The cardiac troponins are biomarkers used for diagnosis of myocardial injury. They are also powerful prognostic markers in many diseases and settings. Recently introduced high-sensitivity assays indicate that chronic cardiac troponin elevations are common in response to cardiovascular (CV) morbidity. Type 2 diabetes mellitus (T2DM) confers a high risk of CV disease, but little is known about chronic cardiac troponin elevations in diabetic subjects. Accordingly, we aimed to understand the prevalence, determinants, and prognostic implications of cardiac troponin T (cTnT) elevations measured with a high-sensitivity assay in patients with T2DM.cTnT was measured in stored, frozen serum samples from 124 subjects enrolled in the Asker and Bærum Cardiovascular Diabetes trial at baseline and at 2-year follow-up, if availabe (96 samples available). Results were analyzed in relation to baseline variables, hospitalizations, and group assignment .One-hundred thirteen (90 %) had detectable cTnT at baseline and of those, 22  subjects had values above the 99th percentile for healthy controls (13.5 ng/L). Levels at baseline were associated with conventional CV risk factors . There was a strong correlation between cTnT levels at the two time-points . Risk for hospitalizations during follow-up increased step-wise by quartiles of hscTnT measured at baseline (p = 0.058).Elevations of cTnT above the 99th percentile measured by a highly sensitive assay were encountered frequently in a population of T2DM patients. cTnT levels appeared to be stable over time and associated with conventional CV risk factors. Although a clear trend was present, no statistically robust associations with adverse outcomes could be found. Troponin T values were associated with several conventional risk factors for CV disease, and concentrations of troponin T remained largely stable throughout the follow-up period. These observations imply that in this patient population, troponin T release reflects underlying and chronic pathophysiological processes. Thus, troponin T may be a useful surrogate marker to define risk among subjects with T2DM, and may facilitate early identification of patients with subclinical CV disease and other high-risk individuals.th percentile found in the present dataset. Our results also support and extend previous investigations of troponin T values in populations with established CV disease [Using the older and less sensitive conventional assay, a previous analysis observed elevated troponin T values in about 0.7 % of the general population . Elevati disease -18. In t disease ,20. We f disease ,22, and We were not able to demonstrate that intensive, structured care translated into lower troponin T values, despite the fact that such interventions improved risk profiles  as described in the primary report from the ABCD trial . PossiblThe follow-up at 2 years allowed us to tentatively probe the prognostic properties of troponin T, although the post hoc design restricts interpretation and some important limitations should be recognized: First, according to the study protocol, hospitalizations were only classified as all-cause or diabetes/CV-related. It would have been instructive to also consider only CV hospitalizations. Second, adjustments for relevant covariates were not possible due to the low number of patients and endpoints. In light of these limitations no inferences can be drawn on the basis of the trend of increasing hospitalizations across quartiles of increasing troponin T values, even though the prognostic value of troponin T that these numbers allude to are in agreement with previous reports from other much larger populations ,17. InteThe larger context of the present study is the increasing use of circulating biomarkers for diagnostic and prognostic purposes - as well as to directly inform treatment decisions - in many fields in medicine. Cardiovascular diseases and associated conditions like diabetes are no exceptions, as new studies are emerging defining novel biomarkers as well as exploring the role of well-known markers in new contexts ,24. In rSome important limitations with this investigation have already been mentioned and others should also be considered. This was a post hoc study performed in a relatively small cohort and due to the low absolute number of patients and events, we were not able to explore our data in multivariable regression models. Clearly, this represents a major weakness of our study. Yet, our findings fit well with the current understanding of the distribution and prognostic properties of troponins in populations with established, or at high risk for, CV disease . It is likely that some of the troponin T elevations seen in our population compared to the reference population are related to increased age, although increases with this assay were not reported as part of the assay validation. In any case, this does not restrict interpretation of the results, as our population is representative of a real-life T2DM population; and that regardless of cause troponin-release confers a higher risk of CV events and death. Measurements of baseline and 2-year troponin T samples were performed at different time-points, which increase the analytical variability and may explain the slight fall in troponin T levels during follow-up.th percentile of a reference population when measured by a highly sensitive assay. Levels of troponin T were stable over time, associated with conventional risk factors, and displayed a graded relationship with hospitalizations although this trend was not statistically significant.In conclusion, in a population of T2DM patients, we found that almost 1 in 5 had troponin T values above the 99Roche Diagnostics  provided reagents for troponin T assays. JH and OEJ were supported by grants from the South-Eastern Norway Regional Health Authority. JH also received support from Aker University Hospital Research Foundation and Center for Heart Failure Research, Oslo, Norway. ASJ has consulted over time for most of the major diagnostic companies. OEJ is an associated post-doctoral researcher at Vestre Viken, Asker and Bærum Hospital  and JH is a PhD student at Oslo University Hospital and the University of Oslo, but both are employees of Boehringer Ingelheim.JH conceived of and designed the study, performed the cardiac troponin T measurements, performed all statistical analysis and wrote the first draft of the manuscript. OEJ was principal investigator of the ABCD study and was responsible for data collection and diagnostic tests. KB and LG supervised the ABCD study and ASJ and DA supervised this substudy. SA and KE participated in collection of data and performed diagnostic tests. SJ performed the cardiac troponin T measurements. All authors contributed to the design of the study, interpretation of the results, and critical revision of the manuscript. All authors have read and approved the final manuscript.


Objective: The purpose of this study was to determine if granulocyte colony-stimulating factor(G-CSF) is normally present in amniotic fluid and then to determine if amniotic-fluid G-CSF levelsare affected by labor and intrauterine infection.Methods: Amniotic fluid was collected from 35 patients in 4 groups: no labor, early labor, late labor, and labor plus chorioamnionitis. G-CSF levels were measured by enzyme-linked immunosorbentassay (ELISA).Results: The mean amniotic-fluid G-CSF concentrations prior to labor were lower than during labor . With chorioamnionitis, the mean levels were elevated compared with normal labor . In early and late labor, G-CSF was higher than prior to labor . The mean concentrations in early and late labor were not different.Conclusions: G-CSF is present in amniotic fluid and increased with labor. When labor is complicated by chorioamnionitis, G-CSF is significantly elevated.


Calorimetry is a nonspecific technique which allows direct measurement of heat generated by biological processes in the living cell. We evaluated the potential of calorimetry for rapid detection of bacterial growth in cerebrospinal fluid (CSF) in a rat model of bacterial meningitis.Streptococcus pneumoniae, Neisseria meningitidis or Listeria monocytogenes. Control animals were injected with sterile saline or heat-inactivated S. pneumoniae. CSF was obtained at 18 hours after infection for quantitative cultures and heat flow measurement. For calorimetry, 10 μl and 1 μl CSF were inoculated in calorimetry ampoules containing 3 ml trypticase soy broth (TSB).Infant rats were infected on postnatal day 11 by direct intracisternal injection with either 8 for S. pneumoniae, 1.3 ± 0.3 × 106 for N. meningitidis and 3.5 ± 2.2 × 104 for L. monocytogenes. Calorimetric detection time was defined as the time until heat flow signal exceeded 10 μW. Heat signal was detected in 10-μl CSF samples from all infected animals with a mean (± SD) detection time of 1.5 ± 0.2 hours for S. pneumoniae, 3.9 ± 0.7 hours for N. meningitidis and 9.1 ± 0.5 hours for L. monocytogenes. CSF samples from non-infected animals generated no increasing heat flow (<10 μW). The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). The lowest detectable bacterial titer by calorimetry was 2 cfu for S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes.The mean bacterial titer (± SD) in CSF was 1.5 ± 0.6 × 10S. pneumoniae and N. meningitidis and <10 hours for Listeria monocytogenes using as little as 10 μl CSF were achieved. Calorimetry is a new diagnostic method allowing rapid and accurate diagnosis of bacterial meningitis from a small volume of CSF.By means of calorimetry, detection times of <4 hours for  Bacterial meningitis requires rapid diagnosis since a delay in antimicrobial therapy results in poor outcome ,2. ExamiStreptococcus pneumoniae, Neisseria meningitidis and Listeria monocytogenes are the main causative organisms responsible for over 80% of all cases of bacterial meningitis in adults [Culture of CSF for the detection of bacterial growth is the gold standard for the diagnosis of bacterial meningitis . Bacterin adults .Calorimetry is a nonspecific technique which allows direct measurement of heat generated by biological processes in the living cell. Microorganisms are reported to produce on average 1–3 pW heat per cell . The expStreptococcus pneumoniae, Neisseria meningitidis and Listeria monocytogenes to evaluate the potential of isothermal calorimetry for rapid and sensitive detection of bacteria in small samples of CSF (i.e. 1 and 10 μL).The present study was performed to demonstrate the proof of principle of calorimetry for the detection of bacterial growth in clinical samples. Here we used an infant rat model of bacterial meningitis due to infection with 6 cfu/ml S. pneumoniae, 3 × 108 cfu/ml N. meningitidis and 2 × 105 cfu/ml L. monocytogenes using a 32-gauge needle. The pathogens investigated in the study show significant differences in virulence and pathogenicity. Therefore the number of bacteria used for infection needed to be adapted to generate reproducible infection and associated disease severity. Thus, for each pathogen the inoculum (cfu/ml) used for intracisternal infection was based on previously established disease models [S. pneumoniae at a concentration corresponding to 2 × 108 cfu/ml (n = 2).An established infant model of bacterial meningitis was used as described previously ,8. The ae models ,10. ContS. pneumoniae (serogroup 3), N. meningitidis (type C) and L. monocytogenes (serotype 4b) were used. The infecting organisms were previously passaged through infant rats in the course of experimental studies [S. pneumoniae and N. meningitidis, or brain heart infusion (BHI) for L. monocytogenes, diluted in fresh medium, and grown to logarithmic phase at 37°C in 5% CO2. The culture broth was centrifuged for 10 minutes at 5,000 × g and the pellet was resuspended in sterile saline to the desired density for intracisternal injection. The accuracy of the inoculum dose was confirmed by quantitative cultures by serial dilution for each experiment. In addition, 10-fold of serial dilutions of test organisms were prepared in concentrations from 106 to 10-1 cfu/ml as quantitative standards for comparison with CSF samples.Clinical isolates of  studies . OrganisAt 18 h after infection, approx. 