examination performed before randomisation. Studies without a standard care arm were included in the systematic review, but were excluded from the meta-analysis.The primary outcomes were (i) neonatal mortality and (ii) moderate or severe neurodevelopmental disability at ≥18 months of age. Secondary outcomes were blood infections within the first week of life, coagulopathy or thrombocytopenia requiring blood products, respiratory failure requiring ventilator support, and hypotension requiring intervention.We used standard Cochrane Neonatal review group  Two review authors (SSP/ST) independently identified the studies to be included, extracted the data, and assessed the study quality based on allocation concealment, blinding of outcome assessment, adherence to intention to treat analysis, and completeness and quality of follow up 2 test .The effect of cooling therapy in settings that lack basic neonatal care may be very different to those with good neonatal care. Hence, we sub-grouped the studies based on the quality of neonatal intensive care support  We examined publication bias using a funnel plot We identified a total of 18 studies on cooling therapy in low-and middle-income countries, of which 11 were excluded . Thus we analysed the data on 567 infants recruited to seven clinical trials . The inclusion criteria for most of the studies were low Apgar score at 5 minutes and evidence of encephalopathy within 6 hours after birth . Four stMost studies used indigenous low technology cooling methods. For whole-body cooling these included ice and frozen gel packs (n = 2) Target rectal temperatures were 33 to 34°C 2 = 16%; p = 0.26). Data on encephalopathy stage was available from six trials – 15% of the infants had mild, 48% moderate and 22% severe encephalopathy.Primary outcome  was available from a total of 567 infants recruited to the seven randomised controlled trials Three studies reported culture positive neonatal blood infections; again no significant difference was seen in infection rates (RR 0.98 (95% CI 0.26 to 3.61)  [15], [2Four studies reported the use of respiratory support; only 12% of infants were ventilated. Although, sedation was not routinely given to all infants undergoing cooling therapy, some studies used morphine The data were inadequate to examine other important short-term secondary outcomes like coagulopathy, respiratory failure, and hypotension. Zhou reported two cases with scalp edema and scleroderma in their study using cooling caps Three studies reported long term neurodevelopmental outcomes. Bharadwaj and Bhat reported adverse neurodevelopmental outcome at age six months in 107 (82%) of the 130 infants in their trial. Two cooled infants and 12 standard care infants had developmental delay  Three trials were in upper mid income countries One study used quasi-randomisation based on whether recruitment was on an odd or even calendar day Although no asymmetry was seen on the funnel plot , only 7 The pooled data from seven low-and middle-income country randomised controlled cooling trials, including a total of 567 infants, did not show a statistically significant reduction in neonatal mortality ; however the confidence intervals were wide, thus clinically important benefits or harm cannot be excluded. Most studies were small and/or of poor quality, and did not evaluate long-term neurological outcomes or other important neonatal morbidities.Our data suggests that there are a number of key differences between high-income country and low-and middle-income country cooling trials with regards to the study population, inclusion criteria, and techniques used. For example, in low-and middle-income country trials most recruited infants were reported to have mild or moderate encephalopathy. More importantly, the vast majority of the low-and middle-income country infants did not require invasive ventilatory support, suggesting that the degree of encephalopathy was much lower. This is in contrast with high-income country cooling trials, and the current practice in high-income countries where the vast majority of infants undergoing cooling also require respiratory support. Alternatively, this may be due to lack of facilities for providing optimal ventilatory support in low-and middle-income countries. Sedation was not routinely used with cooling in many studies, and this may be due to concerns about respiratory compromise.In addition, often babies were hypothermic at randomisation, and the intrinsic hypothermia persisted for a few hours even in the control arm of some studies. This may be due to lack of radiant warmer facilities. Furthermore, unlike high-income country cooling trials, no hyperthermia was reported in the standard care infants. This may have diluted the net treatment effects of cooling.Cooling was administered using a number of low-technology devices; this included ice It is unclear whether the apparent lack of treatment effect, seen in our meta-analysis can be attributed to the use of such low technology devices, and possible inadvertent over or under cooling. Although, one high-income country cooling trial had previously reported the use of ice for whole body cooling The lack of treatment effect  may be also due to the heterogeneity and methodological weakness of the studies included in this meta-analysis. Such a possibility cannot be excluded due to wide confidence intervals of the pooled data. Alternatively, there may be a genuine lack of treatment effect of hypothermia in these settings. There are several arguments that could support this hypothesis. For example, medically induced hypothermia without optimal intensive care may be harmful. It is possible that mechanical ventilation was underused or not optimally monitored in these settings. High concentrations of oxygen Another major concern of cooling therapy in low-and middle-income country relates to the extensive literature on the association of increased mortality with hypothermia, and a potential worsening of sepsis with cooling The lack of outcome data in many studies may be due to the difficulty in undertaking long term follow-up in these settings. Attrition rate in a cooling trial in sub Saharan Africa was particularly high, and only 53% of the infants could be followed up at one year of age A recent National Institute of Child Health and Human Development workshop of cooling therapy experts concluded that the safety and efficacy data on cooling from high-income countries should not be extrapolated to low-and middle-income countries In summary, no reduction in neonatal mortality was seen following selective head or whole body cooling after neonatal encephalopathy in low-and middle-income countries, however, confidence intervals were wide, and significant harm cannot be excluded. Although, low technology devices effectively provided whole body cooling, neuroprotective efficacy and additional resource implications of these devices remain unclear. More importantly, the clinical characteristics of encephalopathic infants in low-and middle-income countries are different to that of high-income countries, and therefore the safety and efficacy data on cooling from high-income countries cannot be extrapolated to low-and middle-income countries. Adequately powered clinical trials are required to before cooling can be considered as a therapeutic option in low-and middle-income countries. One such multi-country trial–‘HELIX’  is currently being set up


Overview. The GiACTA trial is a multicenter, randomized, double-blind, and placebo-controlled study designed to test the ability of tocilizumab (TCZ), an interleukin (IL)-6 receptor antagonist, to maintain disease remission in patients with giant cell arteritis (GCA). Design. Approximately 100 centers will enroll 250 patients with active disease. The trial consists of a 52-week blinded treatment phase followed by 104 weeks of open-label extension. Patients will be randomized into one of four groups. Group A (TCZ 162 mg weekly plus a 6-month prednisone-taper); group B (TCZ 162 mg every other week plus a 6-month prednisone-taper); group C (placebo plus a 6-month prednisone-taper); and group D (placebo plus a 12-month prednisone taper). We hypothesize that patients assigned to TCZ in addition to a 6-month prednisone course are more likely to achieve the primary efficacy endpoint of sustained remission (SR) at 52 weeks compared with those assigned to a 6-month prednisone course alone, thus potentially minimizing the long-term adverse effects of corticosteroids. Conclusion. GiACTA will test the hypothesis that interference with IL-6 signaling exerts a beneficial effect on patients with GCA. The objective of this paper is to describe the design of the trial and address major issues related to its development. Giant cell arteritis  is an inflammatory disease of medium- and large-sized arteries that affects individuals older than 50 years of age . The disGCA and its current treatment strategies carry a substantial morbidity burden. The most feared consequence of the disease is blindness , but mulAttempts to control disease activity and spare CS with other agents have generally failed –11. Tria4-positive T lymphocytes, including IFN-γ producing T helper (Th) 1 cells and IL-17-secreting Th17 cells, orchestrates the formation of granulomatous vascular inflammation  and 162 mg every other week [162q2w]) will be compared to placebo. All patients will receive background CS therapy. Three groups  will follow a prespecified prednisone-taper regimen over 26 weeks, and a fourth group (group D), designed to reflect an alternate standard of care, will receive a 52-week prednisone taper see . The primary efficacy endpoint, sustained remission (SR), will be evaluated at 52 weeks. Remission is defined as the absence of signs and symptoms attributable to GCA and normalization of ESR (<30 mm/Hr) and CRP (<1 mg/dL). Other definitions used in the trial are described in The purpose of Part 2 is to determine the long-term safety and maintenance efficacy of TCZ, to explore a potential requirement for maintenance treatment beyond 52 weeks, and to gain insight into the long-term CS-sparing effect of IL-6R blockade. Those who achieve the primary endpoint will stop their blinded SC injections and be followed for maintenance of response. Patients with persistent disease activity or those who experience a flare after week 52 will have the option to receive open-label TCZ (162 mg weekly) with or without increase in background CS dose at the discretion of the investigator. The duration of open-label therapy will be determined by the investigator according to the patient's clinical condition. The GiACTA research group comprises 100 sites in the USA and Europe. The trial is funded by F. Hoffmann-La Roche, Ltd (Switzerland). Mechanistic studies are supported in part through a grant from the Arthritis Foundation.Each participating clinic will have institutional review board oversight. All participants will give written informed consent.The primary objective of GiACTA is to evaluate the efficacy of TCZ in