It has been estimated that paediatric meningitis without elevated CSF white cell count (pleocytosis) accounts for 0.5–12% of all cases of bacterial meningitis. CSF protein and glucose measurements are therefore essential in management but may be neglected in clinical practice. In order to improve recognition of bacterial meningitis in neonates and to enable adequate management and audit, we investigated whether a systemic inflammatory response in the absence of meningitis is associated with elevated CSF protein and reduced CSF glucose levels. A further aim was to determine whether abnormal levels of these parameters were associated with increased incidence of neurological damage.As part of an audit into management of abnormal CSF findings in neonates, we conducted a retrospective analysis of neonates without meningitis as evident from normal CSF white blood cell counts and negative CSF culture. We compared data from neonates with fever (temperature > 38.0 °C) and/or elevated C-reactive protein (CRP) levels (> 5 mg/l) (possible sepsis) with data from neonates without fever or CRP elevation.We analysed results from a total of 244 neonates. CSF protein levels were 0.89 g/l (SD 0.37) in neonates without fever or elevated CRP (n = 26) and not significantly different from neonates with possible sepsis (n = 218) with 0.92 g/l (SD 0.40). CSF glucose levels in infants with possible sepsis were 2.71 (SD 0.83) mmol/l and not significantly different from infants without sepsis with 2.55 mmol/l (SD 0.34).CSF protein and glucose levels are not affected by a systemic inflammatory response syndrome if there is no meningitis.The online version of this article (10.1186/s12987-018-0095-4) contains supplementary material, which is available to authorized users. Meningitis is a substantial cause of morbidity and mortality in neonates. Abnormal cerebrospinal fluid (CSF) parameters are often used to predict meningitis and determine length and type of antibiotic therapy in neonates with a positive blood culture and negative CSF culture . CurrentHigh CSF protein and low CSF glucose have been associated with increased risk of sensorineural hearing loss in bacterial meningitis , 6. BreaThe aims of his study were to establish baseline data to enable an audit into appropriate management of abnormal CSF biochemistry measurements consistent with meningitis. The knowledge whether sepsis in itself is associated with abnormal CSF parameters without the presence of meningitis would enable those, who had meningitis and in whom appropriate action should be taken , to be distinguished. Aims were also to establish whether CSF protein and glucose in sepsis without elevated CSF cell count and with negative CSF culture and/or PCR is different from CSF in patients without a systemic inflammatory response and different from the normal values of CSF biochemistry as reported in the literature (reference range) for neonates and to establish whether elevated CSF protein and reduced CSF glucose levels were associated with neurological damage. Some of the data presented here has previously been presented as a poster at the 9th Excellence in Pediatrics conference in Vienna in 2017 .All newborn infants (< 28 days of age) who had cerebrospinal fluid analysis recorded in the microbiology database without evidence of meningitis (CSF cell count < 20) over a 2-year period at Luton & Dunstable University Hospital, were included in the study. Patients were excluded from further analysis if bacteria, fungi or viruses were detected in the CSF by culture or PCR or if CSF biochemistry had not been analysed. Patients were divided into those who had a systemic inflammatory response at the time of admission: body temperature > 38.0 °C and/or C-reactive protein > 5 mg/dl  or at any point in time after admission to the neonatal unit or paediatric wards, and those who did not. Organisms isolated in blood or urine culture were recorded. CSF parameters measured in the department for Clinical Biochemistry at the Luton & Dunstable University Hospital NHS Foundation Trust included total protein measured by a turbidimetric method : the samCategorical data  were analysed using Chi square with Yates correction or Fisher’s exact test as appropriate. Continuous data with a parametric distribution  were analysed using Student’s t-test for independent samples and continuous data with features of a non-parametric distribution (CSF white and red cell count) were analysed using Mann–Whitney testing. P-values were 2-tailed and for equal variances not assumed. The statistical software packages used were IBM SPSS version 20.0 and Epi-Info version 7.0 . A p-value of < 0.05 was taken as indicating a statistically significant difference.A total of 244 newborn infants on the neonatal unit or the paediatric wards, who had lumbar punctures with no evidence of meningitis, were included in the study. Evidence of SIR was detected in 218 in that they had pyrexia and/or an elevated CRP at the time of admission. There was no evidence of SIR in 26 patients. A total of 16 patients (6.5%) had pathogens isolated from blood cultures and a total of 10 (4.1%) from urine cultures with no difference between groups with and without SIR. Clinical characteristics of included patients are listed in Table CSF parameters were compared in patients with and without a SIR .One could argue that the group without a systemic inflammatory response was too small to detect a statistically-significant difference. Because our study is to the best of our knowledge the first investigating this topic in this way a sample size calculation was not possible and the number of patients included in each group was the number of eligible patients identified during the study period to avoid any selection bias. Based on our data a sample size calculation based on equivalence showed that to exclude a statistically significant difference at the 5% significance level between groups one would have to include 550 patients (275 in each group) to be 80% certain that the limits of a two-sided 90% confidence interval will exclude a difference in means of CSF protein levels of more than 0.1 g/l (Even without an inflammatory response causing elevated CSF protein levels, systemic sepsis could cause white matter damage in the brain without meningitis. This can be attributed to lipopolysaccharides crossing the BBB and able to cause white matter injury through activation of microglial toll like receptor 4 . The datIn newborn infants, CSF protein and glucose levels are not affected by a systemic inflammatory response syndrome if there is no concurrent meningitis.Additional file 1. Raw data for the newborns included in the report.