15 - 20 μl CSF was obtained by puncture of the cisterna magna and used for quantitative cultures and calorimetry. For quantitative culture, CSF was plated in 10-fold dilutions on 5% sheep blood agar plates (Becton Dickinson). For calorimetry, 10 μl and 1 μl CSF were added to 90 μl and 99 μl saline, respectively , and inoculated in calorimetry ampoules containing 3 ml TSB. Culture broth with 100 μl saline was used as control. Animals were sacrificed after CSF sampling with an overdose of pentobarbital .An isothermal calorimetry instrument  equipped with 48 channels was used to measure the heat flow at 37°C precisely controlled within 0.0001°C. The calorimetric sensitivity according to the manufacturer is ± 0.2 μW. Heat generated or absorbed was continuously measured in air-tightly sealed 4-ml glass calorimetry ampoules, containing the test or control samples; the gas phase consisted of ambient air. Ampoules were sequentially introduced into the calorimetry instrument and remained 15 minutes in the thermal equilibration position before lowering into the measurement position. Heat flow was measured for up to 48 hours at 10 s intervals. After the measurement was completed, the content of each calorimetry ampoule was cultured to confirm the inoculated microorganisms in infected animals or sterility of the negative controls.Calorimetric detection time was defined as the time from insertion of the ampoule into the calorimeter until the growing culture produced an exponentially rising heat flow signal exceeding 10 μW. Peak heat flow was defined as the highest value of the heat power-time curve. Total heat was determined by integration of the area below the heat flow-time curve. Data analysis was accomplished using the manufacturer's software  and Origin 7.5 .At 18 hours after intracisternal injection, all infected rats suffered from meningitis as evidenced by lethargy, diminished weight gain and positive CSF cultures. Table 6 to 10-1 cfu/ml (grey lines). The heat curves of CSF from different individual animals infected by the same organism showed similar characteristics. For example, S. pneumoniae showed a discrete initial peak heat flow at 40 μW, followed by a second peak at about 350 μW. N. meningitidis demonstrated a clear initial peak, followed by a higher peak at approximately 80 μW. Replicates of L. monocytogenes showed three peaks, at ≈ 80 μW, ≈ 300 μW and ≈ 350 μW. The shape of the curves was dependent on the growth medium in the calorimetry ampoule, but independent from the initial bacterial concentration (data not shown). The bacterial titer in CSF at 18 hours after infection correlated with the standard dilutions for S. pneumoniae, but was two orders of magnitude lower for N. meningitidis and three orders of magnitude lower for L. monocytogenes. The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). CSF from animals that had been injected with heat-killed bacteria resulted in power-time curves very similar to those of the medium-controls. The values always remained below the 10 μW positivity limit and continuously decreased towards baseline.Heat signal was detected in CSF samples from all infected animals, whereas those from non-infected animals generated no detectable heat <10 μW). Figure 0 μW. FigS. pneumoniae was detected after a mean ± SD of 1.5 ± 0.2 hours, followed by N. meningitidis after 3.9 ± 0.7 hours and by L. monocytogenes after 9.1 ± 0.5 hours.Table S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes using TSB  in TSB was 1.71 ± 0.37 hours shorter for S. pneumoniae and 1.17 ± 0.87 hours shorter for L. monocytogenes compared to BHI. Similarly, the peak heat flow of CSF in TSB was 174 μW higher for S. pneumoniae and 69 μW higher for L. monocytogenes as compared to BHI.In addition to TSB, BHI was used as an alternative growth medium for the detection of Bacterial meningitis is a medical emergency situation associated with a substantial morbidity and mortality. Despite appropriate antibiotic therapy recent studies document a mortality rate of up to 30% and the occurrence of permanent sequelae due to neuronal injury in up to 50% of the survivors ,2,11. A -6°C [Calorimetry has been widely used in biology, chemistry, pharmacology, biotechnology and ecology because of its high sensitivity, accuracy and simplicity . However-6°C . The exp-6°C . In a re-6°C . The preThe detection principle relies on the heat generation of viable, replicating bacteria from CSF in culture medium that is continuously measured by isothermal calorimetry. The sensitivity of calorimetry compared to standard culture methods is equivalent when identical volumes of sample fluid are used in both methods, as the prerequisite for both detection methods is bacterial multiplication in culture. However, due to the more sensitive and continuous measurement principle of calorimetry (i.e. monitoring of heat production), the detection time is significantly shorter than with conventional cultures, which is based on detection of growth on agar plates or in broth.All infected animals in the present study were diagnosed by calorimetric analysis of punctured CSF samples within hours and with a high discriminative power compared to sterile inflammation or medium controls. The present data are in agreement with the recent finding that the mode of growth observed in bacteria during invasive tissue infection, e.g. growth in cerebrospinal fluid, differs significantly from bacterial growth in liquid medium . The metS. pneumoniae and N. meningitidis and <10 hours for Listeria monocytogenes using as little as 10 μl CSF were achieved. In the clinical situation larger volumes of CSF are usually available from patients, which may further shorten the detection time. This method is particularly well suited in situations where CSF volumes are limited (e.g. in newborns and infants). Calorimetric detection of bacterial growth may thus provide an accurate tool to rapidly diagnose bacterial meningitis with the benefit of low labor intensiveness, few handling steps, potential of complete automation, and minimal contamination and biohazard risk due to the closed ampoule system.In the present approach, detection times of <4 hours for L. monocytogenes after (CSF) or without (standard curves) passages through the animal could be due to adaptation of the pathogen that may be associated with a prolonged lag phases and longer generation times.In addition, we found that TSB is an appropriate medium for rapid detection of all tested microorganisms in this study. The differences in the power-time curves of This study demonstrates that calorimetry is a promising novel approach for rapid and accurate diagnosis of bacterial meningitis, allowing detection of multiplying bacteria in small volumes of CSF (1–10 μl) within hours.Cerebrospinal fluid – CSFTrypticase soy broth – TSBBrain heart infusion – BHIStandard deviation – SDThe author(s) declare that they have no competing interests.All authors read and approved the final manuscript. AT, AS and SLL contributed equally for study design, coordination, interpretation of results and writing of the manuscript. AS, MW and SLL participated in data collection and experimental procedures.The pre-publication history for this paper can be accessed here:


Because pulmonary circulation is the primary vascular target of inhaled particulate matter (PM), and nitric oxide is a major vasculoprotective agent, in this study we investigated the effect of various particles on the NO–cyclic guanosine monophosphate (cGMP) pathway in pulmonary arteries.in vitro to particles or removed from PM-instilled animals for assessment of vasomotricity, cGMP and reactive oxygen species (ROS) levels, and cytokine/chemokine release.We used intrapulmonary arteries and/or endothelial cells, either exposed 2) manufactured particles did not impair NO-mediated relaxation. SRM1648-induced decrease in relaxation response to ACh was prevented by dexamethasone (an anti-inflammatory agent) but not by antioxidants. Accordingly, SRM1648 increased the release of proinflammatory mediators  from intrapulmonary arteries or pulmonary artery endothelial cells, but did not elevate ROS levels within intrapulmonary arteries. Decreased relaxation in response to ACh was also evidenced in intrapulmonary arteries removed from rats intratracheally instilled with SRM1648, but not with fine TiO2.Endothelial NO-dependent relaxation and cGMP accumulation induced by acetylcholine (ACh) were both decreased after 24 hr exposure of rat intrapulmonary arteries to standard reference material 1648 . Relaxation due to NO donors was also decreased by SRM1648, whereas responsiveness to cGMP analogue remained unaffected. Unlike SRM1648, ultrafine carbon black and ultrafine and fine titanium dioxide (TiOIn contrast to manufactured particles (including nanoparticles), urban PM impairs NO but not cGMP responsiveness in intrapulmonary arteries. We attribute this effect to oxidative-stress–independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway may contribute to urban-PM–induced cardiovascular dysfunction. Two majin vitro and in vivo animal and human studies  nanoparticles, which, unlike SRM1648, are relatively free of adsorbed constituents.Therefore, in this study we investigated the influence of PM on the NO–cGMP relaxant pathway in intrapulmonary arteries. We investigated urban PM [standard reference material 1648 (SRM1648)] and manufactured carbon black and titanium dioxide (TiO2α(PGF2α dinoprost), ketamine, and xylazine, from Centravet ; and dihydroethidium (DHE), from Molecular Probes .We obtained drugs and reagents from Sigma Chemical Co. , except 2--diazenolate-2-oxide.diethylammonium salt (DEA-NO) and 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), from Alexis Biochemicals ; 1H-oxadiazoloquinoxalin-1-one (ODQ), from Tocris Bioscience ; prostaglandin FWe purchased SRM1648 from the National Institute of Standards and Technology . Physical and chemical properties of SRM1648 have been previously described . The par2  and fine TiO2  from Sigma. Before experiments, we freshly suspended particles (10 mg/mL) in distilled deionized water. We chose the concentrations of PM for in vitro or in vivo experiments based on the literature (Other particles we used in this study included ultrafine carbon black (ufcb) FW2 and P60 , with average primary particle sizes of 13 and 21 nm, respectively. We obtained ultrafine TiOterature .Guide for the Care and Use of Laboratory Animals , in absence or presence of particles, for 24 hr at 37°C in a humidified atmosphere (95% air/5% carbon dioxide). In some experiments, we added dexamethasone (10 μM), tempol (1 mM), or ascorbate (200 μM) to DMEM for the 24-hr incubation period. We anesthetized some rats by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (4 mg/kg), and intratracheally instilled them with 5 mg SRM1648 or fTiO2 in 500 μL saline (NaCl 0.9%), or with saline alone. After a recovery period (6–72 hr), we euthanized rats and removed lungs.Male Wistar rats  were treated humanely and with regard for the alleviation of suffering, according to the  Animals . We diss Animals . For in Nω-nitro-l-arginine methylester , the reference endothelium-dependent relaxant agent acetylcholine  elicited < 5% relaxation after submaximal precontraction with PGF2α (10 μM).We mounted arterial segments in a myograph as previously described . In some2α (30 nM to 30 μM). After washout, we submaximally contracted them with PGF2α to achieve approximately 50% of the tension obtained with 80 mM KCl. Once we obtained stable contraction, we added cumulative concentrations of ACh, sodium nitroprusside (SNP), DEA-NO, 8-bromo-cGMP (8-Br-cGMP), YC-1, isoproterenol, forskolin, or levcromakalim. 