combination with a six-month prednisone taper to sustain remission through 52 weeks. To meet this endpoint, a patient must maintain disease remission after remission induction by week 12 of randomization, complete the assigned prednisone-taper protocol, and not flare or require escape therapy at any time until week 52. The primary efficacy analysis will compare groups A and B against group C see . We hypoSecondary and exploratory objectives of the trial are to evaluate safety, the impact of TCZ on cumulative CS exposure, long-term remission maintenance beyond 52 weeks, patient-reported outcomes, and to compare the TCZ arms against the 52-week prednisone taper. Pharmacokinetic-pharmacodynamic (PK-PD) studies, immunogenicity assays, and mechanistic investigations will also be performed.The eligibility criteria for the trial are summarized in n = 100); TCZ 162qw + 26 weeks of prednisone + 26 weeks of prednisone placebo (n = 50); TCZ 162q2w + 26 weeks of prednisone + 26 weeks of prednisone placebo (n = 50); and TCZ-placebo + 26 weeks of prednisone + 26 weeks of prednisone placebo (n = 50). TCZ-placebo + 52 weeks of prednisone  and whether the patients present with new onset or refractory/relapsed disease.Two hundred and fifty patients will be randomized through an interactive voice response system (IVRS) in a 2 : 1 : 1 : 1 ratio to the different study arms as follows : TCZ 162Subjects will receive either TCZ or matching TCZ placebo by SC injection once a week. The dose modification guidelines and risk mitigation strategy used for TCZ in RA will be implemented in GIACTA . Measures designed to protect against unblinding are addressed in the Discussion section. The investigator will determine the initial prednisone dose from the time of enrollment based on the disease history (new onset or refractory/relapsing disease), severity of activity , and comorbidities . By definition, patients with refractory/relapsing GCA will have active disease despite previous or concurrent CS therapy. In the last case, an increase in CS dose will be required upon enrollment. Permitted ranges of starting doses for any patient will be 20–60 mg daily. From the dose chosen at baseline, the patients will proceed with a protocol-defined tapering schedule depending on the group assigned . “Pulse”There will be two standardized prednisone-taper regimens: one that extends for 26 weeks  and the other for 52 weeks (group D) . The totThe prednisone tapers will have an open-label phase and a blinded phase . PrednisConcomitant treatment with stable doses of MTX will be allowed only if started more than 6 weeks prior to the study enrollment. The dose of MTX should remain stable and not be increased throughout the screening and 52 week double-blind treatment periods. During these periods, the MTX dose may be reduced or discontinued if necessary for safety reasons, or, if in the opinion of the investigator, it is no longer required to treat the patient's GCA. Other immunosuppressive medications will not be allowed during the study . Patients who cannot follow the protocol-defined prednisone taper due to disease flare will continue in the study under an investigator-defined open-label escape CS regimen in combination with double-blind injections of TCZ/TCZ placebo for the full 52 weeks. Although these patients will be deemed as nonresponders for the primary outcome, their subsequent followup and CS requirement will provide important information for the analysis of secondary and exploratory endpoints. Clinical GCA activity that will be evaluated at every study visit include: fever (≥38°C or 100.4°F); symptoms of PMR; headache; temporal artery or scalp tenderness; visual signs or symptoms such as acute or subacute vision loss due to arteritic anterior ischemic optic neuropathy, transient blurry vision, amaurosis fugax, or diplopia; jaw claudication; new or worsened extremity claudication; and other features judged by the investigator to be consistent with a GCA flare.Laboratory Assessments. Laboratory assessments to be obtained at different timepoints will include: complete blood counts; serum chemistry profile including renal and liver function tests; fasting serum lipids; HbA1c; hepatitis B and C serologies; and ESR and CRP. In order to maintain blinding, the investigator/efficacy assessor will be masked to the results of complete blood counts, serum chemistry, ESR, and CRP.Serological PD-PK Measures and Immunogenicity Assays. Assays designed to evaluate pharmacodynamic and pharmacokinetic parameters of TCZ will include concentrations of CRP, IL-6, sIL-6R, TCZ, and anti-TCZ antibodies.Patient-Reported Outcomes. Planned patient-reported outcomes include the patient global assessment of disease activity (PGA) using a VAS scale and the SF-36, FACIT Fatigue, and EQ-5D questionnaires.Safety Assessments. Review of adverse events, vital signs, concomitant medications, and laboratory data will be performed.Mechanism of Action Studies. Serum, RNA, and DNA samples will be collected at defined timepoints for mechanistic studies.The efficacy outcome measures include the assessment of disease remission based on signs and symptoms of active GCA; ESR and CRP; adherence to the prespecified prednisone taper; the number of flares and the time to disease flare postremission; the cumulative CS dose; and patient-reported quality of life (QoL) assessments.The safety outcome measures include the incidence, nature, and severity of adverse events; laboratory abnormalities , and immunogenicity.achievement of disease remission not later than week 12 after randomization;absence of disease flare through week 52;completion of the assigned prednisone taper protocol;absence of the need for escape therapy (prednisone increase) at any time during the 52 weeks after randomization. Remission is defined as the absence of signs and symptoms attributable to GCA and normalization of ESR (<30 mm/Hr) and CRP (<1 mg/dL).The definition of the primary endpoint, SR at week 52, has four aspects:Patients who do not achieve remission within 12 weeks; do not reach a prednisone dose of zero by the predefined time ; requireThe analysis of the data will be performed on an intention-to-treat basis, including all randomized subjects.For the primary efficacy endpoint, the TCZ treatment groups will be compared to placebo (in combination with 26 weeks prednisone-taper regimen) using a Cochran-Mantel-Haenszel test, adjusting for new onset versus refractory/relapsing disease and starting prednisone dose .For the analysis of secondary endpoints, all continuous endpoints such as SF-36, PGA, and cumulative CS dose will be analyzed using a maximum likelihood repeated measures method to compare the change from baseline to the week 52 value. The analyses will be adjusted for the stratification factors applied at randomization, as well as the baseline value for the parameter being tested. The time to disease flare will be analyzed using a Cox proportional hazards model including stratification factors. The secondary endpoints will be tested using a prespecified fixed sequence method, and adjustments required to control for multiplicity  and to ensure that the alpha level is maintained at 1% will be made. Both TCZ treatment groups will be compared to the 52-week prednisone-taper group for all the study endpoints in an exploratory manner using the methodology as specified previously. Further exploratory analysis considering variables such as temporal artery biopsy status, compliance with 1990 ACR versus modified diagnostic criteria, type of disease flare , and concomitant MTX use will be also performed.The safety analyses will include all randomized patients who received at least one dose of study drug. Patients will be reported according to the treatment received. ρ6-TCZ = 70% versus ρ6-mCS = 30%). In addition, 50 patients will be included in the 52-week prednisone-tapering group (D).A sample size of 100 patients in the A (162qw TCZ) group and 50 patients in both the B (162q2w TCZ) and C (placebo) groups will ensure at least 90% power to detect a difference in the proportion of patients in sustained remission at week 52 for both TCZ arms versus placebo at an alpha level of 0.01 (2 sided). This assumes that the absolute difference in the proportion of patients who are in sustained remission at 52 weeks is equal to 40% . However, we anticipate that data from patients in this fourth arm will provide valuable information regarding the optimal dosing regimens of CS in GCA and help evaluate the risk-benefit ratio of TCZ as a CS-sparing agent.An independent Data and Safety Monitoring Board (DSMB) plans to review accumulated data on safety and efficacy at least twice a year and/or in an event-driven fashion as necessary.Peripheral blood will be collected at different timepoints to evaluate the impact of anti-IL-6R therapy on the concentrations of selected cytokines and chemokines. Genome-wide mRNA expression profiling will be done at baseline and 12 months. The current standard of care for GCA, CS therapy, is a suboptimal treatment strategy. A high percentage of patients relapse upon weaning treatment and more than 85% of the patients suffer from CS-related side effects , 15, 44.a priori in order to prevent the unblinding of treatments that some expected laboratory changes could generate. Finally, although requiring a masked prednisone-taper phase that begins once patients reach 20 mg/day creates certain logistical challenges, this necessity has led to a novel trial design that will accurately assess the potential of TCZ for exerting CS-sparing effects in this disease. We address these issues, one by one. Controlled studies in GCA must address several challenges. First, although management guidelines have been created for large-vessel vasculitis and GCA , 58, the≈60 mg/day). This dose is given until the reversible manifestations of the disease abate and the inflammatory markers normalize (≈4 weeks). The dose is then gradually tapered to reach a maintenance of 10 mg/day by 2.5–6 months, and 5 to 0 mg/day by 12–24 months (and sometimes longer) [Large vessel vasculitis (LVV) guidelines address the initial CS dose but do not standardize the rate of tapering in GCA . The app longer) . Subject longer) . More re longer) .In contrast to the variability of clinical practice that often leads to CS use extending over years, clinical trials have frequently used tapers over a period of six months. Unfortunately, the investigational agents employed to date in these studies have not been successful in reducing the number of disease flares in a meaningful manner, and annual relapse rates in some trials have been as high as 80% , 15. To address the problem of variability in the standard of care, GiACTA will use two prednisone-tapering regimens as control arms. The six-month taper was created for the efficacy analysis, to test the ability of TCZ to maintain disease remission and spare CS. Since many patients in the course of usual care receive courses that are longer than six months, we will also enroll patients into a comparative 