S. mutans, the major causative organism of dental caries. However, physiological activity and cariogenicity of S. mutans affected by Alr remains unknown. The current study examined the biofilm biomass, biofilm structure, extracellular polysaccharide (EPS) synthesis, glucosyltransferase (gtf) gene expression, acid production and acid tolerance in the alr-mutant strain. We found that biofilm formation, biofilm structure, and EPS synthesis was in a D-Ala dose-dependent manner. Biofilm structure was loose in alr-mutant group and the ratio of EPS/bacteria was also elevated. Additionally, the expression levels of multiple gtfs were up-regulated, and acid tolerance was decreased. We also established in vivo models of dental caries and found that the incidence and severity of the caries were decreased in the alr-mutant group in comparison to the parental S. mutans group. Our in vivo and in vitro experiments demonstrate that Alr is essential for the cariogenicity of S. mutans and that Alr might be a potential target for the prevention and treatment of caries.The enzyme alanine racemase (Alr) has been a new target for the development of antibacterial drugs based on the involvement of D-Ala in bacterial cell wall biosynthesis. Our previous study noted that Alr is essential for the growth and interspecies competitiveness of  This function is critical for the growth of bacteria due to their need for D-alanine, an essential component in the biosynthesis of cell wall peptidoglycan in both gram-positive and gram-negative bacteria2. Two kinds of Alr have been identified in bacteria: the alr-encoded racemase, which is constitutive and used for D-Ala biosynthesis, and the dadX-encoded racemase, which is inducible and used for the catabolism of D-Ala4. Lack of expression of the alr gene is lethal when there is no addition of exogenous D-Ala5. Many studies have focused on alanine racemase, which aim to develop antibacterial drugs for multiple bacterial species, such as Pseudomonas aeruginosa6, Acinetobacter baumannii7, Staphylococcus aureus8, Bacillus anthracis9, Lactobacillus plantarum10, and Escherichia coli11.Alanine racemase (Alr) is a bacterial enzyme that catalyses the conversion of L-alanine to D-alanine (D-Ala)12. It is the main cause of tooth pain, tooth loss and can affect overall health by making food intake more difficult13. Therefore, focusing on the factors affecting caries progression and finding efficient strategies for the management of caries is of great significance. Streptococcus mutans (S. mutans) is the dominant bacterium in dental plaque and has long been implicated as the major causative organism of dental caries14. S. mutans is able to ferment carbohydrates and produce acids that reduce the local pH15. In addition, S. mutans embedded in the dental plaque is resistant to host defences because the extracellular matrix inside the biofilms provides protection from harmful factors16. Exploring factors that influence the cariogenic virulence of S. mutans might aid in the discovery of a more effective target for an anti-caries drug. Considering the essential role of D-alanine for the bacterial cell wall, the alr gene is a potential antibacterial target for S. mutans.Dental caries is one of the most common chronic diseases worldwide and is considered a major health concern for professionalsalr-mutant strain was constructed, and the role of alr in cell growth and cell wall integrity was explored. Our data showed that a minimal concentration of D-Ala (150 µg/ml) was required for the optimal growth of the alr-mutant strain. The depletion of D-Ala in the growth medium led to cell wall perforation and cell lysis in the alr-mutant strain17. Another previous study also demonstrated the important role of D-Ala metabolism for the growth of S. mutans18. Given the importance of alr to the growth of S. mutans, the aim of this study was to explore the role of Alr on the physiological activity, including biofilm formation, extracellular polysaccharide (EPS) synthesis, glucosyltransferases (gtfs) gene expression, acids production and tolerance, and cariogenicity of S. mutans.In our previous study, an S. mutans affected by exogenous D-Ala, and the result showed that exogenous D-Ala did not obviously affect the growth of S. mutans  in the 150 µg/ml group. Nevertheless, the mRNA levels of gtfB, gtfC, and gtfD in the alr-mutant cells of the 100-µg/ml group were significantly up-regulated by 41.7-, 117.9-, and 16.2-fold, respectively , slightly dentinal (Ds), moderate dentinal (Dm), and extensive dentinal (Dx). Ds lesions represent the involvement of 1/4 of the dentin between the enamel and the pulp chamber. Dm lesions represent the involvement of 1/4 ~ 3/4 of the dentin region. Dx lesions represent caries progression beyond 3/4 of the dentin region.S. mutans group, the range of caries lesions was larger and deeper in depth as most of the sulcal caries had progressed to the slight dentine (Ds); a few moderate dentinal lesions (Dm) were also observed. The 150 µg/ml D-Ala alr-mutant group, however, had fewer Ds lesions.Photographs of the caries lesions on the teeth of the rats were obtained using a stereo microscope Fig. . No cariS. mutans group (P < 0.05) than the alr-mutant group. In addition, no Dx lesions occurred in either group. Figure alr-mutant strain group.The statistical analysis is shown in Fig. S. mutans17. A minimal concentration of D-Ala (150 µg/ml) was required for the optimal growth of the alr-mutant strain. D-Ala starvation in the growth medium led to cell wall perforation and cell lysis in the alr-mutant strain. Although Alr has been well demonstrated to be closely associated with the survival and cell wall in S. mutans, its cariogenicity and physiological importance in biofilm is yet to be explored. In the current study, we examined the effect of Alr on S. mutans biofilm formation, biofilm structure, EPS synthesis, the expression of genes involved in sugar production, acid production, and acid tolerance, all of which are responsible for caries development. We also observed that Alr is closely associated with caries development. Similar D-Ala dose-dependent manner was observed in alr-mutant biofilm formation, biomass and EPS synthesis, which suggested alr is important for the mutant biofilm structure.To explore whether Alr can represent an effective drug target to modulate the cariogenicity of oral biofilm and benefit the management of dental caries, we firstly determined that Alr is essential for the growth and interspecies competitiveness of alr-mutant was observed at a concentration of 100 µg/ml of D-Ala. The biofilms became denser as the D-Ala concentration increased. The observed biofilm change may also be attributed to the mutant death and cell lysis we have reported previously. A pronounced short chain length of alr-mutant compared to the wild type under the scanning electron microscopy also supported the possibility.The loose biofilm structure of the alr-mutant produced more EPS compared to the parental S. mutans strain with fewer cells in most layers of the biofilm. The EPS/bacteria ratio of alr-mutant was elevated, and the elevated ratio was consistent with the qPCR results of the expression levels of the gtfB, gtfC, and gtfD genes, which might be explained by the regulatory systems inside S. mutans such as two-component signal transduction systems (TCSTS). Some TCSTS e.g. VicRK and CovR are responsible for sucrose-dependent adherence and biofilm formation20. The expression of gtfB/C/D is