2α.We exposed intrapulmonary arteries to KCl (5–100 mM) or PGFWe prepared sections of intrapulmonary arteries and exposed them to the fluorescent dye DHE (2.5 μM) as previously described . We examWe fixed lungs from SRM1648-instilled rats in phosphate-buffered saline (pH 7.4) containing 4% formaldehyde. We stained paraffin-embedded histologic sections (3 μm thick) with hematoxylin, eosin, and saffron (HES) and examined them under optical light microscope.5 cells/mL in MCDB 131 medium, supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin, 2 mM glutamin, 10% (vol/vol) fetal bovine serum, 10 μg/mL vascular endothelial growth factor , and 500 U/mL heparin, 30 mM HEPES, 1 μg/mL endothelial cells growth supplement , and cultured them at 37°C in 5% CO2.We opened intrapulmonary arteries from bovine lung  longitudinally, digested their intimal surface with collagenase, and gently scraped them to remove endothelial cells , exposed or not to SRM1648 (200 μg/mL for 24 hr), at −20°C for subsequent determination of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), or macrophage inflammatory protein-2  using enzyme-linked immunosorbent assay (ELISA) kits . We then transferred arteries to PSS  containing ACh (10 μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (100 μM). After 15 min, we froze arteries, stored them in liquid nitrogen, and then homogenized them in ice-cold trichloroacetic acid (6%). We determined content of cGMP using an ELISA kit . We normalized TNF-α, MIP2, and cGMP levels to tissue protein content, the latter determined by the Lowry method .2α; all data are means ± SE from n rats. We compared concentration–response curves using two-way analysis of variance (ANOVA) and performed other statistical comparisons with one-way ANOVA. We considered differences statistically significant when p < 0.05.We expressed relaxation responses as the percentages of the initial tone induced by PGFn = 25). Such relaxation was slightly lower than that obtained in freshly isolated tissue (in which maximum reaches about 75%), as observed in other arterial models  or after endothelium removal with CHAPS . Neither contractile nor endothelium-independent relaxation maximal capacity of arteries was altered by endothelium removal, because effects of KCl (80 mM) and of the NO donor SNP (10 μM) were not significantly different between CHAPS-treated and untreated arteries (data not shown).After 24-hr preincubation without particles, maximal relaxation to 30 μM ACh was 50.02 ± 3.03% (l models . Neverth2α (30 nM to 30 μM) (data not shown). We observed a significant decrease in ACh-induced relaxation in intrapulmonary arteries after 24-hr exposure to SRM1648 at concentrations ≥ 100 μg/mL (Preincubation of rat intrapulmonary arteries for 24 hr with SRM1648 did not modify contraction to KCl (5–100 mM) or PGF00 μg/mL , 2A. ACh00 μg/mL . Relaxat00 μg/mL .2-adrenergic receptor; ATP (ATP-sensitive K(+) channels) activator].We studied other vasorelaxing agents for comparison. As shown in 2, and fTiO2. As shown in For comparison with SRM1648, we studied the effects of ufcb, ufTiOThe steroidal anti-inflammatory agent dexamethasone  fully prevented SRM1648-induced impairment of the relaxation response to ACh in intrapulmonary arteries, without modifying its relaxant effect in untreated arteries . Exposur2α  and ascorbate (200 μM) failed to modify effect of ACh . Similar2α . Compare2α .2  did not modify ACh-induced relaxation  remained unaffected by SRM1648.The relaxation responses to ACh and NO donors, but not to 8-Br-cGMP, were decreased after exposure of intrapulmonary arteries to SRM1648 (200 μg/mL for 24 hr). In addition to relaxation, ACh-induced cGMP accumulation was also decreased in such conditions. This demonstrates that SRM1648 induced a decrease in responsiveness of smooth muscle to NO, rather than a decrease in endothelial NO production and/or bioactivity, and that SRM1648 impairs the NO signaling pathway upstream of activation of cGMP-dependent protein kinases. YC-1–induced relaxation was not affected by SRM1648, supporting the view that impairment of the NO pathway is likely attributable to a decrease in guanylyl cyclase activation by NO. This argues against a role of decreased expression of guanylyl cyclase in SRM1648-induced impairment of NO-dependent relaxation. In addition, this study demonstrates that impairment is selective for the NO pathway, because other relaxant mechanisms , SRM1648, but not fTiO2, decreased ACh-induced relaxation. Thus, adsorbed components of SRM1648, rather than particulate core, are likely responsible for impaired NO-dependent relaxation. The water-soluble fraction of inhaled PM is more biologically relevant because its components could more easily reach pulmonary vessels than can whole particles or the insoluble fraction  may recover or counteract SRM1648-induced alteration of NO-dependent relaxation. Because the presence of PM was clearly evidenced in lung parenchyma removed 72 hr after SRM1648 instillation, it is unlikely that recovery is related to elimination of particle deposit from lung parenchyma.Several re to PM . This stOxidative stress is a major contributor of the adverse effects of PM . In thisRelease of inflammatory mediators is associated with PM-induced impairment of endothelium-dependent vasodilatation in systemic arteries . In the In conclusion, this study shows that urban but not manufactured PM (including nanoparticles) impairs NO-mediated relaxation, without affecting cGMP responsiveness in rat intrapulmonary arteries. We attribute this result to an oxidative-stress–independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway in pulmonary circulation may favor vasoconstriction, remodeling, and thrombosis, all contributing to enhance arterial resistance, which in turn may have a negative impact on cardiac function.


Arguably, one of the most challenging aspects of liver transplant surgery is the hepatic artery reconstruction. When the donor and recipient arteries are normal, this anastomosis can still be difficult. However, when the recipient artery has been dissected or is small other alternative reconstructions must be considered. Routinely, the donor surgery includes removing the iliac artery and vein specifically to aid in alternative reconstruction techniques. With the increase use of extended criteria donors  the iliac vessel may be unusable because of atherosclerotic disease. This paper describes revisiting an alternative technique for hepatic artery reconstruction during cadaveric liver transplant when the recipient artery has been dissected and the iliac vessels were unusable secondary to arterial plaque from a 75 yo donor. Herein, we describe the successful anastomosis of the celiac artery with aortic patch from the donor directly to the supraceliac aorta of the adult recipient. A 68-year-old female with primary biliary cirrhosis received a liver transplantation from a deceased heart-beating donor for end-stage liver disease with a MELD of 24. Preoperative study showed no evidence of hepatic artery abnormalities. The donor was a 75-year-old female that was pronounced brain-dead after a stroke. The donor's only comorbidity was significant history of cigarette use. During the donor surgery the celiac artery did not have any atherosclerotic disease and the hepatic arterial anatomy was normal. Of note, the iliac arteries were severely diseased with hard circumferential plaque. Liver transplantation was performed by using the Piggy-back technique with restoration of vena caval and portal vein blood flow before the arterial reconstruction. It was then discovered that the native hepatic artery was dissected Figures . In thisThe length of the donor celiac artery was adequate without tension. The gastrohepatic ligament had already been ligated during the hepatectomy and the stomach was retracted laterally. The diaphragmatic cura were divided in a muscle splitting fashion in an attempt to preserve them starting just below the insertion of the aorta through the diaphragm. The supraceliac aorta was exposed inferiorly for approximately 3-4 cm which allowed cross-clamping with two straight vascular clamps above the celiac artery origin. We did not dissect out the aorta circumferentially to avoid the posterior lumbar arteries in that area of the aorta. An arteriotomy was made and enlarged with two 4.8 mm aortic punches. The anastomosis was created with 5–0 prolene in running fashion. Cross-clamp was 17 minutes. Postoperatively, the patient's creatinine rose to 1.6 mg/dL on POD1 and returned to a baseline level of 0.8 mg/dL by POD4. Transaminases reached 1000 (U/L) on POD1 and came down immediately. They did not seem affected by the arterial reconstruction and were probably a result of the age of the donor. The patient was started on TPN from POD1 to POD 9 and had a nasogastric tube until POD 2. TPN was used as a precaution because we were concerned that the patients' age combined with ischemia time to the gut from a supraceliac aortic clamp would cause a significant ileus. She did have some delay in bowel function. We left her nasogastric tube in for two days and then advanced her diet slowly over the next few days. During this time we left her on TPN until she had significant caloric intake by mouth. She tolerated a regular diet on POD 9. She had no evidence of neurological dysfunction [Both the short and long-term outcomes are excellent. She is now a year out and has had no complications including no episodes of rejection. The graft is functioning well and she did not need a retransplant.Successful hepatic artery reconstruction can normally be achieved with the donor celiac artery anastomosied to the recipient hepatic artery. Occasionally, the recipient hepatic artery is insufficient to provide arterial inflow to the graft. At our center, in these cases, we will use the iliac artery from the donor as a jump graft to the infrarenal aorta with good success . HoweverUnfortunately, dissection of the hepatic artery can occur and is a difficult problem to overcome. In this patient, there were no preoperative risk factors such as TACE , hypertension, or vascular disease, except age. The reason for the dissection then could have been technical or from positioning of a retractor during the anhepatic phase of the transplant. Once the dissection was recognized, the team immediately began determining options for arterializations, realizing that the donor iliac vessels were unusable and there were no compatible donor iliac vessels from previous transplants in storage. Because the donor celiac artery was lying on the aorta in perfect position we decided to perform the reconstruction described above. The main issue was dissection of the cura of the diaphragm. The previous hepatectomy had moved the stomach and exposed the cura. We preformed a muscle splitting dissection to preserve the cura. Then we placed two straight vascular clamps on the aorta. After the anastomisis was complete, the cura surrounded the reconstruction. Two case series in the early 1990's described using the supraceliac aorta as arterial inflow to a liver graft. In Shaked et al. they preformed a primary end to side common hepatic artery and supraceliac anastomosis without the use of grafts in eleven adult patients mainly for small arteries <3 mm or for hepatic artery thrombosis . HenneinWhile the modest experience thus far is generally good, direct anastomosis to the supraceliac aorta with the donor's aortic carrel patch should not be forgotten as an alternative anastomosis when the recipient and donor's anatomy dictate. With the increased use of older donors the iliac vessels are often not usable. There is a definite increased morbidity with this reconstruction, but, it can be performed successfully to provide adequate arterial inflow to liver grafts in difficult situations.