12-month prednisone-tapering arm (group D). It should be noted that the study will not be powered to find a statistically significant difference between the TCZ arms and the 12-month prednisone-tapering arm, but this group will provide substantial information on the natural history of CS-treated disease and serve for exploratory analysis. To prevent unmasking due to the different tapering protocols, both the patients and investigators will be blinded to the dose of prednisone given after the daily dose has been reduced to 20 mg/day (week 7) through the end of Part 1 (week 52). The ideal agent for a chronic immune-mediated disease would restore tolerance and induce drug-free remissions following treatment. More realistically, a useful agent for GCA should be able to maintain disease remission, while decreasing the toxicity associated with CS therapy. CS-free sustained remission at 52 weeks, cumulative CS dose, CS-related AEs, CS-TCZ-free sustained remission, and quality of life measures comprise a set of meaningful endpoints that will rigorously test the efficacy of TCZ in GCA. Furthermore, assuming an absolute treatment effect difference of 40% between the experimental and the control groups, and setting the alpha level at 0.01 for the analysis of the primary endpoint will assure robustness of the results if the trial meets its primary outcome. We anticipate that the vast majority of subjects enrolled in GiACTA will meet the ACR 1990 classification criteria for the diagnosis of GCA . HoweverTo prevent unblinding due to expected rapid normalization of inflammatory markers or the occurrence of specific laboratory abnormalities sometimes observed during TCZ therapy , a safety assessor independent of the investigator/efficacy assessor will monitor the results of the following laboratory parameters: ESR, CRP, total white blood cell count (WBC), absolute neutrophil count (ANC), platelet counts, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations. The safety assessor will determine the indication for dose modification of TCZ (or TCZ placebo) following guidelines and risk mitigation strategies used for TCZ in the RA studies. We have considered the possibility that blinding the investigators to the levels of acute phase reactants could create problems during the longitudinal follow-up, since many clinicians rely at least in part on levels of these markers as guides to the degree of ongoing inflammation. As an example, the ESR and CRP values may help in differentiating non-specific musculoskeletal pain from a flare of PMR during CS dose reduction. However, the levels of acute phase reactants can be misleading, with both false-negative and false-positive results. Overtreatment with CS following excessive emphasis on the values of inflammatory markers places patients at risk for additional CS-related morbidity. For this reason, once patients are enrolled, only clearly defined signs and symptoms will be used as parameters of disease activity, and only investigators with expertise in the care of patients with GCA will participate.To our knowledge, this is the first clinical trial in any disease to incorporate blinded CS regimens into its design. This strategy, which will permit an unbiased evaluation of the CS-sparing effects of TCZ, may be extrapolated to trials in other immune-mediated conditions, thereby helping to address an important need in the area of inflammation. The largest trial performed to date in GCA offers important opportunities for exploratory substudies. In this regard, analyzing the effect that IL-6 signaling blockade has on different components of the Th1, Th17 and Treg pathways may elucidate the mechanisms of action of TCZ and increase the understanding of the immunopathogenesis of this disease. Moreover, genome-wide transcriptome analysis may identify important pathogenic factors, new biomarkers, and gene signatures associated with critical clinical outcomes .In summary, the GiACTA study will assess the safety and efficacy of TCZ for the treatment of GCA. The use of CS dose blinding is unique in the field of clinical trials of immune-mediated disease and may be extended to other investigations in which the identification of a CS-sparing agent is important. Successful achievement of the primary outcome in this trial would mark the first demonstration of an effective alternative to continuous CS use as a strategy for maintaining remission in GCA.


A gluten-free diet (GFD) is currently the only available treatment for patients with celiac disease (CD). Several clinical trials have demonstrated that most celiac patients can tolerate a medium-high quantity of oats without any negative clinical effects; however, the inclusion of oats in GFD is still a matter of debate. In this study, Italian children with CD were enrolled in a 15-month, randomized, double-blind, placebo-controlled multicenter trial. Participants were randomized in two groups following either A-B treatment , or B-A treatment . A and B diets included gluten-free (GF) products  with either purified oats or placebo. Clinical data  and intestinal permeability tests (IPT), were measured through the study period. Although the study is still blinded, no significant differences were found in GSRS score or the urinary lactulose/mannitol (L/M) ratio between the two groups after 6 months of treatment. These preliminary results suggest that the addition of non-contaminated oats from selected varieties in the treatment of children with CD does not determine changes in intestinal permeability and gastrointestinal symptoms. Celiac disease (CD) is an immune-mediated disorder, triggered in genetically susceptible individuals by ingested gluten, the alcohol-soluble complex present in wheat, rye, and barley. The clinical spectrum of CD is extremely variable, including (a) typical CD, with the classical features of intestinal malabsorption; (b) atypical CD ; (c) silent CD, ; (d) potential CD, showing positivity of celiac serology associated with a normal  intestinal mucosa at the small intestinal biopsy. The cornerstone of treatment is the lifelong exclusion of gluten-containing cereals from the diet, the gluten-free diet (GFD). in vitro, suggesting that there are differences between oat varieties in relation to their safety/toxicity for people with CD ). Differences between groups were assessed by the Mann-Whitney U test. The Wilcoxon matched pairs test was used to calculate the differences for pair data. A p = 0.14). One hundred-seventy one children received at least 6 months of treatment, 75 patients received A treatment (group A), and 96 received B treatment (group B). Clinical and biochemical data from these subgroups were considered for the purpose of this preliminary analysis. Overall, 306 children  were enrolled in the study . Patients’ enrollment was concluded in March 2013. Fifty-five out of 154 patients enrolled in group A-B (35.7%) and 42/152 patients in group B-A (27.6%) dropped out from the study within the first 6 months of treatment  and 6 months of treatment .Furthermore, comparing the 6-month change in urinary L/M ratio (Δ-L/M), no difference was found between groups A and B, suggesting no relevant impact of a 6-month period of either oats or placebo-added GFD on the intestinal permeability test .The 6-month Δ L/M value did not show a significant trend according to the different age groups (corresponding to different amount of oats or placebo) a,b.In a large group of CD children that had not previously consumed oats, we found that the prolonged intake of a considerable amount of daily oats did not determine any relevant change in terms of clinical symptoms and intestinal permeability, even though the results were analyzed in a blind fashion.Gastrointestinal symptoms were evaluated using the GSRS, that is an interview-based rating scale consisting of 15 items, validated and widely used for assessment of GI symptoms both in adults and children ,22,23,24i.e., the IPT. The integrity of the intestinal barrier can be evaluated by the IPT, by quantifying the amount of orally administered substances recovered in urine. Mannitol (monosaccharide) and lactulose (disaccharide) are largely used as permeability probes due to their different intestinal permeability index. Mannitol is absorbed through enterocyte membrane’s pores (transcellular pathway), while lactulose is absorbed through tight junctions (paracellular pathway). Ratio between the percentage of lactulose and mannitol excretion represents the permeability index that reflects the intestinal epithelium integrity. The non-invasive functional IPT is a sensitive tool for both triage of active celiac disease, as well as for monitoring celiac patients on a GFD [Besides a few exceptional cases reported in the literature , previouon a GFD . In pation a GFD ,28. Convon a GFD . Furtheron a GFD  or 6-weeon a GFD  gluten cIn our study, IPT was chosen as a non-invasive surrogate measure to detect minimal damages of the small intestinal mucosa at different time points. Thus far, no study has been performed to investigate the effects of prolonged oats ingestion on the intestinal permeability test in patients with CD. We did not find significant changes in urinary L/M ratio either in group A or B. This finding is further supported by the comparison of the Δ L/M in the two groups, again failing to show any difference between groups. However, the final results of our large study will give more information about the possible effect of oats ingestion on the results of the intestinal permeability. More data will also be available on the correlation between permeability indexes and serological CD markers. The daily amount of suggested intake of oats products was calculated to guarantee medium-high amounts of oats (up to 40 g/day for older children). Although it was not possible to analyze the data according to the amount of ingested oats, the evaluation of the IPT results in the three age groups (corresponding to the different quantity of study products suggested) again showed no significant differences between groups. Overall, the lack of changes in intestinal permeability suggests that the varieties of gluten-uncontaminated oats chosen for production of these oat-based items are safe. The sample analyzed for the purpose of this analysis represents more than 50% of the enrolled study group. However, this analysis has not considered the original cross-over design of the protocol. Such a limitation implies caution in the interpretation of the results.The preliminary results of this investigation suggest that the oats varieties used for producing the gluten-uncontaminated products used in this study are safe when administered for a 6-month period of time. These preliminary conclusions are based on both clinical data and the results of a non-invasive test for intestinal mucosa barrier function (IPT). The final analysis of the data collected in this study will provide conclusive data on the safety and the acceptance of these oat-based products in Italian children in treatment with the GFD.