positively regulated by VicR and/or repressed by CovR20. The cell lysis of the alr-mutant was sensed by TCSTS, which subsequently turned on the up-regulation pathway to EPS synthesis for survival as high EPS levels lead to a thick biofilm, causing resistance to immune factors or harmful metabolites16. There was no adequate D-Ala in the 100 μg/ml group, and alr-mutant exhibited strongest EPS-producing ability, indicating stronger survivability in the worst environment. Therefore gtfs expression was highest at 100 μg/ml. However, EPS synthesis was highest in the mutant treated with 200 μg/ml D-Ala. We speculate that the high concentration of D-Ala served as an additional carbon source that the bacteria utilized to produce polysaccharide. As previously mentioned, S. mutans is able to metabolize carbohydrates that leads to acid accumulation and a subsequent pH declination in the dental biofilm21. EPS provide a limiting barrier for acid diffusion by generating acidic microenvironments within the dental plaque. This dual-effect leads to a continuous pH decline to the critical pH, below which tooth hard-tissue demineralization begins and dental caries occurs23. Although the alr-mutant produced high EPS levels and limited acid diffusion, the distinctly weak acid tolerance  and was routinely cultured in brain heart infusion broth  at 37 °C aerobically (95% air/5% CO2). The alr-mutant strain17 was cultured in BHI plus 100, 150, or 200 µg/ml of D-Ala (Sigma). The medium was supplemented with 1% sucrose  when needed. 1 × 107 CFU/ml of S. mutans or the alr-mutant strain was used for both in vitro and in vivo studies.24.After culturing in 96-well microtiter plates for 24 h, the biofilms were gently washed three times with phosphate buffer saline (PBS), fixed with 95% methanol for 30 min, washed three times with PBS, stained with 0.5% crystal violet for 30 min and washed three times with PBS. The crystal violet was extracted with 200 µl of 100% ethanol. The extract was evaluated at 600 nm using a spectrophotometer25. For planktonic cells, cells were pelleted by centrifugation at 4000 rpm for 5 min, washed twice with PBS and re-suspended. An aliquot (5.0 µl) of bacterial suspension was deposited on a sterile glass slide and air-dried at 37 °C. Samples were then fixed, dehydrated, immersed and dried26. After coating with gold-palladium, samples were analysed in a scanning electron microscope  at 5000×, 10000×, 20000× magnification.After culturing in 24-well microtiter plates for 24 h, the biofilms (grown on glass slides) were gently washed three times with PBS, fixed with glutaraldehyde (2.5%) overnight at 4 °C, washed three times with PBS, dehydrated using a series of ethanol rinses , immersed for 10 min in 100% ethanol and dried in a desiccator28.Biofilms cultured on glass slides were grown in BHIS medium and supplemented with 1 µM Alexa 647 red fluorescent dye  and protected from light. Alexa 647 are labelled dextran conjugates and were incorporated into EPS. After incubation for 24 h, the biofilms were washed with PBS three times and incubated with 1 µM SYTO 9 green fluorescent dye  at room temperature for 20 min. SYTO 9 bound to bacterial nucleic acid and represented bacteria in biofilms. Then the biofilms were washed with PBS three times and dried. ProLong Gold Antifade Reagent was dropped on the biofilms, and images were obtained using a confocal laser scanning microscope . The Alexa Fluor 488 and 647 fluorescent channels were selected to detect green and red fluorescence respectively. The whole procedure was performed away from light31. Briefly, the biofilms were harvested after incubation in BHIS medium for 24 h, washed three times with PBS, and stabilized using the RNAprotect Bacteria Reagent . Then, the biofilms were suspended in 180 µl of lysis buffer  with 10 µl of mutanolysin  and 15 µl of proteinase K (Qiagen), incubated with agitation at 37 °C for 120 min, and sonicated for 5 cycles . The biofilm RNA was extracted and purified using the RNeasy Mini kit (Qiagen). The RNA concentration was measured using a NanoDrop 2000 . A high-capacity cDNA reverse transcription kit with random primers  was used to synthesize cDNA from 10 µg RNA. PCR reactions comprised two micrograms of cDNA, S. mutans primers (0.375 µM) and the Fast SYBR green master mix (Applied Biosystems) and were run using an ABI Prism 7000 sequence detection system for quantitative PCR. The primers were 16 S, gtfB, gtfC, and gtfD31. The values of 2−∆∆Ct were analysed to calculate the gene expression fold changes21.The qRT-PCR protocol has been described in detail in our previous studiesS. mutans glycolysis was measured using the method described by Xu et al.32. The bacteria were harvested at the mid-logarithmic growth phase, washed with PBS and resuspended (OD600 nm = 0.5) in 0.5 mM potassium phosphate buffer consisting of 1.25 mM MgCl2 and 37.5 mM KCl (pH = 6.5). Glucose was added to the bacterial suspension, and the final concentration was adjusted to 1% (wt/vol). The glycolytic activity of S. mutans and the alr-mutant strains resulted in a decrease in pH, and the pH levels were monitored at 15-min intervals over a period of 120 min.The role of Alr in S. mutans acid tolerance was also measured using the method described by Xu et al.32. The bacteria were harvested at the mid-logarithmic growth phase, collected by centrifugation and resuspended (OD600 nm = 0.2) in TYEG medium  buffered with 40 mM phosphate-citrate buffer solution (pH = 5.0) and incubated at 37 °C aerobically for 2 h. After incubation at pH 5.0, samples were removed for viable counts.The role of Alr in in vivo study was approved by the ethics committee of West China School of Stomatology, Sichuan University (WCCSIRB-D-2014-072), and all experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The S. mutans (n = 7), alr-mutant , and blank (n = 2). Upon arrival, animals were determined to be free of any indigenous oral microorganism by feeding the rats antibiotics, namely, ampicillin, chloramphenicol and carbenicillin (1.0 g/kg), for 3 consecutive days33. After a washout period of 3 days, in which the residual antibiotics in the oral cavity were removed by feeding rats antibiotic-free distilled water, rats were challenged with 1.0 × 107 CFU/ml S. mutans or alr-mutant strain suspensions for 3 consecutive days . All rats were fed the National Institutes of Health cariogenic diet 2000 and 5% sucrose water34. For the alr-mutant group, D-Ala was added to the water (150 µg/ml). The experiment lasted for 27 days, after which the rats were sacrificed. The jaws of the rats were aseptically dissected, and the caries status was scored using the Keyes method35.Sixteen specific pathogen-free (SPF) male Wistar rats, aged 21 days, were randomly divided into three groups: in vivo study. The data were statistically analysed using the SPSS 21.0 software. P < 0.05 was considered statistically significant.Each experiment was independently repeated at least three times. One-way analysis of variance (ANOVA) was used to analyse the crystal violet staining, acid tolerance, and qRT-PCR. Independent t-tests were used for the The datasets generated and analysed during the current study are available from the corresponding authors on reasonable requests.Supplementary information