Polyorchidism is defined as the presence of more than two testes. The management of this rare condition is still debatable, particularly when it is an incidental finding at surgery.We present the case of an 8-year-old boy with triorchidism found incidentally during an elective orchidopexy. This supernumerary, ectopic and atrophic testis was removed to avoid an increased risk of malignancy.Risk of malignancy justifies the removal of an atrophic and ectopic testis in triorchidism. However, it would appear safe to preserve a viable intrascrotal supernumerary testis found incidentally at surgery provided that the patient is followed up in the long term. Polyorchidism is a rare condition defined as the presence of more than two histologically proven testes . TriorchThe management of supernumerary testes is still debatable, particularly when found incidentally in association with undescended testes, testicular torsion, hydrocoele or inguinal hernia. We report a rare case of triorchidism in an 8-year-old boy that was found during an elective orchidopexy and discuss current views on the management options.An 8-year-old boy presented with an undescended left testicle. Although bilateral testes were present at birth, the left testis was reported as retractile at the age of 3 years. On examination, the right testis was normal in size and well placed in the scrotum, the left testicle was palpable in the groin and the left scrotum was well developed but empty.At operation, a normal sized testis with normal morphology was found in the left inguinal canal attached to normal testicular vessels and a vas deferens. A smaller, atrophic testis was found more distal in the suprascrotal space also attached to a normal appearing vas deferens and vessels. The two vasa deferentia arose from a single vas at the internal ring. The findings were consistent with Leung's type C polyorchidism . The norPolyorchidism is a rare condition that may be found incidentally during a groin exploration. There is no consensus in the literature regarding the management of this condition, particularly when the supernumerary testis is viable, thus posing a surgical dilemma when found incidentally.Authors proposing a conservative approach argue that infertility is a common finding in patients with polyorchidism  and presOther authors argue that the majority of accessory testes have histologically reduced or absent spermatogenesis and propose that the increased risk of malignancy warrants removal of the supernumerary testis, particularly if it is non-viable, undescended or ectopically located ,7. This This report illustrates the unexpected presentation of polyorchidism during elective groin procedures and the management challenge considering the risk and benefits of excising a supernumerary viable testis.The authors declare that they have no competing interests.SA presented the case history, researched the topic and helped draft the manuscript. JT reviewed the literature and drafted the manuscript. KL was the supervising consultant who performed the surgery, obtained consent for publication and edited the manuscript. All authors read and approved the final manuscript.Written informed consent was obtained from the patient's next-of-kin for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Critical needs for treatment trials in gastroesophageal reflux disease (GERD) include assessing response to treatment, evaluating symptom severity, and translation of symptom questionnaires into multiple languages. We evaluated the previously validated Reflux Disease Questionnaire (RDQ) for internal consistency, reliability, responsiveness to change during treatment and the concordance between RDQ and specialty physician assessment of symptom severity, after translation into Swedish and Norwegian.Performance of the RDQ after translation into Swedish and Norwegian was evaluated in 439 patients with presumed GERD in a randomized, double-blind trial of active treatment with a proton pump inhibitor.The responsiveness was excellent across three RDQ indicators. Mean change scores in patients on active treatment were large, also reflected in effect sizes that ranged from a low of 1.05 (dyspepsia) to a high of 2.05 (heartburn) and standardized response means 0.99 (dyspepsia) and 1.52 (heartburn). A good positive correlation between physician severity ratings and RDQ scale scores was seen. The internal consistency reliability using alpha coefficients of the scales, regardless of language, ranged from 0.67 to 0.89.The results provide strong evidence that the RDQ is amenable to translation and represents a viable instrument for assessing response to treatment, and symptom severity. Symptom-focused questionnaires have an important role in clinical trials of gastroesophageal reflux disease (GERD) management. This is especially the case given that symptom relief is a major goal of treatment for patients with GERD , and thaThe Reflux Disease Questionnaire (RDQ), a 12-item self-administered questionnaire, was designed to assess the frequency and severity of heartburn, regurgitation, and dyspeptic complaints and to facilitate the diagnosis of GERD in primary care . The psyIn this study, the performance of the RDQ was assessed in a clinical treatment trial for patients with GERD. Whereas prior work on the RDQ was completed with patients seen in primary care, the current investigation was undertaken in the context of a multicenter, double-blind, randomized study in which Scandinavian patients with heartburn as the predominant symptom were treated with esomeprazole for 2 weeks. We extended the earlier psychometric work on the RDQ by investigating its responsiveness to changed symptom status as a result of therapy in a large clinical population of patients diagnosed as having GERD. The concordance between the RDQ evaluations of symptom severity was compared to those offered by specialty physicians. The success of the translation of the RDQ into Swedish and Norwegian was also evaluated.Adult patients presenting with presumed GERD symptoms were recruited from 35 endoscopy units across Sweden and Norway ,5. IncluAll patients underwent an endoscopy and pH monitoring, including assessment of Symptom Association Probability (SAP). A diagnosis of GERD was considered 'proven' when either endoscopy (LA grade A or B) and/or pH monitoring  or SAP (95% or more during the 24 hr pH monitoring) was positive.The RDQ is a self-administered questionnaire in which subjects are asked to report the frequency and severity of their upper gastrointestinal symptoms. There are three subscales that evaluate regurgitation, heartburn, and dyspepsia . The heaThe OTE, a validated scale, rates the change in symptoms on a 15-point scale  . At the At both clinic visits, a clinical trial assessment interview and a physical examination were conducted by the investigators. Patients were asked about the severity of their heartburn, regurgitation, dysphagia, epigastric pain, and nausea over the three days prior to each clinic visit, this inquiry being structured by the trial case record form for each visit and graded 0 = none, 1 = mild, 2 = moderate, and 3 = severe.The RDQ was translated into Norwegian and Swedish according to international principles . The traThere were three specific analytical objectives: 1) assessment of the responsiveness to treatment of selected RDQ scales; 2) assessment of the concordance between RDQ- and physician-generated ratings of disease severity; and 3) assessment of the internal consistency reliability of the translated versions to verify the consistency of the concepts. Responsiveness was determined by comparison of RDQ data to global symptom change reported on the OTE question. The change from baseline should be larger for patients who were 'better' according to OTE than for those who were 'worse or 'unchanged'. Student's t test for paired samples, the standardized response mean , and the134 subjects from Norway and 305 from Sweden with a mean age of 51.4 (13.5) years were enrolled at 35 sites. The baseline characteristics and clinical information for patients with data from both a baseline and subsequent visit are provided in Table After two weeks of trial therapy, patients were told to assess symptoms during the previous seven days and completed the RDQ a second time. Responsiveness was first examined by collapsing responses on the OTE question to four possibilities , as described above. A progressive increase was seen in the change score moving from the worse to much better categories, regardless of the treatment group Table . Table 3The observed effect sizes ranged from a low of 1.05 (dyspepsia) to a high of 2.05 (heartburn). As anticipated, the responsiveness of the heartburn scale was highest and the dyspepsia scale the lowest of the three scales.It must be noted that a sizable change in scores was observed for those in the placebo group who indicated they were at least a good deal better on the OTE item.Esomeprazole in either dose was markedly more effective than placebo in improving the GERD score p < 0.0001)  surpassed the accepted level of 0.70 .The RDQ was developed to facilitate the identification of GERD in primary care and this was the setting in which its psychometric properties were established . This stThe responsiveness of the RDQ scales to treatment was observed to be quite high by all three methods of analysis. The observed effect sizes outstripped conventional thresholds for superior responsiveness . While, An important aspect of a useful symptom questionnaire is its ability to capture nuances in various disease symptom complexes. Evidence that an instrument is able to capture severity would be particularly useful because disease severity often directs different courses of treatment. The purpose of the current study was to evaluate how well the RDQ tracked physician ratings of disease severity for regurgitation, heartburn, and dyspepsia. The results demonstrated that the RDQ is quite sensitive to symptom severity as measured by specialty physicians. The fact that the concordance between the two sources was more pronounced at the follow-up visit may be due to several factors, e.g. practice effects (on both the patient's and physician's part), compression of symptom evaluations at the follow-up visit in response to treatment (most patients got better), and/or a more comparable time referent between both data sources at the follow-up. The results of a recent paper argue for the use of a self-report survey to supplement investigator obtained data in this critical step, especially if the primary outcome measurement tool is going to be a self-report symptom measure . In thisDevelopment of subjective, self-report questionnaires for symptom assessment requires rigorous psychometric evaluation. Development and psychometric evaluation of instruments includes item selection and multitrait scaling, internal consistency of items that combine into a dimension , as wellThis study provides evidence that the RDQ is amenable to translation into Norwegian and Swedish, and that it represents a viable instrument for assessing symptom severity and response to treatment in clinical trials of patients with GERD.The authors declare that they have no competing interests.Ola Junghard and Tore Lind are AstraZeneca employees, and Ingela Wiklund was an employee at the time of the study.FJ was the principal investigator in the study, and was involved in the design. MS, TB, TL, OJ, and JD were involved in the analysis, reporting and writing up of the manuscript. All authors read and approved the final manuscript.