Misfolded proteins accumulating outside the bacterial cytoplasmic membrane can interfere with the secretory machinery, hence the existence of quality factors to eliminate these misfolded proteins is of capital importance in bacteria that are efficient producers of secretory proteins. These bacteria normally use a specific two-component system to respond to the stress produced by the accumulation of the misfolded proteins, by activating the expression of HtrA-like proteases to specifically eliminate the incorrectly folded proteins.S. lividans causing secretion stress permitted the identification of a two-component system (SCO4156-SCO4155) that regulates three HtrA-like proteases which appear to be involved in secretion stress response. Mutants in each of the genes forming part of the two-genes operon that encodes the sensor and regulator protein components accumulated misfolded proteins outside the cell, strongly suggesting the involvement of this two-component system in the S. lividans secretion stress response.Overproduction of alpha-amylase in S. lividans, a bacterium that has been repeatedly used as a host for the synthesis of homologous and heterologous secretory proteins of industrial application.To our knowledge this is the first time that a specific secretion stress response two-component system is found to control the expression of three HtrA-like protease genes in  Streptomyces bacteria As in most bacteria, the Sec pathway is the main route for secretory proteins exported across the cytoplasmic membrane in Escherichia coli bacterium by the CpxRA two-component system and the extra-cytoplasmic sigma factor σEThis secretion stress is sensed in the Gram-negative Bacillus subtilis, bacterium secretion stress is sensed at least by the CssRS two-component system (a homologue of the E. coli CpxRA) cssS or cssR-null mutants are viable, the high level of alpha-amylase (AmyQ) secretion results in a significant reduction in the growth rate of the mutant strains htrA or htrB do not have a noticeable effect on growth or secretory protein yield In the Gram-positive Streptomyces lividans, in particular, has often been used as a host for the secretory production of homologous and heterologous proteins In order to adapt to their natural environment, largely formed of insoluble polymers, Streptomycetes produce and secrete large quantities of suitable proteins Streptomyces lividans when alpha-amylase is oversynthesised.Hereby we report the identification of a novel CssRS-like two-component system governing the expression of three HtrA-like proteases, HtrB, HtrA1 and HtrA2 that seem to form part of the secretion stress response in Streptomyces coelicolor A3(2) Streptomyces lividans, a search for the S. lividans homologue to the two-component system of the B. subtilis CssRS was performed. From the several potential equivalent systems found, the SCO4156-SCO4155 proteins, showing 34% and 21% identical residues in stretches of 170 and 301 residues respectively to the corresponding B. subtilis counterparts, were selected, due to them being the only two-component system with a gene encoding a HtrA-like protease (SCO4157) adjacent to it, a similar chromosomal organisation to that of B. subtilis. SCO4156-SCO4155 seems to form a unique transcriptional unit where the stop codon of the potential SCO4156 transcript overlaps with the start codon of the SCO4155 transcript, a situation widely accepted as a strong suggestion of both genes forming a single transcriptional unit, as is the case with the equivalent B. subtilis and E. coli genes. SCO4157 shares 32% identity with the HtrB protease of B. subtilis in a stretch of 271 residues. In a further search for the best homologues to the B. subtilis HtrA-like protease three additional candidates were identified: SCO2171, SCO3977 and SCO5149, with 35%, 35% and 36% identity in stretches of 288, 297 and 284 residues, respectively. A similar degree of identity was obtained in all cases when the deduced amino acid sequences of the different proteins were compared to the E. coli equivalents. Thus, the two component system SCO4156-SCO4155 was named CssRS and proteases SCO4157, SCO2171, SCO5149, SCO3977, were named HtrB, HtrA1, HtrA2 and HtrA3, respectively, and henceforth will be referred to as such.Based on the complete genome sequence of S. lividans ΔcssS and S. lividans ΔcssR mutant strains having disrupted cssS or cssR genes respectively were constructed, as indicated in Materials and Methods.cssR or cssS was down regulated in their respective mutant strains, as expected. Expression of htrB, htrA1 and htrA2 was also down regulated in each of the two mutant strains  at 24 hours of growth. The expression of  strains , stronglBacillus amyloliquefacies activates the CssRS two-component system in B. subtilisStreptomyces lividans, the pAMI11 plasmid harbouring the S. lividans amlB gene under the control of its own promoter was propagated in the wild type and cssS, cssR mutant strains, to generate S. lividans TK21 (pAMI11), S. lividans ΔcssS (pAMI11) and S. lividans ΔcssR (pAMI11), respectively. The mutant strains did not seem to show differences in growth rate in liquid medium with that of the wild type strain, as described in B. subtilisamlB in the mutant strains seemed to cause some minor constraint in the growth rate of the mutant strains when compared to the wild type strain equally overexpressing amlB, which is far from what was reported for B. subtilis, and particularly in the case of the B. subtilis CssR deficient strain oversynthesising AmyQ High-level production of alpha-amylase (AmyQ) of cssS, cssR and the three HtrA-like protease genes was analysed by qRT-PCR in the S. lividans wild type and the CssR and CssS deficient strains overproducing AmlB. The results showed that all genes were induced at 24 h of growth (amlB overproduction in the mutant strains was unable to trigger induction of any of the genes (not shown), again confirming the regulatory role of the cssRS operon on the S. lividans secretion stress response.The expression of f growth , whereasS. lividans cell by the Sec pathway. Proteins secreted via the Sec route are translocated in a misfolded manner and need to be properly folded before attaining their functional state in the supernatant. Overproduction of misfolded proteins triggers the induction of the CssRS two-component system and synthesis of the specific set of HtrA-like proteases. Some specific proteins secreted outside the cell in a fully-folded state use a minor secretion route, the so-called Tat pathway. Therefore, when overproduced, these proteins are unlikely to induce the CssRS system, and the S. coelicolor agarase (DagA) has been shown to be one of the proteins able to use the Tat route dagA gene was propagated in multicopy in S. lividans (S. lividans (pAGAs5) strain), the relative level of expression of the cssR, cssS and the three HtrA-like encoding genes remained unaltered (not shown), thus confirming that the likely accumulation of a misfolded secretory protein of the Sec route, such as AmlB, was responsible for the induction of the cssRS two genes operon and, subsequently, that of the three genes encoding the HtrA-like proteases.AmlB is a secretory protein that is transported and secreted outside the S. lividans TK21 (pIJ486) carried out as a control (not shown).The synthesis and secretion of the overproduced AmlB protein in the wild type and the CssR or CssS deficient strains were monitored by Western blot analysis. No precursor or mature forms of alpha-amylase were revealed by the anti-AmlB serum as associated to the cellular fraction of the wild type or the mutant strains, and AmlB was exclusively detected in the cultured cell supernatants in all cases , stronglThe observed molecular size (59 kDa) for the mature alpha-amylase was in accordance with that predicted from the derived amino acid sequence. The maximum level of mature AmlB appeared at the exponential phase of growth of the wild type bacterium, decreasing