Rabbit TT diameters were more variable than those of mouse (p < 0.01) and the smallest TT detected was 41 nm in mouse and the largest 695 nm in rabbit. These estimates are consistent with TT diameters derived from the more limited sampling of high-pressure frozen samples by electron tomography . Other measures of TT abundance and geometry  were also derived. On the physiological time scale of E-C coupling (milliseconds), the average TT electrical space constant is ~ 175 μm in rabbit and ~ 120 μm in mouse and is ~ 50% of the steady-state space constant. This is sufficient to ensure reasonable electrical uniformity across normal cells. The image processing strategy and shape-based 3D approach to feature quantification is also generally applicable to other problems in quantification of sub-cellular anatomy.Transverse-axial tubules (TTs) are key structures involved in cardiac excitation-contraction coupling and can become deranged in disease. Although optical measurement of TTs is frequently employed to assess TT abundance and regularity, TT dimensions are generally below the diffraction limit of optical microscopy so determination of tubule size is problematic. TT diameter was measured by labeling both local surface membrane area and volume with fluorescent probes , correcting image asymmetry by image processing and using the relationship between surface area and volume for a geometric primitive. This method shows that TTs have a mean (± SEM) diameter of 356 ± 18 nm in rabbit and 169 ± 15 nm in mouse ( •Living cardiomyocytes were dual-labeled with fluorescent surface and volume probes.•A novel 3D image processing strategy enabled calculation of t-tubule diameter.•The method shows rabbit and mouse t-tubules have quite different morphologies.•Mean diameters of rabbit and mouse t-tubules were 360 and 170 nm, respectively.•Estimated electrical space constants are sufficient to ensure electrical uniformity. The ventricles were then minced and filtered to isolate single cells. The cells were centrifuged and re-suspended in storage solution (see below).All procedures were performed in accordance with the Animals (Scientific Procedures) Act (UK). Ventricular myocytes were enzymatically isolated from the hearts of male mice  or rabbits , as described previously 2.22, 0.4 NaH2PO4, 10 d-glucose, 4.2 HEPES, 20 taurine and 10 creatine, pH = 7.4. For rabbit cells, the solution was similar, except that it contained 4.5 KCl, 3.5 MgCl2 and 5 HEPES. The cell storage solution was a low Ca Kraftbrühe (KB) medium to relax cells and prevent possible TT compression by cell contraction, which contained: 100 L-glutamic acid, 30 KCl, 10 HEPES, 1 EGTA, 5 Na pyruvate, 20 taurine, 20 glucose, 5 MgCl2, 5 succinic acid, 5 creatine, 2 Na2ATP, 5 ß-OH butyric acid. All experiments were performed at room temperature in KB medium.The standard solution used for mouse cell isolations contained (in mmol/L): 130 NaCl, 5.4 KCl, 1.4 MgCl2.3Cell membranes were labeled with 5 μM FM4-64  for 5 min, washed, then bathed in 400 μM calcein as an extracellular marker (Sigma-Aldrich). This dye selection allowed good separation of fluorescence signals, although other dye pairs could be used with suitable correction of cross-talk between channels if necessary. Cells were imaged using a LSM 880  with an Airyscan detector set to super-resolution mode, and water-immersion objective with 40 × magnification and 1.2 numerical aperture. Dyes were excited with the 488 nm Argon laser line, and fluorescence recorded at 495–550  and > 605 nm (FM4-64). Volume images were recorded at 12-bit resolution with voxel size set to 60 nm in the focal plane (x-y) and ~ 180 nm along the optical axis (z) to ensure oversampling.2.4A novel method was developed to assess TT width in living myocytes. The algorithm was written in MATLAB R2015a , and ImageJ  was used to aid the analysis. (Codes can be obtained by contacting the authors.)V:SA. For any given shape V:SA increases with size, so V:SA can be used to compare object sizes with no explicit assumptions beyond shape similarity. V:SA can be calibrated to physical dimensions using a geometric model, as described below.The improved method relies on microscopic measurement of the local TT luminal volume  and membrane area  to calculate the volume to surface area ratio, V:SA. Since V:SA increases linearly with t-tubule diameter (DTT), it can be used to monitor changes in average DTT. As pointed out previously, use of confocal fluorescence intensity signals assumes a spatially invariant and symmetric probe point spread function (PSF) V:SA method with a flow diagram showing the image processing steps. Briefly, calculation of the TT V:SA involved segmentation of cell interior and surface, background subtraction, conversion of the normal microscope asymmetric PSF to a spherical PSF, and data normalization to remove experimental variations in dye concentration and to permit calibration to size units.The ratio of the calcein to FM4-64 signal intensities is a measure of A TT skeleton was produced by applying an Otsu threshold to produce a binary image mask, which was then skeletonized by thinning  signal was normalized to that recorded from in the perfusion bath, and the FM4-64 (surface membrane) signal to that at the cell surface sarcolemma (SS). 2.52 at 2000 bar. Frozen samples were freeze-substituted in 1% osmium tetroxide and 0.1% uranyl acetate (in acetone), and subsequently embedded in Epon resin. All sections were stained with uranyl acetate and lead citrate; sections for tomography were additionally coated with 15 nm colloidal gold particles. Sections (80 nm) for EM were analyzed using 100 kV Tecnai 12 transmission electron microscope  fitted with a TVIPS F214 digital camera. Sections (380 nm) for ET were analyzed using 200 kV Tecnai T20 transmission electron microscope fitted with an Eagle 4k × 4k camera (FEI Company). For ET, images were acquired in a double tilt series (with 90° sample rotation between series), between ± 70° using the Saxton tilt increment scheme TT shape and dimensions were also measured with 3D electron tomography (ET) and electron microscopy (EM), for comparison with our optical method. Isolated cells were re-suspended in the standard physiological solution to which 10% BSA was added, left to sediment into a pellet, and then high-pressure frozen 2.6The FM4-64 signal was also used to quantify some features of TT organization. TT length was obtained from the 3D skeleton (see above), while tubule orientation was calculated from the Eigenvector of the image 2.7p < 0.05, and ** is used to indicate p < 0.001.Mean data is presented with one standard error of the mean (SEM), unless otherwise stated. Statistical testing was performed with Prism v6.01 , using the Mann-Whitney Test, or Spearman rank correlation, unless otherwise stated. Statistical significance * was defined by 33.1V:SA method to determine TT width in living myocytes. p < 0.001). Closer inspection reveals that some TT invaginate from the SS at grooves (arrowed), confirming that FM4-64 labels the TT membrane, while calcein labels the TT lumen (which is continuous with the extracellular space). Punctate labeling, closer to the cell center corresponds to TTs running orthogonal to the image plane, as seen in 3D data sets . The group mean V:SA  was 0.139 ± 0.007 (SEM).Rabbit ventricular myocytes, which are known to have wider TTs than mice, were used to assess the utility and accuracy of the ata sets . Fig. 1Bata sets . The divV:SA corresponds to a TT diameter of 0.356 ± 0.018 μm in rabbit ventricular myocytes  and shows that the low molecular weight volume dye was not physically excluded from the TT lumen.Using a cylindrical model of TT shape see , this V:myocytes . The val3.2V:SA method to analyze TTs in mouse ventricular myocytes is shown in V:SA . The group mean V:SA  was 0.066 ± 0.006 (SEM), indicating that DTT in mouse ventricular myocytes is approximately half that seen in the rabbit (p < 0.001). Using our cylindrical calibration, this V:SA corresponds to a mean DTT of 0.169 ± 0.015 μm (p < 0.01).Application of the  in V:SA C was gen in V:SA C, suppor0.015 μm . It is a3.3DTT in rabbit cardiomyocytes  was twice as large as that in mouse cells  (n = 12) in mouse and 0.75 ± 0.01 (n = 29) in rabbit. These values were not different between species (p > 0.62).