HIV-1 invades the central nervous system (CNS) in the context of acute infection, persists thereafter in the absence of treatment, and leads to chronic intrathecal immunoactivation that can be measured by the macrophage activation marker, neopterin, in cerebrospinal fluid (CSF). In this review we describe our experience with CSF neopterin measurements in 382 untreated HIV-infected patients across the spectrum of immunosuppression and HIV-related neurological diseases, in 73 untreated AIDS patients with opportunistic CNS infections, and in 233 treated patients.In untreated patients, CSF neopterin concentrations are almost always elevated and increase progressively as immunosuppression worsens and blood CD4 cell counts fall. However, patients with HIV dementia exhibit particularly high CSF neopterin concentrations, above those of patients without neurological disease, though patients with CNS opportunistic infections, including CMV encephalitis and cryptococcal meningitis, also exhibit high levels of CSF neopterin. Combination antiretroviral therapy, with its potent effect on CNS HIV infection and CSF HIV RNA, mitigates both intrathecal immunoactivation and lowers CSF neopterin. However, despite suppression of plasma and CSF HIV RNA to below the detection limits of clinical assays (<50 copies HIV RNA/mL), CSF neopterin often remains mildly elevated, indicating persistent low-level intrathecal immune activation and raising the important questions of whether this elevation is driven by continued CNS infection and whether it causes continued indolent CNS injury.Although nonspecific, CSF neopterin can serve as a useful biomarker in the diagnosis of HIV dementia in the setting of confounding conditions, in monitoring the CNS inflammatory effects of antiretroviral treatment, and give valuable information to the cause of ongoing brain injury. The AIDS dementia complex (ADC) or HIV-associated dementia (HAD) was recognized as a novel central nervous system (CNS) disorder early in the AIDS epidemic  and subsHowever, interest in neopterin and other soluble immunological biomarkers in blood waned with the development and widespread clinical use of quantitative assays of HIV-1 RNA that provided a valuable practical guide to the pace of disease progression and the effects of treatment. In parallel attention to CSF immunological biomarkers, including neopterin, declined after it was shown that HIV RNA levels could be measured in the CSF of most untreated patients and that high levels could often be detected in ADC/HIVE ,9,10. AtMore recently, several factors have converged to suggest that it might be worthwhile to revisit immunological CSF biomarkers in general, and neopterin, in particular. One of these again parallels considerations of systemic HIV disease and relates to the renewed appreciation of the importance of immune activation in systemic disease pathogenesis and progression . A numbeIn this review we examine changes in CSF neopterin concentrations in the different stages of systemic HIV infection and HIV-related neurological disease in untreated patients and the impact of treatment. To examine and illustrate these issues, we have aggregated a cross-sectional experience derived from four clinical sites  that span a broad range of subjects who have been examined in the context of natural history, treatment and clinical studies. Some of these patients have been reported as part of smaller previous reports ,15-19, bWe will first briefly review the biology of neopterin and its use as an indicator of macrophage activation in HIV CNS infection and disease. We will then describe our experience with this measurement in the aggregate cross-sectional cohort and also present some longitudinal subject examples before considering what these findings indicate and how neopterin might be used in the future. While CSF neopterin has been known to be elevated in HIV infection and further increased in ADC/HIVE and CNS opportunistic infections for more than two decades ,5, most in vitro, but importantly tumor necrosis factor-α (TNF-α) can accelerate neopterin synthesis when initiated by IFN-γ [Neopterin is a biochemical product of the guanosine triphosphate pathway that is both cell-restricted and inducible by immune-inflammatory stimuli. It is produced primarily in monocyte/macrophage and related cells and the most important stimuli are interferons, especially Th1-type cytokine interferon-γ (IFN-γ)  by macrophages ,25, whicThere is often a good correlation between blood and CSF neopterin concentrations in patients with HIV infection. An early study also demonstrated a significant correlation between blood neopterin concentrations and the loss of brain tissue expressed as the ventricle-brain ratio measured by computed tomography . The parA practical further aspect of relevance in considering the use of CSF neopterin as a disease biomarker in comparisons to others, including the cytokines that regulate its production, is its stability in biological fluids, due to its rather polar chemical character, ready diffusibility, and long half-life. By contrast many cytokines  have short half-lives with biological activities that rely on effects on neighbouring cells in close proximity rather than at a distance and that might not be as well reflected in lumbar CSF. Neopterin appears to provide a more stable indicator of the aggregate macrophage activation in the CNS compartment. It is also easily and reliably measured with commercially available ELISA or RIA  that have been shown to yield comparable results . AdditioTo provide a view of the CSF neopterin changes across the spectrum of HIV infection and HIV-related CNS injury within the context of other biomarkers, we examined a cross-sectional sample derived from four clinical centers that included HIV seronegative subjects, untreated neuroasymptomatic HIV-infected subjects grouped according to blood CD4+ T cell, ADC neurological diagnoses, two groups of treated HIV-patients and five groups with CNS opportunistic diseases.neuroasymptomatics, NA) were stratified by blood CD4+ T cell counts and included: 53 subjects with CD4+ counts <50 cells/μL (mean age 38.9); 69 subjects with CD4+ counts 50-199 cells/μL (mean age 38.6); 69 with counts 200-349 cells/μL (mean age 38.8); and 108 with CD4+ counts >350 cells/μL (mean age 37.5). Untreated patients with ADC were divided into 30 with Stage 1 (mean age 38.9) and 53 with Stage 2-4 severity (mean age 40.1). Treated subjects included 150 with plasma HIV RNA suppressed below 50 copies/mL (referred to as treatment successes) (mean age 43.4) and 83 with >50 copies/mL (treatment failures) (mean age 45.3) after >6 months of treatment. The 73 subjects with CNS opportunistic diseases  included 16 with progressive multifocal leukoencephalopathy (PML), 13 with cytomegalovirus encephalitis (CMV-E), 18 with toxoplasmic encephalitis (toxo), 16 with cryptococcal meningitis (crypto) and 10 with primary CNS lymphoma (PCNSL). CSF neopterin was measured by either EIA or RIA using the BRAHMS kit and following the manufacturer's instructions. The assays were performed in Innsbruck  and San Francisco ; while formal quality control comparison between those two laboratories was not done, samples in this and subsequent studies performed in duplicate at both sites were in close agreement (<12 percent variance). Multiple group comparisons were analyzed by Kruskal-Wallis test and Dunn's multiple comparison post hoc tests, two-group comparisons used the Mann Whitney test, while correlations among variables across groups used Spearman's test, all performed with Prism 5 (GraphPad Software).The 53 HIV-seronegative subjects in San Francisco who volunteered for study lumbar puncture (LP) as controls were derived from a similar background to the HIV-infected subjects in San Francisco (mean age 43.9 years); 43 (81%) were male, similar to the proportion in the HIV-infected subjects. The untreated HIV-infected subjects without overt neurological disease  in untreated HIV infection across the spectrum of CD4+ T cell decline. Indeed, CSF neopterin increased as blood CD4+ cells fell, rising from a mean of 17.9 nmol/L (SD 17.7) in those with CD4+ counts >350 cells/μL, to 21.0 nmol/L (SD 14.2) with CD4+ cell counts of 200 - 349 cells/μL, and seeming to plateau in those with 50 - 199 and < 50 cells/μL, with means of 28.7 and 26.2 (SDs 17.2 and 17.1), respectively Figure . ClearlyThe changes in blood neopterin Figure  showed amild but definite impairment in daily activities (Stage 1), to moderate impairment with inability to perform the more demanding aspects of daily life (Stage 2), severe with major intellectual or motor incapacity and slowing (Stage 3), or end stage disease with nearly vegetative state and only rudimentary comprehension and responses (Stage 4)This group included patients defined by impairment in their cognitive-motor functional status in daily life and confirmed by bedside examination  and classified as ADC stages 1-4 as previously defined . In brieIn the patients with ADC stage 1-4, there was a notable jump in CSF neopterin Figure  comparedBlood neopterin was also higher in the ADC patients, but did not show the same increase as CSF. Thus, whereas the increases in blood and CSF levels of neopterin were of similar magnitude in the neuroasymptomatics , the increase in blood neopterin in the ADC patients was notably less marked than in the CSF. Blood neopterin was higher in the ADC 2-4 than in the NA with CD4+ cell counts ≥ 200, but not in those below 200.Because data on the CSF concentrations in CNS OIs in HIV infection are limited, we included subjects with five different OIs in this analysis. None of the patients were on antiretroviral treatment at the time of CSF collection. The diagnosis was confirmed by positive CSF PCR for JC virus in progressive multifocal leukoencephalopathy (PML) and for cytomegalovirus (CMV) in CMV encephalitis, by response to treatment for toxoplasmosis, by CSF cryptococcal antigen or culture, and for primary CNS lymphoma (PCNSL) by histological confirmation or presumptively by combining CSF Epstein-Barr virus PCR, thallium 201 SPECT and lack of response to antitoxoplasmic treatment Figure . As a grBecause of the heterogeneity of this OI aggregate group, we further examined the individual OIs and compared them to two groups with similar CD4 counts, the NAs with <200 and the ADC 1-4 . As shown in Figure Thus, overall, OIs can confound diagnosis of ADC using CSF neopterin, particularly in the case of CMV encephalitis which may also be difficult to distinguish by neuroimaging and non-focal clinical findings, emphasizing the importance of CSF CMV PCR in this differential diagnosis. The other OIs can usually be distinguished by clinical and neuroimaging findings. The relatively low CSF neopterin in PML is consonant with the paucity of inflammation pathologically. Since none of these patients exhibited clinical or radiographic immune reconstitution inflammatory syndrome (IRIS), it will be of interest in the future to examine whether neopterin or other CSF immune inflammatory markers might help to understand and clinically distinguish and monitor this disorder .Treated patients were defined as those receiving at least three antiretroviral drugs (cART). They were divided into success and failures, according to plasma HIV-1 levels above or below 50 copies/mL. CSF neopterin was, in general, markedly reduced in the two treated patient groups compared to the untreated subjects, showing that combination therapy has a potent effect on intrathecal immunoactivation. However, as previously reported ,19, thesWhile the ANOVA analysis that include all of the groups did not show a difference between the successes and