when the culture reached the stationary phase .The maximum level of extracellular mature enzyme was mainly detected at the exponential phase of growth in the CssR- and CssS-deficient strains , althougS. lividans secretion stress response.To investigate whether the alpha-amylase accumulated in the supernatant of both mutant strains was properly folded, the activity of the enzyme was measured and compared to that of the alpha-amylase present in the supernatant of the wild type strain. The alpha-amylase secreted in both mutant strains retained one third of the activity of that of the wild type strain at the exponential phase of growth, being inactive at later phases of growth . The difThe existence of quality systems to avoid the accumulation of misfolded proteins outside the cytoplasmic bacterial membrane is of paramount importance, since this protein accumulation is a major obstacle for the effective production of many secreted homologous or heterologous proteins in Gram-positive bacteria Streptomyces lividans, a well-known efficient secretory strain.We have identified a two-component system and three proteases that appear to participate in the elimination of misfolded proteins in Streptomyces two-component systems that seem to share homology with the Bacillus subtilis CssRS proteins we selected three possible two-component systems as candidates, two of them encoding the two sensor proteins with the highest degree of homology (SCO5283-5282 and SCO2142-2143) and SCO4156-SCO4155, whose genes were the only ones close to a potential HtrA-like coding gene within a chromosomal organisation similar to that of the Bacillus subtilis cssRS two genes operon. The expression of the possible two-component systems was analysed in a strain of S. lividans oversynthesising alpha-amylase, S. lividans TK21(pAMI11). Only the SCO4156-SCO4155 operon was upregulated while the expression of the other two remained unaltered (data not shown), strongly suggesting that the upregulated operon was likely to be the one involved in the S. lividans secretion stress response. In fact, the transcriptional studies performed in the CssR deficient strain revealed the polar effect exerted on the cssS expression by the cssR mutation, thus suggesting the functionality of both genes as a single transcriptional unit, as is indeed the case in the B. subtilis and E. coli equivalent operons Among the 24 B. subtilisThe presence of an HtrA-like protease forming part of the secretion stress response has been found in several Gram-positive strains other than B. subtilis harbours two genes encoding HtrA-like proteins in its genome and both genes are modulated by a two–component system whose expression is triggered by the presence of secretory misfolded proteins S. lividans two genes cssRS operon, although novel, may not be too surprising. In this regard, it is worth mentioning that overproduction of the DegU regulator in S. coelicolor, a closely related strain to S. lividans, does trigger overexpression of the major Sec route secretory proteins S. coelicolor CssRS two-component system and the HtrA-like genes resulted also up regulated in the DegU overproducer strain  and cssSdisrup_Rv (5′GGTGAATTCTTGTCCAGGATGTTGACCAC 3′) were used to amplify a 627 nt DNA fragment from the S. lividans TK21 genome. To construct the cssR mutant strain, oligonucleotides cssRdisrup_Fw ( 5′GTTGGATCCACCTGGTCGTCCTGGACAT 3′) and cssRdisrup_Rv (5′GGTGAATTCACGGCCTTCAGGATCTGCT 3′) were used to amplify a 421 nt DNA fragment from the S. lividans TK21 genome. Both fragments were inserted into plasmid pOJ260 BamHI and EcoRI sites to generate plasmids pOJS and pOJR respectively, later used to conjugate E. coli to Streptomyces, as described E. coli ET12567 carrying the non-transmissible “driver” plasmid pUZ8002 was used for conjugation S. lividans ΔcssS and S. lividans ΔcssR, containing the disrupted genes cssS and cssR, respectively, were selected upon verification of the disruption by PCR amplification and Southern blot hybridization analysis.S. lividans gene amlB and a frame-shift-mutated thiostrepton resistance gene which was used to transform the S. lividans TK21, S. lividans ΔcssS and S. lividans ΔcssR protoplasts to generate S. lividans TK21(pAMI11), S. lividans ΔcssS (pAMI11) and S. lividansΔcssR (pAMI11).Plasmid pAMI11 S. coelicolor agarase gene (dagA) and a frame-shift mutation in the thiostrepton resistance gene S. lividans TK21 to obtain S. lividans TK21 (pAGAs5). Plasmid pIJ486 was propagated into the S.lividans TK21, cssS and cssR mutants to generate the corresponding isogenic strains.Plasmid pAGAs5 is a pAGAs1 Total RNA was isolated from bacteria growing cultures using the RNeasy midi Kit (Qiagen). Cell lysates were extracted twice with phenol-chloroform before being loaded onto RNeasy midi columns for RNA purification. DNA potentially contaminating the RNA preparations was removed by incubation with RNase-free DNAse (Ambion) and its absence was tested by quantitative real time PCR amplification in the absence of reverse transcriptase. Complementary DNA was synthesised using the High Capacity Archive kit (Applied Biosystems). Quantitative real time PCR (qRT-PCR) was performed using SYBR Green technology in an ABI Prism 7300 Sequence Detection System (Applied Biosystems). Samples were initially denatured by heating at 95°C for 10 min. A 40-cycle amplification and quantification program was then followed (95°C for 15 sec and 60°C for 1 min) by a single fluorescence measurement per cycle, according to the manufacturer's recommendations. Subsequently a final extension cycle  was performed. Three biological samples from the different bacterial cultures were amplified in triplicate in separate PCR reactions. All PCR products were between 50 and 150 bp in length.5′-GGACAAGCTGGCGAACTC -3′) and HRDBR (5′-CCTCCAGCAGGTGGTTCT -3′) were used to amplify the hrdB transcript carried out as an internal control to quantify the relative expression of the target genes. The hrdB transcript has currently been used as a reference to normalise the relative expression of Streptomyces genes A melting curve analysis was conducted after amplification to distinguish the targeted PCR products from the non-targeted ones. The melting curves were obtained by heating at temperatures ranging from 60°C to 95°C at a rate of 0.2°C per sec, with continuous fluorescence scanning. Oligonucleotides HRDBD  followed by incubation with HRP-conjugated protein A (Invitrogen Laboratories) diluted 1∶10,000 in PBS containing 5% (w/v) skimmed milk for 40 min at room temperature For Western blot analysis, proteins were fractionated by SDS-PAGE in 10% (w/v) acrylamide gel 4, as described To determine extracellular alpha-amylase activity, the supernatants from 20-ml aliquots of bacterial cell cultures at the indicated phases of growth were concentrated by precipitation with ammonium sulphate brought to 80% saturation; the precipitated protein was collected by centrifugation at 13,000×g for 30 min and dissolved in 20 mM phosphate buffer (pH 7). The activity of alpha-amylase activity was estimated by determining the amount of reducing sugar released from starch. The assay was carried out by adding supernatant sample and starch solution 1% (w/v) treated with NaBHThe protein concentration in the different samples was determined using the BCA protein assay kit (Pierce), as indicated by the supplier.Table S1Quantitative RT-PCR analysis of the two-component system and the three genes encoding HtrA-like proteases in the S. coelicolor DegU overproducer strain. Quantitative RT-PCR analysis of the two-component system and the HtrA- like protease genes in the S. coelicolor DegU overproducer strain . The results correspond to the mean of at least three biological replicates, standard deviation are shown. hrdB was used as the reference gene to quantify the relative expression of the target genes. Oligonucleotide primers used to amplify the transcripts are indicated in (DOC)Click here for additional data file.