< 0.001) C. These 3.4V:SA method also provides information about the organization of the TT network, including TT density and TT orientation, both of which have been shown to have important implications for E-C coupling. TT length per cell volume, determined from the FM4-64 signal, was 0.70 ± 0.04 μm/μm3 in murine cardiomyocytes. Further segmentation of the TT network using Eigenvectors enabled us to identify transverse and axial components . This analysis revealed that transverse tubules comprised ~ 37% and axial tubules ~ 11% of total tubules in mice.The optical data used for the 3, mouse: 0.70 ± 0.04 μm/μm3, p < 0.001, TT abundance in rabbit myocytes was 39% of that in mouse (rabbit: 0.27 ± 0.02 μm/μm44.1V:SA) can be calculated and compared across groups (e.g. rabbit vs. mouse as shown here). In addition, we show how the V:SA may be converted to physical dimensions, using a simple, reasonable geometric model of TTs. Our V:SA method revealed that rabbit TTs were generally twice as wide as those of mice, with mean DTT of ~ 0.36 and ~ 0.17 μm, respectively . As E decreases from 1.0 (a circle), V:SA decreases for a given width. However, this effect is small at E > 0.6. For example, at E = 0.75 for the same V:SA value in rabbit (E = 0.73) in reasonable agreement with our ET measurements By using ET, we avoid the problem of the plane of section making TTs appear to be elliptical even if they have circular cross-sections. Despite the almost 2-fold difference in rabbit and mouse TT diameter  both hadn rabbit D TT widt4.3DTT of 0.05–0.12 μm DTT of 0.24–0.28 μm DTT was ~ 0.2 μm DTT to increase and a 50% contour measurement could also add a part of the PSF FWHM to the measure. With these effects in mind, we suggest that there is reasonable concordance with our measurements, but it should be noted that our method is faster and samples more tubules without explicit tubule-by-tubule selection and measurement.In murine ventricular myocytes, an early study using chemical fixation and subsequent stereological analysis of EM images reported 2/μm3, and the percentage of cell volume occupied by TTs ranged from 0.8 to 3.2% DTT of 0.17 μm  that determines electrical uniformity along t-tubules. From the well known steady state (dc) cable equation2 we estimate the membrane resistivity Rm to be ~ 7 kΩcm2. The lumen electrolyte resistance RL is probably similar to that of physiological saline  dependent and the space constant is given by:m is the membrane time constant. On the physiological (ms) time scale , the space constant would decrease by ~ 50% to ~ 170 μm and ~ 120 μm for rabbit and mouse, respectively. This suggests that TT membrane potential should be quite uniform unless TTs become disconnected or damaged in some way so as to decrease the membrane input resistance.The ~ 50 Ωcm  suggesti4.6V:SA ratio of spheres.The current method cannot distinguish between two closely apposed fine t-tubules and a highly flattened larger t-tubule. Nevertheless, as a wide-scale analysis method it can give useful information by revealing possible differences between samples, even if the cause of the underlying difference may need to be explored by higher resolution methods. There is a potential for this general method to be employed in conjunction with a third, spectrally well-separated dye, to examine the relationship between TT geometry and local function (e.g. Ca release using a Ca indicator). Finally, the method is not limited to ventricular muscle but can be exploited wherever changes in surface area to volume ratios may be of interest. For example, changes in cable properties in neural circuits may also be due to changes in the diameter of dendrites/axons which could be amenable to the method shown here. In addition, estimates of the volume/size of synaptic (or other membrane) vesicles could also be obtained from consideration of the 5DTT of ~ 360 nm and ~ 170 nm, respectively. Since it is known that there is extensive t-tubule remodeling in various disease states, the methods described here could provide powerful insight into subcellular remodeling of a key cellular structure.By quantifying the signals which mark the surface and interior volume of subcellular structures, and applying a suitable geometric model, it is possible to measure the physical dimensions objects well below the conventional diffraction limit. This method is generally applicable to all confocal microscopes, and produces large volumes of quantitative data relatively simply. We have shown that the t-tubules of rabbits and mice are quite different in arrangement and size, with mean Supplementary materialImage 1The following are the supplementary data related to this article.


Luteinizing hormone (LH) regulation of the ligand, natriuretic peptide precursor type C, and its receptor, natriuretic peptide receptor 2 (NPR2), is critical for oocyte maturation; however, the mechanism is not fully understood. Macrophage colony-stimulating factor (M-CSF) has recently been shown to be involved in oocyte maturation and ovulation. In the present study we determined whether or not M-CSF plays a role in the intermediate signal that mediates LH regulation of NPR2 in resumption of oocyte meiosis.Immature female C57BL/6 mice were injected i.p. with 5 IU of equine chorionic gonadotropin (eCG) to stimulate follicle development. After 44–48 h, the eCG-stimulated mice were injected i.p. with an ovulatory dose of 5 IU of human chorionic gonadotropin (hCG). The ovaries were excised at selected times. Pre-ovulatory follicles (POFs) and cumulus-oocyte complexes were cultured in different media. Immunohistochemical and quantitative real-time PCR analyses were used to assess the expression of M-CSF, M-CSF receptor (M-CSF-R), and NPR2. The presence of germinal vesicle breakdown (GVBD) was examined under a stereomicroscope to morphologically evaluate resumption of oocyte meiosis.NPR2 was mainly expressed in cumulus cells of pre-ovulatory follicles, while M-CSF and M-CSF-R were expressed in both mural granulosa and cumulus cells. The levels of M-CSF/M-CSF-R and NPR2 decreased within 4 h after treatment of hCG. M-CSF not only reduced the expression of NPR2 mRNA via its receptor (M-CSF-R), but also increased the proportion of GVBD in oocytes.M-CSF serves as an intermediate signal, thus inducing a vital decrease in the NPR2 levels in cumulus cells, and regulates the process of LH-induced resumption of meiosis. In female mammals, oocytes grow and undergo meiosis over a prolonged period of time , 2. OnceOocytes resume meiosis when the luteinizing hormone (LH) surge causes dramatic changes in pre-ovulatory follicles (POFs) , 17. It Macrophage colony-stimulating factor (M-CSF), a hemopoietic growth factor with a classic function of controlling the proliferation and differentiation of macrophages, has recently been shown to be involved in oocyte maturation and ovulation –30. We hImmature (22–25 days old) female