failures, a simple comparison between these two groups suggested a significant difference (p = 0.0004 using Mann Whitney test) consistent with the view that more effective viral control had an effect on CSF neopterin. However in neither group did the levels of CSF neopterin clearly relate to the penetration and efficacy of their antiviral drugs, at least as measured by the CNS penetration-effectiveness (CPE) score or rank as proposed and recently revised by Letendre and colleagues ,41. We aThese results also bring up the issue of normal levels of CSF neopterin. For this study our controls were taken from a population with a similar range of risks and background conditions as the HIV-infected subjects. This may account for the higher mean level in this group than in other control group studies. In an earlier study of 24 healthy volunteers CSF, neopterin concentrations ranged between 3.2 and 5.5 nmol/l (mean 4.2 nmol/L) with the RIA method (Henning/BRAHMS Berlin) . In 47 cFour longitudinal case examples shown in Figure The first patient (A) illustrates the rapid decrease in CSF immune activation and HIV RNA in some patients with ADC treated with potent cART. It also shows the dissociation of intrathecal and systemic macrophage activation at baseline. The response to therapy shows that the high level of CSF neopterin was 'driven' by HIV infection, since it improved as quickly as viral replication was inhibited.Patient A was 33 years old when he presented with Stage 2 ADC in January, 2000 with both cognitive and motor (including spastic gait) impairment. This was his presenting manifestation of HIV infection which was diagnosed at the same time with a blood CD4+ T cell count of 133 cells per μL. He was treated with abacavir, 3TC, nevirapine, and ritonavir-boosted indinavir with rapid HIV RNA response in both blood and CSF (top panel). After a transient increase, his high CSF neopterin also fell rapidly, and over the year of follow-up reached a near normal level (6.4 nmol/L). While his blood neopterin was also elevated and fell, the magnitude at baseline and subsequent change were far less than the CSF. Over the same period he improved clinically and was able to return to acting school, albeit with mild residual gait stiffness; his performance measured by an aggregate Z score n four quantitative neurological performance tests (QNPZ-4) improved from -4.56 to -1.86 [to -1.86 .The second patient (B) again illustrates the potent effects of cART on CSF neopterin. It also illustrates a phenomenon reported in other 'failing' patients - CSF HIV RNA levels may remain disproportionately reduced in the face of drug resistance and poor adherence . Also, aPatient B was 47 years when diagnosed with ADC Stage 2 in January, 2000, again with a substantially higher CSF than blood neopterin level. Dementia was his presenting manifestation of HIV infection and the blood CD4+ T cell count was 130 cells per μL The CSF neopterin response was a little slower then in the previous case when he was treated with ritonavir-boosted indinavir, zidovudine and lamivudine. It remained elevated at 21.3 nmol/L at one year when the CSF (and blood) HIV RNA had reached the limit of detection (40 copies per mL). Subsequently, his treatment adherence varied (dashed line in top panel) and his plasma HIV RNA rose above his pre-treatment level. The CSF HIV RNA level, though detectable, did not rise proportionately, nor did his CSF neopterin which remained modestly elevated, but not to the baseline level. Over the effective treatment period his mental status improved clinically.The third patient (C) again illustrates the dissociation of CSF and blood neopterin levels, even without overt CNS disease, and the response to cART over a very long period and after treatment interruption.Patient C was 51 year in January, 1997 when diagnosed with a symptomatic primary HIV infection with a CD4 cell count of 250 cells per μL. Treatment was given immediately with indinavir, zidovudine and lamivudine. He has been followed for 12 years with yearly lumbar punctures, including a period of treatment, drug holiday, and resumed treatment. As he stopped his treatment, the CSF neopterin rose to 40.5 nmol/L and when new treatment with efavirenz, abacavir and lamivudine was given, the CSF neopterin concentration fell to just above normal (6.9 nmol/L).10 CSF HIV RNA increase and preceded his clinical presentation. This suggests not only an increase in CNS infection, but a switch in its character to a type that associates with brain injury. In this case, the change in CSF neopterin might have served as a helpful indicator of the development of ADC.The final patient (D) illustrates a steady rise in CSF neopterin that was dissociated from his relatively stable blood neopterin, proportionally exceeded his logPatient D was 41 years old when he first entered a longitudinal natural history  study in July, 2002. He had a history of drug abuse and psychiatric disease, both of which obscured his underlying HIV-related neurological impairment as he began to develop neurological disease over the second year of follow-up; this also contributed to his refusal to begin cART. While his blood and CSF HIV RNA levels gradually increased over the initial two years of his course, this also did not lead to starting therapy. His CSF neopterin rose steeply during the second year of follow-up at a time when was judged neurologically stable until he presented with increasing confusion and was diagnosed with ADC Stage 1 and a blood CD4+ T cell count of 267 cells per μL (his nadir). He was hospitalized and began treatment with zidovudine, 3TC and nevirapine. His CSF neopterin decreased rapidly, then more gradually, reaching 6.4 nmol/L at the end of follow up. CSF and plasma RNA were at the limit of detection. He also recovered clinically with eventual restoration to normal activities with his QNPZ-4 improving from -2.28 before treatment to 0.45 after.Together the presented data, along with earlier studies, show that neopterin is produced in the intrathecal space (higher CSF than blood concentrations) and that increased CSF concentrations of this pteridine indicate a nearly universal state of enhanced macrophage activation within the CNS in HIV infection. Its elevation with infection and rapid decrease with treatment show that it is ultimately driven by HIV infection. However, in ADC patients, the levels of CSF neopterin rise above those in neuroasymptomatic patients with comparable systemic and CSF HIV RNA concentrations and pleocytosis. One speculation is that to some extent neopterin is produced in the meningeal and perivascular spaces in relation to local infection, but that in ADC and its underlying substrate, HIVE, the character of infection and its capacity to produce neopterin changes as infected and uninfected macrophages and microglia are activated. Extending this hypothetical framework, the augmented neopterin in these patients may indicate autonomous compartmentalized HIV infection within CNS macrophages , whereasGiven the changes in CSF neopterin and its relation to the critical process of immunoactivation within the CNS, one can ask whether there might be a role for measurement of this CSF biomarker in clinical practice, including diagnosis, prognosis and treatment evaluation related to CNS injury.When HIV-infected patients present with neurological abnormalities, the character of symptoms and signs leads to appropriate evaluations for opportunistic infections, malignancies, vascular diseases and other afflictions using neuroimaging and other modalities. Absence of focal clinical or neuroimaging toxoplasmosis/CNS lymphoma findings, and negative CSF analysis for CMV, other herpes virus, JCV, EBV and cryptococcus supports the ADC/HIVE diagnosis.To assess the value of CSF neopterin in this setting, we used the cross-sectional study results shown in Figure Thus, measuring the CSF neopterin has diagnostic value in ADC, though not to a degree to provide sufficiently certain diagnosis on its own. The reasons for this uncertainty relate principally to the overlap of the neuroasymptomatic subjects into the range of the ADC subjects, particularly the ADC 1 group. There are several explanations for this beyond a true biological overlap in CSF neopterin. These include imprecision of clinical diagnosis in classifying our subjects. Thus, some of the neuroasymptomatics may indeed have had incipient or unrecognized brain injury. Case D provides an example where CSF neopterin elevation indeed predicted clinical presentation. On the other hand, some patients diagnosed as ADC might have suffered other conditions. At the present time there is no objective 'gold standard' for this diagnosis.Is it possible to use CSF neopterin concentrations as a prognostic marker? In a prospectively studied cohort of 35 neurologically asymptomatic HIV-infected patients, CSF neopterin above 20 nmol/l had almost 7 times the risk of developing ADC, but the risk did not increase further when CSF neopterin was above 40 nmol/L . These pThe goal for antiretroviral treatment is to eliminate mortality and morbidity related to organ dysfunction, including the CNS, related directly or indirectly to HIV infection. We generally measure this efficacy using the surrogate, plasma HIV RNA level. However, there is growing concern that CNS morbidity can continue despite treatment that suppresses plasma, and even CSF, viremia, at least as measured by conventional clinical assays . Can CSFCombination ART has a profound effect on CSF viral load and neopterin levels as shown above and reported previously . Hence, Persistent low-level neopterin production might per se be associated with chronic CNS damage, as expression of intrathecal immune activation and also because of its probable neurotoxic effect. Given the long lifespan of HIV-infected persons, these effects might, in the long-term, combine with CNS insults typical of the older age and contribute to functional neurological impairment.Combination antiretroviral drug treatment has had a dramatic effect on morbidity and mortality of HIV infection, including those involving the CNS and the previously most common of these, ADC/HIVE. The study of CSF neopterin has contributed to our understanding of CNS HIV infection and its consequences. As attention now turns to the potential CNS effects of lower grade immunoactivation before or in the presence of treatment, continued study of CSF neopterin may turn out to be helpful in this next phase of understanding pathogenesis and designing and evaluating therapeutics. We do not understand the consequences of chronic immune activation in these settings and whether interventions can be tailored to reduce any deleterious effects. Hence, further studies are needed to evaluate this as well as whether particular antiretroviral drug combinations may more effectively minimize such immune activation. Although nonspecific, we think that neopterin concentration is a useful biomarker in monitoring this CNS immune activation and its potential consequences, and in evaluating the effects of different antiviral and even adjuvant strategies that have proved so difficult to assess using neurological symptoms or signs, neurocognitive performance or CSF viral loads. CSF neopterin may prove to be a valuable surrogate to address these important issues.The authors declare that they have no competing interests.LH, PC, RWP, MG, BJB, SS, and AB collected CSF samples and made the subject evaluations. DF and RWP were responsible for the biochemical analyses. The study was planned and interpreted and the data were reviewed and revised by all the authors. LH and RWP prepared the manuscript. All authors read and approved the final manuscript.