To explore if stimulus–response (S-R) characteristics of the silent period (SP) after transcranial magnetic stimulation (TMS) are affected by changing the SP definition and by changing data presentation in healthy individuals. This information would be clinically relevant to predict motor recovery in patients with stroke using stimulus–response curves.t-tests. The Bonferroni correction was used to adjust for multiple testing.Different landmarks to define the SP onset and offset were used to construct S-R curves from the biceps brachii (BB) and abductor digiti minimi (ADM) muscles in 15 healthy participants using rectified versus non-rectified surface electromyography (EMG). A non-linear mixed model fit to a sigmoid Boltzmann function described the S-R characteristics. Differences between S-R characteristics were compared using paired sample For the BB, no differences in S-R characteristics were observed between different SP onset and offset markers, while there was no influence of data presentation either. For the ADM, no differences were observed between different SP onset markers, whereas both the SP offset marker “the first return of any EMG-activity” and presenting non-rectified data showed lower active motor thresholds and less steep slopes.The use of different landmarks to define the SP offset as well as data presentation affect SP S-R characteristics of the ADM in healthy individuals. Transcranial magnetic stimulation (TMS) of the human motor cortex is a noninvasive technique to assess the integrity of the corticospinal motor pathways. Transcranial magnetic stimulation of the primary motor cortex elicits a motor evoked potential (MEP) as an excitatory effect that can be recorded by surface electromyography (EMG) of the target muscles. In pre-activated muscles, TMS also induces a transient suppression of the EMG-activity after the short-latency MEP, i.e. the silent period (SP), as an inhibitory effect -4. Both level of muscle pre-activation [Comparison of the studies focusing on the role of the SP in post-stroke motor recovery is difficult due to variability in patients’ characteristics and the time post stroke, but also due to differences in the applied methodology. Indeed, the technique to elicit and assess the SP is all but standardized . Althougtivation -15, it vtivation .In this perspective, Kimiskidis and associates  designedYet, even if the SP is studied independently of the corticomotor threshold, lack of uniformity regarding the landmarks to define the SP onset and offset may influence its duration . In addiEighteen healthy individuals participated in this study. Because it has been reported that the threshold for eliciting a SP in upper-extremity muscles on the dominant side is lower than on the non-dominant side , only riParticipants were comfortably seated in a chair with their right forearm and hand supinated and supported by a custom built device Figure . The elbFirst, EMG-activity was recorded from the biceps brachii muscle (BB). The forearm and hand were supinated and EMG-activity of the BB was recorded while participants performed elbow flexion against a fixed frame. Secondly, to obtain isometric abductor digiti minimi (ADM) muscle contractions, right digit V abduction was performed against a fixed frame, while digits II-IV were immobilized by Velcro straps. For measurements of both proximal and distal muscles, participants were instructed to exert maximum force for 3 seconds during 3 trials. The maximal voluntary EMG-activity was defined as the mean EMG-amplitude achieved during these 3 trials.Transcranial magnetic stimulation of the motor cortex was performed through a 90 millimeter circular coil powered by a Magstim 200 magnetic stimulator (Magstim Company Limited). The vertex was located and marked directly on the scalp. The coil was positioned in a tangential plane near the vertex at approximately 45 degrees to the sagittal line  and fixed in this position through a mechanical arm. A counterclockwise inducing current flow was used to activate the left hemisphere.During the TMS sessions, the participants performed constant isometric muscle contractions at 50% of their maximal voluntary EMG-activity to ensure optimal facilitation of motoneurons ,25. VisuThe EMG was recorded in two files. In one file the data were stored after rectification, whereas the raw (non-rectified) data were stored in a different file. At each stimulus intensity, the SP duration was determined from the average of 5 trials. The active motor threshold was defined as the minimum stimulus intensity needed to elicit a recordable SP from the target muscle in at least 3 out of 5 trials. The SP duration was defined as the latency between SP onset and SP offset in the EMG recordings. If no SP could be detected in the averaged data, a duration of 0 milliseconds was assigned.In both the non-rectified and rectified EMG data, the landmarks to define the SP onset can potentially be defined as stimulus onset, MEP onset, or MEP offset. However, in many healthy individuals, and even more pronounced in patients with stroke, the MEP does not show a clear-cut ending. As a result, the MEP offset is difficult to visually assess. Because the stimulus and MEP onsets appear much more distinctly in the EMG tracings, they were both used to mark the SP onset in this study.As for the SP offset, the landmarks were defined as (1) the earliest reoccurrence of any EMG-activity, including burst activity (i.e. short peaks of reappearing EMG-activity followed by EMG silence) (SP1), 2) the return of continuous EMG-activity (SP2), and (3) the return of continuous EMG-activity to pre-stimulus levels (SP3). In the non-rectified data the pre-stimulus level was determined as the root mean square (RMS) amplitude of the EMG activity during the time period from 150 to 25 milliseconds before stimulus delivery. In the rectified data the pre-stimulus level was determined as the mean amplitude during the same time segment :where the three parameters of the function are:and wherei reflecting the plateau value of participant i1) ai1 reflecting the active motor threshold of participant i, and2) bi2 reflecting the slope parameter (steepness) of participant i,3) byij is the SP duration of participant i at stimulus intensity j,Sj is the stimulus intensity j, and εij is the normally distributed residual with mean zero and variance σ2 of participant i at stimulation intensity j.y) and the independent variable was the stimulus intensity (S). The plateau value (a) was treated as random effect to allow subject-specific regression coefficients. The slope parameter (b2) and the active motor threshold (b1) were treated as fixed (subject independent) regression coefficients. Differences between the models with random effects and models with fixed effects were tested for statistical significance, using the likelihood ratio test. Note that this test is based on the full likelihood function of the hierarchical models [t-tests were used to test differences in stimulus–response characteristics between the pair wise comparisons of the selected landmarks to define SP onset and offset, as well as between rectified and non-rectified data. The Bonferroni correction was used to adjust for multiple testing. The required 2-tailed significance level was set at 0.05. All statistical analyses were performed with the statistical software package SAS version 8.2 .A non-linear mixed regression model was used to fit the individual data to the sigmoid Boltzmann function. The dependent variables were the SP durations of the ADM and BB muscles  were used to construct the stimulus–response curves  and SP3  on the other hand, in the rectified EMG. Similar results were found when the non-rectified EMG data were used. Lower active motor thresholds and less steep slopes could be observed in SP1 compared to SP2 and SP3. No differences in these Boltzmann parameters were observed between SP2 and SP3.Using either the MEP onset or the stimulus onset as the SP onset marker did not show any effect on the Boltzmann parameters Table . As for t-test: p = 0.002; slope, paired-sample t-test: p = 0.022) or SP3  were used to define the SP offset.In addition, the non-rectified data showed lower active motor thresholds and less steep slopes compared to the rectified data when either SP2  affect the SP stimulus (TMS) - response (EMG) characteristics. In healthy individuals, these effects differ between proximal arm and distal hand muscles. Given the importance of using stimulus–response curves to measure parameters of corticomotor excitability for both prognostication and treatment evaluation after stroke, the ultimate goal of this study was to provide a feasible basis for standardizing the procedure for visually guided manual assessment of SP data in a clinical setting. Among other difficulties affecting the reproducibility of TMS studies , variabiIn healthy subjects, no differences were found in the stimulus–response characteristics using either the stimulus onset or the MEP onset to mark the SP onset. So both the stimulus onset and MEP onset can be used as a clear-cut SP onset marker in healthy individuals. If either the stimulus onset or the MEP onset is used to mark the SP onset, the duration of the MEP is included in the SP duration. In conditions that lead to a prolonged MEP, this could mask concurrent shortening of the SP duration. In patients with stroke, increased MEP latencies have been found in the sub-acute phase post stroke, shifting towards more normal values with progressive stages of recovery ,28. If tIn the BB, it was difficult to accurately define the SP1 offset especially at the lower stimulus intensities. Because a complete EMG-silence was not observed in many participants, the landmark to define SP1 was often absent or at least difficult to define at low stimulus intensities. As a result, the active motor thresholds varied substantially, as can be observed in Table Particularly in the BB, re-occurrence of EMG-activity below pre-stimulus levels was seen before the EMG-activity returned to the pre-stimulus levels, specifically at higher stimulus intensities (80-100%). As a result, if SP2 is used to define the SP offset, lower active motor thresholds and less steep slopes were seen compared to SP3. These differences, however, did not reach statistical significance. This early EMG activity observed in the BB at higher stimulus intensities might be due to fluctuations in contraction force in the period immediately after TMS, such that some responses might have been evoked when the active contraction force was more than the targeted pre-activation level . BecauseThe SP evoked in the muscles of the