C57BL/6 mice  were injected i.p. with 5 IU of equine chorionic gonadotropin (eCG) to stimulate follicle development. After 44–48 h, the eCG-stimulated mice were injected i.p. with an ovulatory dose of human chorionic gonadotropin . The ovaries were excised at selected times after injection and processed for immunohistochemical analysis and quantitative real-time PCR. For cell culture of POFs and cumulus-oocyte complexes (COCs), the eCG-stimulated mice were euthanized and the ovaries were excised without hCG injection. All chemicals were purchased from Sigma-Aldrich  unless otherwise stated. All animal procedures were approved by the guidelines of the Nanjing Medical University Administrative Panel on Laboratory Animal Care.2, 5% CO2, and 90% N2 for the indicated time in the presence or absence of hCG (5 IU/ml). At the end of the culture period, follicles were collected for quantitative RT-PCR analysis to measure hCG regulation of NPR2 transcript levels.The POFs were dissected stereomicroscopically from the ovaries of eCG-stimulated mice, as previously described , 33. The2, 5% CO2, and 90% N2 at 37 °C. After culture, COCs were collected for quantitative RT-PCR to calculate the NPR2 transcript levels. The presence of germinal vesicle breakdown (GVBD) was examined under a stereomicroscope.COCs were obtained by puncturing the POFs in the ovaries from eCG-stimulated mice. After isolation, COCs were washed in the medium and cultured for 2 h. The culture medium was MEM-α supplemented with 100 mg/ml of FBS, 100 U/ml of penicillin G, and 100 μg/ml of streptomycin sulfate with or without 30 nM NPPC. At least 10 COCs per treatment group were cultured. Cultures were maintained under a controlled atmosphere of 5% OMouse ovaries, cultured follicles, and COCs were collected in 350 μl of RNeasy lysis buffer. The tissues and cells were stored at −80 °C until analysis for mRNA expression. Total RNA was isolated from frozen samples using the RNeasy micro-RNA isolation kit , as recommended by the manufacturer’s instructions. Reverse transcription and real-time PCR was then carried out to quantify the steady-state mRNA levels of NPPC, NPR2, M-CSF, and M-CSF-R using an ABI 7500 real-time PCR instrument . The housekeeping gene, Rpl19, was considered the internal control. The primers for real-time PCR of NPPC, NPR2, M-CSF, M-CSF-R, and Rpl19 are listed in Table 2O2 for 20 min to inactivate intrinsic peroxidase activity, and incubated with ethylene dinitrilo tetraacetic acid buffer for 10 min for antigen retrieval. Washes were carried out with automation phosphate buffer. Sections were incubated for 2 h at 4 °C with rabbit anti-mouse antibody diluted 1:200 in buffer containing 5% bovine serum albumin. Sections were next incubated with horseradish peroxidase-labeled goat anti-rabbit antibody for 50 min at room temperature. Staining was achieved using diaminobenzidine chromogen. The staining reactions were stopped with distilled water, and sections were dehydrated and mounted with neutral balsam.The excised ovaries were fixed in 10% formalin. After dehydration, the fixed ovaries were embedded in paraplast, then sectioned at 5-μm intervals onto Superfrost Plus microscope slides. For immunohistochemical staining, sections of ovaries were deparaffinized and rehydrated, treated with 3% HSlides were examined under a microscope with a 200 × objective. The obtained images were captured and examined by Image Pro Plus 6.0 software . The integrated optical density (IOD) was calibrated, and the area of interest was set. The mean optical density was defined as the IOD divided by the total area examined.P value of less than 0.05.Statistical analyses were carried out using SPSS software . Data are presented as the mean ± SEM. Differences between experimental and control groups were analyzed by ANOVA test. Statistical significance was set at To investigate if M-CSF, M-CSF-R, and NPR2 signaling is involved in regulation of oocyte meiosis in POFs. We analyzed M-CSF, M-CSF-R, and NPR2 localization in ovarian sections from eCG-stimulated mice. Immunohistochemistry analysis revealed that M-CSF and M-CSF-R are expressed in both mural GCs and cumulus cells Fig. -d, whileWe further elucidated the importance of M-CSF, M-CSF-R, and NPR2 signaling in LH-induced resumption of oocyte meiosis. Using immunohistochemical techniques, we determined the changes in expression of M-CSF, M-CSF-R, and NPR2 in the ovaries of eCG-stimulated mice at 0 (48 h after eCG treatment), 0.5, 1, 2, and 4 h after injection with hCG Fig. . The meaTo study the effect of hCG on the kinetic curve of NPR2 mRNA levels in POFs, we examined the expression of NPR2 mRNA after 0, 0.5, 1, 2, and 4 h of culture after hCG treatment. The hCG (5 IU/ml) significantly decreased NPR2 mRNA levels by one-half at 0.5 h, and nearly 90% at 4 h of culture. In the control group without hCG treatment, the levels of NPR2 mRNA in POFs were slightly increased after POFs were cultured for 1 h. Then, the NPR2 mRNA levels were only slightly decreased after POFs were cultured for 4 h Fig. . The imaThe effect of M-CSF/M-CSF-R signaling on NPR2 mRNA expression and oocyte maturation was determined using cultured COCs isolated from POFs of eCG-stimulated mice. Isolated COCs spontaneously resume meiosis because mural GCs containing the inhibitory molecule, NPPC, is removed. Therefore, a culture system supplemented with 30 nM NPPC was adopted to maintain meiotic arrest in oocytes . M-CSF . M-CSF sIn the present study, we focused on the effect of M-CSF in the regulation of oocyte meiosis, and identified some crucial roles, including: (a) NPR2 was mainly expressed in cumulus cells of POFs, while M-CSF and M-CSF-R were expressed in both mural GCs and cumulus cells; (b) the levels of M-CSF/M-CSF-R and NPR2 decreased within 4 h after hCG treatment; and (c) M-CSF not only reduced the expression of NPR2 mRNA via its receptor (M-CSF-R), but also increased the proportion of GVBD of oocytes, which indicates that M-CSF is an intermediate signal, inducing a vital decrease in NPR2 levels in cumulus cells, and regulates the process of LH-induced resumption of meiosis.2). Reportedly, the expression of M-CSF was enhanced by E2 in luteinized GCs in a dose-dependent manner in vitro [2 maintains cumulus cell expression of NPR2 and inhibits the resumption of meiosis in mouse oocytes in vitro [2 has a certain role to the resumption of oocyte meiosis and ovulation. Therefore, the decreased level of M-CSF after hCG treatment may be due to the decreased levels of E2. Although the hypothesis is theoretically feasible, it is still necessary to further investigate whether or not a decrease in M-CSF during ovulation is related to a decrease in the E2 level.M-CSF extensively participates in the processes of ovulation , 29. Femin vitro . E2 mainin vitro , and theRecent studies have revealed that cGMP stimulated by NPR2 from cumulus cells diffuses into oocytes via gap junctions and controls cAMP concentration through inhibition of PDE3A activity, indicating that higher NPR2 levels in cumulus cells is responsible for oocyte meiotic arrest by maintaining high cAMP levels in oocytes , 4–7. OuWe conclude that M-CSF is an intermediate signal, inducing a vital decrease in NPR2 levels in cumulus cells, and regulates the process of LH-induced resumption of meiosis. Although further research is needed, our findings bring forth powerful evidence to interpret “multiple pathways” that mediate LH regulation of NPR2 in the process of resuming oocyte meiosis.