Sheep are seasonal breeders. The key factor governing seasonal changes in the reproductive activity of the ewe is increased negative feedback of estradiol at the level of the hypothalamus under long-day conditions. It has previously been demonstrated that when gonadotropin secretions are inhibited during long days, there is a higher concentration of estradiol in the cerebrospinal fluid (CSF) than during short days. This suggests an involvement of the CSF and choroid plexus in the neuroendocrine regulatory loop, but the mechanisms underlying this phenomenon remain unknown. One possible explanation of this difference in hormonal content is an effect of concentration or dilution caused by variations in CSF secretion rate. The aim of this study was thus to investigate changes in the CSF turnover rate related to light-dark cycles.The turnover rate of the CSF was estimated by measuring the time taken for the recovery of intraventricular pressure (IVP) after removal of a moderate volume (0.5 to 2 ml) of CSF (slope in mmHg/min). The turnover rate was estimated three times in the same group of sheep: during a natural period of decreasing day-length corresponding to the initial period when gonadotropin activity is stimulated (SG1), during a long-day inhibitory period (IG), and finally during a short-day stimulatory period (SG2).p < 0.05). The turnover rate in SG2: 183.16 ± 23.82 μl/min was not significantly different from SG1: 169. 23 ± 51.58 μl/min , but was significantly different from IG: 71.33 ± 16.59 μl/min (p = 0.016).The time taken and the speed of recovery of initial IVP differed between groups: 8 min 30 sec, 0.63 ± 0.07 mmHg/min(SG1), 11 min 1 sec, 0.38 ± 0.06 mmHg/min (IG) and 9 min 0 sec, 0.72 ± 0.15 mmHg/min (SG2). Time changes of IVP differed between groups (ANOVA, p < 0.005, SG1 different from IG, This study shows that the turnover rate of CSF in ewes changes according to the light-dark cycle; it is increased during short day periods and reduced in long day periods. This phenomenon could account for differences in hormonal concentrations in the CSF in this seasonal species. The choroid plexus has two main functions: it provides a barrier between the blood and the cerebrospinal fluid (CSF), and it is the major secretory source of CSF ,2. CSF aSheep in temperate latitudes are seasonal breeders. Among the different seasonal cues, photoperiod is the most reliable parameter and is used by animals as an indication of the season. The photoperiodic information is transduced into neuroendocrine changes through variations in melatonin secretion from the pineal gland. As a result of these variations in ewes, the female sheep, the key phenomenon governing seasonal changes in gonadotropin is an increase in the negative feedback exerted by estradiol within the hypothalamus during long days . We prevThe experiments were conducted in accordance with Authorization N°37801 for Animal Experimentation and Surgery from the French Ministry of Agriculture, following the European Community Council Directive 86/609/EEC. Surgery and postoperative care were conducted in certified facilities .Six adult crossbred Romanov X Ile-de-France ewes aged 7 ± 1 yr were ovariectomized and received a 2 cm subcutaneous estradiol implant [ implant . These iR; 12 μg/kg b.w., Abbott, Rungis, France) in order to perform tracheal intubation and was then maintained with isoflurane (3% in O2). Following the previously described method [R, Nycomed Ingenon SA, Paris, France) was injected into the lateral ventricle through a cannula, delimiting the ventricular system on lateral and dorsal X-ray images. These lateral and dorsal images allowed us to place the cannula accurately within the third ventricle. The cannula  was constructed from a stainless steel luer-lock needle  and cut to a length of 40 mm. It was inserted into the third ventricle and the outflow of CSF through the cannula once inserted was checked. The cannula was then sealed and covered with a plastic cylinder. After surgery, the animals were allowed to recover for 4 days in a specifically designed room . During the recovery period, they received antibiotics  and combined anti-inflammatory and diuretic treatment  daily.Food and water was withheld for 24 h prior to premedication with atropine sulphate . General anesthesia was induced by intravenous injection of barbiturate  initially set to zero. IVP measurements, averaging high and low values due to heart beat, were recorded every minute. After stabilization, a volume of 0.5 to 2 ml of CSF was removed with a 2 ml syringe via a three-way tap by gentle aspiration limited to 30 s and this volume was weighed to ensure precise measurement. Care was taken to ensure that the ventricle did not collapse; this was done by feel and as soon as any resistance to flow was felt we terminated the aspiration. The IVP (in mmHg) was then recorded for 15 min or more. Occasionally, the transducer was connected to a paper recorder and the IVP recorded in real time (not shown). After that, the sheep were allowed to recover from anesthesia and returned to the indoor, light-controlled pen.The IVP of the ewes was recorded first during the decreasing natural photoperiod at the September equinox (12 hL/12 hD), a time of year when gonadotropin secretion is stimulated (SG1). Thereafter, they were kept in artificial long days for a sufficient duration to ensure inhibition of gonadotropin secretion (IG) through increased negative feedback of estradiol at the level of the hypothalamus . After 9High and low IVP was measured continuously and averaged every minute. From the lowest IVP recorded, we measured the time taken to reach the initial pressure (T), and computed the average speed of recovery after CSF removal (slope in mmHg/min). The measurements were usually ended after 15 min. However, in two cases during the IG session, and one during SG2, IVP did not return to the initial values within this period. In these cases, the recording continued up to recovery (18 to 20 min) and a value of 15 min was attributed to the animals for computation. We computed the TOR of the CSF in μl/min by dividing the extracted volume of CSF by T. Analysis of differences between groups for the time changes of IVP after CSF removal was carried out using ANOVA, with effect of photoperiodic treatment and time as independent variables. Analysis of differences between groups for TOR was carried out using the Mann-Whitney non-parametric test. Values are given as mean ± SEM.2 = 0.348), but was correlated with the speed of recovery (r2 = 0.8171). The times (T) taken to reach the initial pressure were 8 min 30 sec ± 1 min 23 sec, 11 min 1 sec ± 54 sec and 9 min 0 sec ± 1 min 37 sec, for SG1, IG and SG2, respectively. IVP recoveries were quasi-linear from 2 to 15 min, with rates of recovery of 0.63 ± 0.07, 0.38 ± 0.06 and 0.72 ± 0.15 mmHg/min for SG1, IG and SG2 respectively. ANOVA showed a significant interaction for the time changes of IVP after CSF removal between treatment groups , with a statistically significant difference between SG1 and IG, p < 0.05. As shown in figure p = 0.41), but differed significantly from TOR in IG, 71.33 ± 16.59 μl/min (p = 0.016).When the cannula was opened to connect the pressure transducer, slight CSF leaking <0.2 ml) was occasionally, but not systematically observed, indicating IVP values very close to or equal to atmospheric pressure when the head of the animal was in the sphinx position. After removing the CSF, IVP in all animals first decreased within seconds and thereafter started to increase rapidly. When it reached the initial value, the IVP did not stabilize but went on increasing up to about 2 mmHg during the recording session. The kinetics for recovery of IVP differed between groups Figure . The ext ml was oOur results demonstrate for the first time that IVP recovery after CSF removal varies with the photoperiodic status of the ewe. These results are in accordance with established data from other species, including human, suggesting a change in TOR in non-pathological situations, namely aging ,19,24,25TOR values in sheep (112 μl/min: , 82 μl/mThe previous studies were performed with un-anesthetized sheep or goats -30. We aDaily changes in the TOR of human CSF have been described, with increased TOR at night. This nocturnal increase in CSF production is inhibited by a β1-receptor antagonist, a type of receptor stimulating melatonin secretion in the pineal gland . Our expUsing a light-dark cycle which reproduces the photoperiodic modulation of reproductive activity, this study has shown that the turnover rate of the CSF in the ewe is higher during periods of short day length and reduced in periods of long day length. This phenomenon could account for the higher concentration of estradiol in the CSF, previously observed during long days when the estrous cycle is blocked by increased negative feedback of this steroid in the hypothalamus. Our study highlights the active role of the choroid plexus and CSF in neuroendocrine regulation.IG: inhibitory period of gonadotropin activity; IVP: intra-ventricular pressure; PKC: Protein kinase C; SG1 and SG2: first and second stimulatory periods of gonadotropin activity; TOR: CSF turnover rate.The authors declare that they have no competing interests.JCT conceived and designed the project and drafted the manuscript. JCT, DL and SB carried out the experiment and collected data. BM was involved with interpretation and mathematical processing of data and drafted the manuscript. JCT and BM revised the manuscript. All the authors read and approved the manuscript.