upper limb originates largely from activation of cortical inhibitory interneurons, although also spinal mechanisms are involved in the early part ,12. WhilFor SP3 the EMG data were analyzed relating the resumption of EMG-activity to the level of pre-stimulus EMG-activity . There aIn the ADM, the non-rectified data showed lower active motor thresholds as compared to the rectified data for both SP2 and SP3. Rectifying the data alters the appearance of the data and might reduce the differences between successive data points. As a result, the first resumption of EMG activity below pre-stimulus levels might be more difficult to identify in the rectified data, especially in low-voltage ADM muscle activity. In the BB the pre-stimulus levels of EMG activity are more pronounced compared to the ADM and, therefore, less difficult to identify. However, the raters reported that marker placement was easier to perform in rectified data compared to non-rectified data, especially when using any resumption of continuous EMG activity as the SP offset marker.Three subjects failed to complete the experiment due to discomfort and pain. In these subjects only the assessment of the ADM muscle was performed. The stimulus–response characteristics obtained from the ADM muscle in these 3 participants did not differ from the results of the other participants. In our study isometric contraction levels of 50% of maximal voluntary EMG-activity have been used to ensure maximal facilitation of the MEP ,25. EspeThis study indicates that the most feasible method to manually assess the SP duration in healthy individuals is the time interval from either stimulus onset or MEP onset to the return to the level of continuous pre-stimulus EMG-activity in rectified data. Among other difficulties affecting the reproducibility of TMS studies, variability due to different definitions of SP onset and offset can be overcome by establishing a standard among researches. Because the total amount of investigated subjects was limited, and because the method of measuring SP was based on ease of practice rather than on reliability data, there is still not enough evidence to make definitive statements about standardization. Moreover, manually assessing TMS data remains subject to inter-rater variability ,37. MeasADM: Abductor digiti minimi muscle; BB: Biceps brachii muscle; EMG: Electromyography; MEP: Motor evoked potential; RMS: Root mean square; SD: Standard deviation; SDres: Within subject  standard deviation; SE: Standard error; SP: Silent period; SP1: The earliest reoccurrence of any EMG-activity, including burst activity; SP2: The return of continuous EMG-activity; SP3: The return of continuous EMG-activity to pre-stimulus levels; S-R: Stimulus–response; TMS: Transcranial magnetic stimulation.The authors declare that they have no competing interests. There is no conflict of interests between the authors and others, and there were no sources of support or funding involved in this study.AK participated in the design of the study, carried out the study, and drafted the manuscript, CB participated in the draft of the manuscript, JH participated in the design of the study, performed the statistical analysis, and participated to draft the manuscript. AG and DS both participated in the design of the study as well as in the draft of the manuscript; JP participated in the design and coordination of the study, and drafted the manuscript. All authors read and approved the final manuscript.


Electromyography (EMG) pattern-recognition based control strategies for multifunctional myoelectric prosthesis systems have been studied commonly in a controlled laboratory setting. Before these myoelectric prosthesis systems are clinically viable, it will be necessary to assess the effect of some disparities between the ideal laboratory setting and practical use on the control performance. One important obstacle is the impact of arm position variation that causes the changes of EMG pattern when performing identical motions in different arm positions. This study aimed to investigate the impacts of arm position variation on EMG pattern-recognition based motion classification in upper-limb amputees and the solutions for reducing these impacts.With five unilateral transradial (TR) amputees, the EMG signals and tri-axial accelerometer mechanomyography (ACC-MMG) signals were simultaneously collected from both amputated and intact arms when performing six classes of arm and hand movements in each of five arm positions that were considered in the study. The effect of the arm position changes was estimated in terms of motion classification error and compared between amputated and intact arms. Then the performance of three proposed methods in attenuating the impact of arm positions was evaluated.With EMG signals, the average intra-position and inter-position classification errors across all five arm positions and five subjects were around 7.3% and 29.9% from amputated arms, respectively, about 1.0% and 10% low in comparison with those from intact arms. While ACC-MMG signals could yield a similar intra-position classification error (9.9%) as EMG, they had much higher inter-position classification error with an average value of 81.1% over the arm positions and the subjects. When the EMG data from all five arm positions were involved in the training set, the average classification error reached a value of around 10.8% for amputated arms. Using a two-stage cascade classifier, the average classification error was around 9.0% over all five arm positions. Reducing ACC-MMG channels from 8 to 2 only increased the average position classification error across all five arm positions from 0.7% to 1.0% in amputated arms.The performance of EMG pattern-recognition based method in classifying movements strongly depends on arm positions. This dependency is a little stronger in intact arm than in amputated arm, which suggests that the investigations associated with practical use of a myoelectric prosthesis should use the limb amputees as subjects instead of using able-body subjects. The two-stage cascade classifier mode with ACC-MMG for limb position identification and EMG for limb motion classification may be a promising way to reduce the effect of limb position variation on classification performance. Myoelectric signals recorded with electrodes on the skin surface overlying the residual arm muscles have been used in control of motorized upper-limb prostheses for several decades -23. A siIn most reported studies of EMG pattern recognition systems for multifunctional prosthesis control, subjects generally took a seated position with a tested arm resting on a plate surface such as chair arm or table and multi-channel EMG signals were acquired with a number of surface electrodes placed on either the muscles of forearm and hand for an able-bodied subject or the residual muscles for an upper-limb amputee. One portion of the acquired EMG data was used to train a classifier and then remaining portion was loaded into the trained classifier for calculating the offline classification accuracy in identifying a number of arm and hand movements -18,21-23In order to achieve the high classification accuracy of EMG pattern recognition approach for control of a multifunctional myoelectric prosthesis, it is required that the contraction of the targeted muscles could produce the repeatable EMG patterns for doing a movement. This is not the case in doing daily activities that would need the user’s arm position to be various. Keeping arm in different positions and performing a movement may require different forearm muscles to be contracted. Thus when doing identical movements in different arm positions, the arm muscles may generate disparate EMG patterns. Therefore, with a classifier trained by EMG recordings in one specific arm position, the EMG pattern changes may erode the classification accuracy of movements. This raises a question: whether does the variation of arm positions significantly affect the control performance of multifunctional myoeletric prosthesis? If the answer is yes, the following question is: how to reduce the impact of arm position variations?Most recently, a study has been conducted by a research group to address these issues ,20. TheiFive subjects  with unilateral transradial (TR) amputation aged from 22 to 43 years participated in the study. Their post-amputation times varied from 2 years to 10 years. The length of their residual forearms ranges from 5 cm to 14 cm . They wear either a myoelectric prosthesis or a cosmetic prosthesis everyday. The protocol of this study was approved by the Shenzhen Institutes of Advanced Technology Institutional Review Board, China. All subjects gave the written informed consent and provided permission for publication of photographs with a scientific and educational purpose.In this study a commercial wireless biological signal acquisition system  was used to record surface EMG and ACC-MMG signals with eight sensors. Each sensor integrates one bipolar EMG electrode with one tri-axial ACC-MMG accelerometer, which could provide one-channel EMG recording and simultaneously one-location ACC-MMG recording. Note that the ACC-MMG signal, a three-dimensional measure of a sensor location, is composed of three sub-signals in x, y, and z axis, respectively. So with 8 sensors, 8-channel EMG signal recordings and 8-position ACC-MMG signal recordings with 24-channel could be simultaneously obtained. For each subject, six of the eight bipolar EMG sensors were placed around the apex of the muscle bulge, 1–2 cm distal to the elbow crease and another two sensors were placed on the distal end over flexor muscle and extensor muscle, respectively, as illustrated in Figure For each subject, experiment was comprised of five sets. In each set, the subject was instructed to perform seven classes of frequently used forearm movements at a moderate and repeatable force level with his/her arms in one of five typical arm positions considered in the study. The seven classes of forearm movements were wrist flexion (WF), wrist extension (WE), wrist supination (WS), wrist pronation (WP), hand open (HO) and hand close (HC), plus a “no movement” (NM) class. Five typical arm positions commonly used in daily life activities Figure  were conSubjects were prompted to elicit contractions corresponding to the target motion class displayed in a video. Each movement contraction was sustained for 4 s to generate myoelectric signals and the rest time between subsequent contractions was 5 s. Each of seven movements was repeated 10 times per set. Thus each set produced 40-sec EMG and ACC-MMG recordings per movement. To avoid