Anterior and posterior circulation atherosclerosis differ in vascular risk factors and stroke mechanisms. However, few studies have compared the pathological features between these lesions. Using a series of intracranial artery specimens, we characterized the intracranial atherosclerotic lesions and compared pathological features among different arteries of the intracranial vasculature.Intracranial large arteries of 32 consecutively recruited autopsy cases of Chinese adults aged 45 years or older were examined pathologically using routine histology and immunostaining, to characterize the pathological features of the atherosclerotic lesions. We analyzed middle cerebral arteries (MCAs) (both left and right), vertebral arteries (VAs) (side more affected), and basilar arteries (BAs).p < 0.05). MCA had more eccentric (vs. concentric) plaques than VA  and BA . Lumen thrombi were more frequent in BA, and calcification most commonly occurred in VA atherosclerotic lesions.Progressive atherosclerotic lesions were present in 91(71%) of the 128 arteries examined. Features of complicated plaques were infrequently detected: plaque hemorrhage was encountered in 12%, neovasculature in 12%, lumen thrombi in 13%, macrophage infiltration in 20%, and calcification in 25% of arteries. Luminal narrowing of MCA was the most severe, followed by VA; the BA least stenotic (37 ± 25 vs. 30 ± 24 vs. 20 ± 20%, all Intracranial atherosclerotic plaques were commonly present in this sample, but the lesions generally lacked features of complicated plaques. MCA lesions had demonstrable differences compared with VA and BA lesions. Further studies are needed to determine whether these characteristics indicate a distinctive atherosclerotic phenotype for the intracranial vasculature. Intracranial atherosclerotic disease (ICAD) is gaining greater clinical prominence as one of the most common causes of ischemic stroke worldwide, especially in Asians , 2. CurrRecent studies demonstrated that anatomical location is an important determinant of arterial morphometric characteristics , 13, andn = 13 (41%); infection or sepsis, n = 3 (9%); other natural causes , n = 13 (41%); unnatural causes , n = 3 (9%).All large intracranial arteries were retrieved from 32 autopsied specimens from December 2003 to June 2005 at the Prince of Wales Hospital, Hong Kong , who werIntracranial large arteries  were extracted for each of the 32 subjects. Each artery was divided into three equal parts. Each part was then dissected and decalcified overnight in 10% formic acid, followed by perfusion fixation in fresh 30% formaldehyde. Serial sections of the isolated arteries were cut transversely at 4-mm intervals, embedded in paraffin. Bilateral VA, paralleling left and right VA for individual cases, were embedded into one paraffin block together, without special labeling left or right ride. For an individual autopsy case, the side of VA with a more severe degree of atherosclerotic lesions was used in the analysis. Sections 5-µm thick were cut and stained with hematoxylin–eosin (H&E) and Victoria blue. Immunohistochemical staining was performed to evaluate the degree of macrophage infiltration, using standard avidin-biotin techniques and commercially available antibodies for CD68  at a dilution of 1: 200.Two pathologists (Chun Bo Niu and Hai Lu Zhao), who were blinded to clinical data, examined the histological sections from all segments using standard light microscopy. For each large artery, only the atherosclerotic lesion with the maximum grade of phenotype was subjected to histological and immunohistochemical evaluations. For the 32 autopsy cases, the most stenotic locations from each of the left MCA, right MCA, VA (choosing the artery with more severe lesions), and BA were analyzed for a total of 128 intracranial arteries.According to the revised American Heart Association criteria , the lesLesions in which the atherosclerotic plaques were distributed along the entire circumference of the internal elastic laminar were identified as concentric plaques, and those with a disease-free wall present were defined as eccentric plaques, according to prior work . The occP.” The original luminal area “A” was determined by the formula: A = P2/4π. The area of atherosclerotic plaque was then traced and determined by the software automatically (recorded as “Ai”), and the percentage of luminal narrowing was determined by the formula (Ai/A)*100%. This method eliminated errors due to luminal collapse that might occur during processing.The histological sections were photographed with a Leica DC 200 digital microscope. The degree of luminal narrowing was measured using Image-Pro Plus according to the method described by Leung et al. . The intt-test for continuous data. A value of p < 0.05 was considered to be statistically significant.Data analyses were conducted with the SPSS 20.0 software package . Comparisons among MCA, VA, and BA groups were assessed by McNemar’s test for categorical data and paired-samples n = 23, 72%). Nine cases (28%) were smokers. Hypertension was present in nine cases (28%) and diabetes in six (19%). Cardiovascular diseases including ischemic heart disease, ischemic stroke, and hemorrhagic stroke were found in 9 (28%), 14 (44%), and 2 (6%) cases, respectively (Table The 32 cases had a median age of 71 years (range 45–97 years) and most were men  had pre-atherosclerotic intimal lesions, whereas 91 (71%) showed progressive atherosclerotic lesions, including 15 (12%) pathologic intimal thickening, 72 (56%) fibrous cap atheroma, and 4 (3%) fibrocalcific plaque . Compared with posterior circulation arteries (VA and BA), anterior circulation arteries (MCA) had a higher prevalence of eccentric lesions  and a lower prevalcne of lumen thrombi . VA plaques showed a higher frequency of calcification compared with BA plaques . Plaque phenotype did not show significant difference between anterior and posterior circulation arteries. Other components were detected similarly between anterior and posterior circulation plaques.Plaque morphology and components were compared among MCA, VA and BA. Statistical analysis comparing left and right MCA showed no significant differences. We therefore chose the MCA with more severe lesions for each patient 32 MCAs) and compared the plaque characteristics with 32 VAs and 32 BAs. As shown in .” The protocol was approved by the “The Joint CUHK-NTEC CREC.” Consent forms were signed by the subjects’ relatives.WY, LZ, and AP-H were involved in data collection, analysis interpretation, and/or manuscript writing. MF, YX, KW, and XC were responsible for the conception of the study, study design, manuscript writing, and/or final approval. CN and HZ were responsible for the pathological slides review. HN was responsible for the autopsy examination. All authors approved the final report.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hematopoietic stem cell transplantation (HSCT) is an important treatment option for children with severe and refractory sickle cell disease (SCD) with debilitating clinical complications. HSCT with cells from the bone marrow of a HLA-identical sibling used in SCD has a low mortality risk, high cure rate, and high event-free survival rate after a median follow-up of 5-6 years. However, matched donors are found in only about 20% of the patients. A boy aged 8 years with SCD had a sister, <2 years old, a fully compatible donor. The boy met all eligibility criteria to undergo HSCT, and he was suffering from cognitive and neurologic impairment due to ischemic events. A Bioethical Committee jointly discussed the ethical issues on this case after a pediatric evaluation released the very young sister for donation. The justification was that the sister would benefit from the donation too because of the greater likelihood of survival and cure and less suffering of her brother. The parents were informed about the risks and benefits for both children, and the family was psychologically evaluated. After their consent, HSCT was performed and the patient is cured from SCD. The complication for the donor was the need for blood transfusion. Sickle cell disease (SCD) is a severe inherited disease which affects multiple organs in the body, causing acute and chronic complications, with significant morbidity and reduced life expectancy –3. HematGene therapy might be an option for SCD cure, mainly for patients who do not have HLA-identical relative donors, but the treatment is still in the early stages of testing , 8, 10.HSCT with cells from the bone marrow of a HLA-identical sibling used in SCD has a low mortality risk (5–10%), high cure rate (90%), and high event-free survival rate after a median follow-up of 5-6 years (85–100%) , 6, 13. The objective of this study is to present a bioethical dilemma in the case of a young boy undergoing HSCT for SCD.Our patient is a SCD patient, a boy aged 8 years, living in São Paulo, Brazil. He met all eligibility criteria to undergo HSCT  1, 2, 77, 8.He had an ischemic stroke, the major cause of morbidity in SCD , at 8 moAfter seven years of monthly transfusion program, he presented an iron overload, with heart disease and mild diastolic congestive heart failure. Even though these blood transfusions have prevented neurologic events, they have brought about significant end-organ toxicity in this patient. This was expected , since gThe clinical and laboratory features supported HSCT indication, and the patient has an HLA-matched sister (10 × 10). Postponing his transplant could lead to increased cardiac involvement, which would increase the morbidity of BMT.His sister, a girl aged 1 year and 5 months, was identified as a possible donor. She was evaluated by two different pediatricians, one from the Hematology Department