Identifying finger and wrist flexion based actions using a single channel surface electromyogram (sEMG) can lead to a number of applications such as sEMG based controllers for near elbow amputees, human computer interface (HCI) devices for elderly and for defence personnel. These are currently infeasible because classification of sEMG is unreliable when the level of muscle contraction is low and there are multiple active muscles. The presence of noise and cross-talk from closely located and simultaneously active muscles is exaggerated when muscles are weakly active such as during sustained wrist and finger flexion. This paper reports the use of fractal properties of sEMG to reliably identify individual wrist and finger flexion, overcoming the earlier shortcomings.p value, indicative of the significance of the relationships between each of these parameters with the wrist and finger flexions. Classification accuracy was also computed using the trained artificial neural network (ANN) classifier to decode the desired subtle movements.SEMG signal was recorded when the participant maintained pre-specified wrist and finger flexion movements for a period of time. Various established sEMG signal parameters such as root mean square (RMS), Mean absolute value (MAV), Variance (VAR) and Waveform length (WL) and the proposed fractal features: fractal dimension (FD) and maximum fractal length (MFL) were computed. Multi-variant analysis of variance (MANOVA) was conducted to determine the p value for the proposed feature set consisting of FD and MFL of single channel sEMG was 0.0001 while that of various combinations of the five established features ranged between 0.009 - 0.0172. From the accuracy of classification by the ANN, the average accuracy in identifying the wrist and finger flexions using the proposed feature set of single channel sEMG was 90%, while the average accuracy when using a combination of other features ranged between 58% and 73%.The results indicate that the The results show that the MFL and FD of a single channel sEMG recorded from the forearm can be used to accurately identify a set of finger and wrist flexions even when the muscle activity is very weak. A comparison with other features demonstrates that this feature set offers a dramatic improvement in the accuracy of identification of the wrist and finger movements. It is proposed that such a system could be used to control a prosthetic hand or for a human computer interface. Controlling devices such as prosthetic or robotic hand requires automated identification of the command. To provide the user with a natural feeling and benefit of the dexterity of the hand, the (intended) finger and hand movements of the user have to be identified. One method to determine movement and posture is by estimating the strength of contraction of associated muscles based on the electrical activity of the muscles. The benefit of this over other sensing techniques is that it is suitable for people who have suffered amputation of their hands and the control can be based on the user's intention. A recent survey on myoelectric prosthesis by Oskoei and Hu  has repoSurface electromyography (sEMG) ,3 is a nThe fundamental principle underlying the identification of actions and gestures using sEMG is by measuring the strength of contraction of the associated muscles. This may be done in time or frequency domain or a combination. Various analogous measures such as root mean square (RMS), integral of the signal, auto-regression, signal length and wavelet coefficients have been used to identify the movement and/or posture ,9-13. ThTo identify actions that are a result of multiple simultaneously active muscles require an estimation of the relative strength of contraction of the different muscles. Researchers have used artificial intelligence and genetic algorithms ,10,13,14Recent work  has compOne general limitation of the established and widely reported features of sEMG is that these are unreliable at low levels of contraction due to low signal-to-noise ratio . At low When the muscle activity is low, the density of motor unit action potentials (MUAP) can be used to determine the strength of muscle contraction ,20. The Another proposed measure of strength of muscle activity is the fractal dimension (FD) of sEMG ,24-28. FBased on the fundamental model of sEMG , MUAPs oA new feature, the maximum fractal length (MFL) of the signal, as a measure of the muscle activation has been proposed. This is the intercept of the fractal relationship with the length of the curve at lower scale Figure . This isAuthors have identified the maximum fractal length (MFL) as a measure of the strength of contraction ,33. WhilThe aim of this work was to develop a sEMG based technique that can accurately identify the basic hand and wrist gestures which may be used by people in special circumstances to communicate or give commands. This requires determining suitable features of sEMG that can be used to identify the actions even when the muscle activity is low. It is also important to differentiate between activities of different muscles.SEMG is the electrical recording resulting from the interferential summation of MUAPs at the surface. All MUAPs are similar at the source ,20-23,34σ = 2.05) years; mean weight 70.6 (σ = 6.56) kg; and mean height was 170.6 (σ = 7.42) cm. The participants' inclusion criterion was; (i) healthy with no history of myo or neuro-pathology, and (ii) no evident abnormal motion restriction. All participants in this study were right-handed. Experiments were conducted after receiving approval from University Ethics Committee for Human Experiments. Each participant was given an oral and written summary of the experimental protocol and the purpose of the study and then was required to sign a consent form prior to participation.Five subjects  volunteered to participate in this study. Mean age was 26.6 Channel 3: Flexor Carpi Ulnaris (FCU)Channel 4: Flexor digitorum superficialis (FDS)th order butterworth band pass filter from 20 to 450 Hz. The sampling rate of the system is 1024 samples/second for each channel. Prior to placing the electrodes, the skin of the participant was prepared by shaving (if required) and exfoliation to remove dead skin. Skin was cleaned with 70% v/v alcohol swab to remove any oil or dust from the skin surface. The skin impedance during the recording was measured and in all cases was less than 6 KΩ. Standard electrode placement procedures were followed [DELSYS , a proprietary sEMG acquisition system, was used for recording sEMG. This system has bipolar differential electrodes units with each unit having two parallel bars with fixed inter-electrode distance of 10 mm Figure  and a prfollowed ,37.M1 - All fingers and wrist flexion, M2 - Index and Middle finger flexion, M3 - Wrist flexion towards little finger, M4 - Little and ring finger flexion. The flexions were performed without any resistance and as were convenient to and easily reproducible by the participant.At the start of the experiment, the participants were given a demonstration towards maintaining finger and wrist flexion. Prior to the recording, the participants were encouraged to familiarize themselves with the experimental protocol and with the equipment. SEMG was recorded from the four electrodes when the participant maintained the specific wrist and finger flexions: The recordings from four channels were used to compare with other similar studies, however for the single channel analysis, only channel 2, located closest to the elbow • Mean absolute value (MAV),• Variance (VAR)• Waveform length (WL).N is the number of samples in a segment and xi is the signalwhere The next step was the computation of the proposed set of features; Fractal Dimension (FD) and Maximum Fractal Length (MFL) of sEMG. This is described below:FD was calculated using Higuchi algorithm ,39 for nx(n) = X(1), X(2), X(3), ....., X(N) as follows:The first step for computing the MFL requires the computation of the length of the curve,  denotes the Gauss' notation and both k and m are integers. m = initial time; k= time interval; i = 1 to where k, ⟨L(k)⟩ is defined as the average value over k sets of Lm (k). If ⟨L(k)∝kD-⟩, then the curve is fractal with the dimension D. Maximum fractal length (MFL) was determined from the plot  at the smallest scale. The slope of the line gives the fractal dimension (FD). The computation MFL and FD is shown in Figure The term t Figure  as the aA sliding window of 1024 samples corresponding to one second was used for computing the features of sEMG recorded from each flexion. This corresponds approximately to one hertz, which is faster than the normal speed of human finger actions. While this paper reports 1024 sample window, experiments were also conducted where the window size was 512 samples and the outcome was identical. SEMG corresponding to the isometric contraction was analyzed and sEMG corresponding to the action was removed by removing the first and the last one-second of the contraction.After removing the first and the last second data of each flexion, 5 seconds long recordings corresponding to each flexion were selected. With a window size of 1024 samples, this provided 5 segments for each flexion. The data of all 5 segments and for all the 12 repetitions for each flexion  was considered for statistical analysis. The data of the 5 subjects were analyzed together. Hence the total sample size of (12*5*5*4 =) 1200 was used for the statistical analysis. It was observed that inter-experimental variations were very significant for RMS compared with other features. Hence the RMS was normalized by taking a ratio of all the channels with respect to channel 1. This resulted in channel 1 becoming redundant and effectively reduced the four channels to three. While computing MFL, the threshold, T was considered for data from every subject individually. The threshold, T did not vary much with the subjects as it was computed from sEMG when the participant performed no hand movement (when the muscle is at rest).Multi-variant analysis of variance (MANOVA) was conducted to determine the significance of the relationships and to obtain the two out of four most representative channels for each of the features. MANOVA identifies a linear combination of the variables, called the canonical variable, that has the highest multiple correlations with the groups, and these canonical variables provide the order of level of correlation. Statistical analysis was performed to determine the significance of separation of the different features to identify the different actions. A combination of various variables and multiple channels were taken:• Two channels (selected using MANOVA) for each of the 6 features.• Four channels for each of the 6 features.• Single channel with FD paired with each of the other 5 features.The next step was the classification of each of the feature set listed above using Artificial Neural Network (ANN). The ANN with two hidden layers and 20 neurons for each hidden layer was simulated. The Sigmoid threshold function with a learning rate of 0.05 was used to reduce the likelihood of local minima. The ANN analysis was repeated for each feature sets, with input to the ANN being the sEMG features and the target being the associated actions. ANN was trained using 50% of the data and tested with the other 50% which has not been used for training. Ten cross validation was performed by changing the training and testing data. Classification accuracy was computed as the average accuracy based on the results from cross validation testing. The data of the proposed set of features, FD and MFL of a single channel  was plotted for visualization to assess the separation of the different classes. This provides a qualitative analysis of the data.Figure p inversely indicates the significance of separation of the classes.From table p for FD and MFL combination is 0.0001, the value of p for other combinations ranges between 0.02 and 0.199. The F value results also indicate that the most suitable feature set is FD and MFL of a single channel. This demonstrates that FD and MFL of a single channel are suitable for identifying the four different hand gestures.The results of statistical analysis of single channel sEMG for FD paired with each of the other features are tabulated in table The average classification accuracy of multiple (two and four) channels of the six features for identification the associated actions are shown in table The accuracy using a combination of FD and MFL obtained from single channel was even better than when using all four channels, where the accuracy of identification of the actions ranged between 70% and 90%. This indicates that FD and MFL combination of single channel sEMG was significantly more accurate in identifying finger and wrist flexions compared with any other feature that was tested.SEMG is a measure of the muscle activity that has been used by many researchers to identify control commands for controlling prosthetic hands and for human machine interface. One shortcoming in the use of sEMG for identifying control commands is the unreliability when the muscle activity is weak and there are multiple active muscles. This is because of the presence of background noise, other artefacts and cross-talk. This study has overcome the above limitations and developed a technique that can reliably identify control commands even when the strength of sEMG is weak, and there are multiple active muscles such as during finger and wrist flexions.This study has demonstrated that the combined use of FD and MFL of single channel sEMG recorded from the forearm is the most accurate feature set to identify finger and wrist flexion movements when compared with the established features reported in literature. While the features set (FD and MFL) accurately identified finger and wrist flexion movements with average accuracy of 90.7% by comparison the accuracy of identification using other features of the signal reported in literature  ranged fBased on the experimental outcomes of this study, it is concluded that a combined use of FD and MFL of single channel sEMG is suitable for reliably identifying various finger and wrist flexion actions without being sensitive to inter-experimental variations and does not require strict electrode positioning. While comparable accuracies are obtainable using number of channels, a single channel is desirable because of lower complexity and it being suitable for amputees who may not have a large area of the forearm available for multi-channel sEMG recording. Such a system can be used for controlling the individual fingers of a prosthetic hand for amputees. The system may be suitable for other applications such as human computer interface for the elderly and for people in special circumstances such as defence.The authors declare that they have no competing interests.SPA has conducted the experiments, developed the signal processing technique and performed the data analysis. He has also written the first draft of the manuscript. DKK has designed the experiment, and discussed and developed the underlying concepts for the technique. He has also done the proof-reading, and finalized the manuscript. All authors have read and approved the manuscript.


Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D