muscle and mental fatigue, subjects were allowed to have a rest for 10–15 minutes between sets. During experiments EMG and ACC-MMG data were simultaneously acquired with a sampling rate of 4000 Hz for EMG signals and 296.3 Hz for ACC-MMG signals. With 8 EMG-ACC-MMG hybrid sensors, the 8-channel EMG signal recordings and 24-channel (3×8) ACC-MMG signal recordings  could be simultaneously obtained from residual arm and intact arm, respectively.Delsys Inc.). Considering that the major power (about 95%) of surface EMG signals is often below 400–500 Hz, the EMG signal recordings were down-sampled to 1 kHz to simplify data processing. To remove the slow variations in the EMG signals caused by the motion artifacts such as electrode shift and cable movement, the EMG signal recordings were digitally filtered with a five-order Butterworth high-pass filter at a 5 Hz cut-off frequency.EMG and ACC-MMG signals were acquired with the maximum sampling rates of the commercial bioelectric signal acquisition system  was used for feature extraction ,21,22. TA simple linear discriminant analysis (LDA) ,8,14-23 For each subject, the features from the first half of EMG or ACC-MMG recordings (20-sec) were concatenated as the train data set to train a LDA classifier; the features from the second half of EMG or ACC-MMG recordings (20-sec) were also combined together as the test data set to estimate classification performance of the trained classifier. Classification error was used to evaluate the motion identification performance in this study, which is defined as:The average value of classification errors in identifying all seven classes of movements was calculated as the overall classification error for a subject.For each subject, in order to assess the sensitivity of EMG pattern recognition for motion classification to the five arm positions, a LDA classifier was built for each arm position, totally producing five single arm position classifiers. Then the five test feature sets corresponding to the five arm positions were fed into each of the five trained classifiers to calculate the classification errors, respectively, resulting in an overall classification error matrix (5×5) in which the diagonal elements represented the intra-position classification errors and the non-diagonal elements were inter-position classification errors. Similarly, an overall classification error matrix also was obtained for each subject when ACC-MMG signals were used for motion classification.With an attempt to attenuate the impact of arm position variation on classification performance, three possible solutions were proposed in the study as follows:Training a classifier with both EMG and ACC-MMG data from a single position - By training a classifier with the combination of EMG and ACC-MMG signal recordings at each of five arm positions, we looked for if involving more training information associated with arm movements could significantly increase the robustness of the classifier against the impact of arm position variation.1)Training a classifier with EMG data from multiple arm positions - In order to investigate if using EMG data from two or more arm positions to train a classifier could substantially reduce the influence of arm position variation on the classification performance, a LDA classifier was trained with the concatenated EMG data from multiple arm positions. With five arm positions, the possible combination number was 10 for two- and three-position, 5 for four-position, and 1 for five-position combinations. Totally, 26 multi-position classifiers would be built. And then each of trained multi-position classifiers was tested using the concatenated EMG data from all the five positions.2)Using a cascade classifier - The cascade classifier was composed of two sequential classifiers, as shown in Figure 3)Generally speaking, using more signal recording channels could get more motion information for better performance of movement classification, but may increase the complexity of computation and analysis that may lead to slow discrimination response. A pilot analysis was performed in this study to investigate the feasibility of using a reduced number of electrodes without compromising classification accuracy. With a straightforward exhaustive search algorithm  we also t-test was used to assess the statistical difference between the means of compared classification errors and the level of statistical significance was set to p<0.05.In this study, a paired p-value=0.11). However, the ACC-MMG signals had much higher inter-position classification error with an average value of 81.1±1.5% over the arm positions and the subjects, in comparison of the EMG signals with an average value of 29.9±3.2%; the difference was significant . This suggests that ACC-MMG signals are much more sensitive to arm position variation in comparison to EMG signals.The average overall classification error matrix over all the five subjects was calculated for their amputated arms and showed in Figure P1. The average intra-position classification error over the five arm positions from amputated arm was about 1.0% lower than that from intact arm, as shown in the most right column of Figure p-value<0.01). The average inter-position error across all arm positions and subjects were 29.9±3.2% for amputated arm and 40.9±3.4% for intact arm  in classifying seven classes of arm and hand movements, but significantly different average inter-position classification errors (around 30% versus 81%). The significant difference between intra-position and inter-position classification errors indicate that the classification performance of either EMG classifier or ACC-MMG classifier could be substantially affected by the arm position changes, which is consistent with the results of the reported able-body subject study ,20. The With a quite high average inter-position classification error, ACC-MMG recordings should be much more sensitive to arm position variations; this indicates that ACC-MMG is good at classification of different arm positions, but poor at motion classification. It is noteworthy that EMG recordings from amputated arms produced the low average intra-position (about 1% low) and inter-position (about 10% low) classification errors, in comparison to those from intact arms. This may be the most important finding of the current study. Note that most previous studies ,20 used With an attempt to look for a suitable method for attenuating the influence of arm position variations on EMG pattern-recognition prosthesis control, three possible solutions were examined in this study. The first solution was to build a single-position classifier trained with the combining information of EMG and ACC-MMG. This hybrid classifier yielded lower average intra-position classification error (5.6%) than the EMG classifier (7.3%) or the ACC-MMG classifier (9.9%), but produced a quite high average inter-position classification error (around 74%). The lower intra-position classification errors indicate that including more information representing muscles activities into training data set could generally get better classification performance, which is consistent with the reported study . The higAnother solution considered in this study was to train a multi-position classifier. Since ACC-MMG is very sensitive to arm position variation, the multi-position classifier was only trained with EMG. By adding EMG data from more arm positions into training set, the effect of arm positions on EMG patterns could be involved in the trained classifier, which would increase the classifier’s generalization or robustness. This was proved by the experimental results of the study. The more the arm positions in EMG training set were involved, the more the classification performance was improved Figure . When thThe third solution examined in the study was a two-stage cascade classifier. Taking the high sensitivity of ACC-MMG to arm positions and EMG to movements, the position classifier was trained with ACC-MMG data and the movement classifier was trained with EMG data. Table Note that the classification performance was evaluated by the classification errors that were calculated by post-processing EMG recordings (offline) and was not a direct measure of real-time performance in clinic. Generally speaking, the high offline classification errors may decay the accuracy and reliability of a multifunctional prosthesis control in real time application. Three real-time performance measures have been proposed  to gain The current study used the transradial amputees as subjects who are the final user of myoelectric prostheses to assess the effects of arm position variation on EMG and/or ACC-MMG pattern-recognition based motion classification in limb amputees and evaluated the performance of three proposed solutions in reducing the impact of arm positions. For amputated arms, the average inter-position error of EMG classification across all the five arm positions and the five subjects was around 22% higher than the average intra-position error. This indicates that the performance of EMG pattern-recognition based method in classifying movements strongly depends on arm positions. This dependency is stronger in intact arm than in amputated arm, which suggests that the investigations associated with practical use of a myoelectric prosthesis should be conducted with the limb amputees as subjects instead of able-body subjects. Using eight-channel ACC-MMG signals as input of arm position classifier could achieve an average classification error as low as 0.7% across five arm positions and five subjects; even though using two-channel ACC-MMG signals, the position classification error slightly increased to 1%. Thus ACC-MMG signals would be very suitable for the arm position identification. With ACC-MMG and EMG data as the input signals of arm position and movement classifier, respectively, the two-stage cascade classifier could obtain the best performance in attenuating the impact of arm position variation among three proposed solutions. This suggests that the cascade classification strategy may be promising for the accurate and reliable control of EMG pattern-recognition based prosthetic systems in practical use.The authors declare that they have no competing interests.YG performed experiment design, subject recruitment, data collection and analysis, interpretation of the results, and drafting of the manuscript, PZ was involved in interpretation of the results and the manuscript preparation, GL oversaw the study and was involved in each stage of the study, including critical revision of the manuscript. All authors read and approved the final manuscript.