at Hospital Israelita Albert Einstein and the second pediatrician from another hospital, the Child Cancer Institute . After these evaluations, the sister was released for donation.For HSCT, bone marrow donation may be performed by surgical harvesting of the bone marrow or by apheresis collection. The patient's surgical harvesting has the benefit of lower risk of graft-versus-host disease (GVHD) after transplantation .To perform the surgical harvesting, it would be necessary to collect a volume equivalent to 15 ml/kg of the recipient patient, in order to achieve the sufficient amount of progenitor cells, minimizing the risk of graft failure. In the literature, there is a recommendation that each collection does not exceed the maximum volume of 20 ml/kg of the donor , 18.In this case, the patient weighs 26 kg and his sister weighs 9 kg. Thus, it would be necessary for the procedure for bone marrow donation to be carried out 2 or 3 times, in order to reach the sufficient quantity of cells to HSCT , 18.Bone marrow collection could be performed under general anesthesia through multiple punctures to the posterior iliac crest, with low risk of severe adverse events , 19, 20.As this donor is compatible with her brother (here reported) and her sister, both diagnosed with sickle cell anemia and indicated a bone marrow transplant, we decided to collect the 20 ml/kg donor volume to maintain a fraction stored for future transplantation of sister, reducing the need for additional collections.These considerations led to the consultation of the Bioethical Committee of the hospital as follows.The reason for the consultation of the bioethics committee was the ethical conflict that their parents would be subjected to, on consenting to the procedures, due to their difficulty in understanding their son and daughter's risks and benefits.For the hematologists, it could be very difficult to decide about the risks involved in bone marrow donation because of the young age of the donor: the girl is only 1 year and 5 months old. She was the only possible donor for the patient; however, to do so, she would be submitted to three bone marrow surgical harvesting procedures, in order to obtain enough material for her brother.The HSCT Team requested an ethics consultation with the Bioethical Committee (BC) to obtain necessary help on deciding what to do about the donor age and how to support family decision . The BC At the time of this specialist meeting, the sister donor had already been evaluated by two pediatricians of another hospital, who were in favor of the procedure. Their parents had already signed the informed consent after a meeting with HSCT Team, and all of the donation risks had been explained.The considerations presented to them were as follows: unfortunately, studies showed the most unfavorable results and more complications with unrelated donors for HSCT in SCD, so the patient's sister was in fact the best option , 12. GenSome studies have shown psychological benefits to donors, such as increased self-esteem, pride in donating, a greater sensitivity to the needs of others, increased family union, feeling like a better person, and an increased meaning and worth of life –25. SpecAlthough some studies have shown that HSCT donation is safe, with only temporary and modest discomfort (1 severe adverse event/453 donations), there is a higher risk of needing a blood transfusion, fatigue, wound infection, and pain. Also, cardiovascular complications can occur after general anesthesia is used for catheter insertion (stem cell harvest) or for bone marrow collection from the hip , 27, 28.The risk of death from HSCT donation is considered low: 1/10.000 donations . In our Hospital Israelita Albert Einstein is a HSCT referral center;The donor would have a multidisciplinary pediatrician team to do the prior transplant evaluation and the systematic follow-up after that;The donor and her family would have psychological evaluation and support prior to and after to transplant;Donor risk would not be significant, based on scientific studies and clinical practice in our hospital;Without the transplantation, there was a severe risk of death and complications in our SCD patient at that time. The boy could not wait for his sister to get older;There were not any medically equivalent histocompatible adult donors among relatives who would be able to donate;There is a strong and positive emotional relationship between the donor and the recipient;There was a reasonable probability that the recipient would benefit from the HSCT;Clinical emotional and psychosocial risks to the donor would be reduced;Parental permission consent was obtained after the parents had been informed of the risks and the benefits of the procedure.Our decision for the donation was based on the recommended criteria bellow , 28, 30:After three months of BC decision, the first bone marrow donation was carried out by surgical harvesting of the bone marrow. The donor had to receive a blood transfusion, and this was the only donation complication. Despite the fact that the hematology team programmed three donation procedures, only two donations were really performed, because the volume of the bone marrow collected was enough.8/kg and CD34 positive cells of 4.42 × 106/kg.The first bone marrow collection was performed on November 30, 2015, with a volume of 191 ml and total nucleated cells of 1.34 × 10We choose to cryopreserve the product and perform a new collection in order to reach the minimum dose of nucleated cells, reducing the risk of failure of primary grafting.8/kg and CD34 cells of 5.06 × 106/kg. The donor did not have any significant side effects and she is still healthy until now.The second collection was performed on February 23, 2016, after a reevaluation of the donor, which verified complete recovery after the first collection. The collected volume was 173 ml, with total nucleated cells of 1.97 × 10The HSCT was performed without significant complications and our patient has been cured from SCD. Four months after his HSCT, he had 85% of donor-host hematopoietic chimerism, which is sufficient to overcome a genetic defect [Our BC discussion was based on the “four principles approach to medical ethics”: autonomy, beneficence, nonmaleficence, and justice , 32. primum non nocere principle, because it is an altruistic and solidary practice, without a lot of risks for donors [Firstly, we discussed the risk-benefits for the procedure with the multidisciplinary team, in a BC meeting, based on the nonmaleficence principle , 33. We  beneficence and the nonmaleficence principles were respected in this point of view [Although studies have shown that iron overload caused by regular transfusions can be controlled by an oral iron chelator , our pat of view .Parents should protect and represent their children, and, in the case of HSCT sibling donation, they must be assisted by health care providers to decide about it . In someThe justice bioethical principle, the fair distribution of goods and services , in HSCTAn important factor to consider in such cases is also the competent support from relatives and/or caregivers during the pre- and post-HSCT period . In our Finally, our patient, his donor, and their parents were able to comprehend the procedure, and they had the educational, cultural, family, and social background according to the “rule of descending order.” This rule suggests that patients have less knowledge than the physician, justifying our efforts to inform them adequately .The accepted justification for permitting a minor sibling to donate bone marrow stem cells was that the donor, the sister, would benefit from it because of the greater likelihood of survival and cure and less suffering for her brother. After discussing all essential points with the multidisciplinary team, the BC considered that all important conditions were satisfied in order to provide more benefits and fewer risks to the HSCT patient and his donor. Thus, the donation and HSCT were recommended by the BC.


Troponin I is one of the most commonly tested biochemical markers in the emergency room (ER) and in the hospital setting. Besides coronary artery disease (CAD), demand ischemia with underlying tachycardia, anemia, hypertensive emergency, congestive heart failure, kidney disease, sepsis, and pulmonary embolism have also been reported to cause troponin I elevations. Few reports have excluded patients with CAD, and no study has summarized the proportion of these factors relative to an increased troponin I level.The aim of this retrospective study was to investigate the level of contribution of causative factors in troponin I elevation. Charts of patients tested for troponin I during an ER visit or during hospitalization were collected. Patients with known CAD, abnormal stress tests, cardiac catheterizations, or discharge without an adequate cardiac evaluation were excluded. Logistic regression was used to identify predictors of elevated troponin I levels.P < .001). In multivariate logistic regression, sepsis, CHF, age, Hb, and HR were independent predictors of troponin I . A simple clinical scoring system was generated with 1 score on patients with age ≥ 60, Hb < 10 g/dL, and HR ≥ 100 beats per minute (bpm). The prevalence of elevated troponin I was 4%, 16%, 38%, and 50% for patients with scores of 0, 1, 2, and 3, respectively. In patients without sepsis and CHF, the chances of elevated troponin I were 2%, 11%, 28%, and 43%.A total of 586 patients were investigated in this study. Age, hemoglobin (Hb), heart rate (HR), glomerularfiltration rate, atrial fibrillation, congestive heart failure (CHF), and sepsis were significant predictors of elevated troponin I by analysis in univariate logistic regression , and heart rate (HR) can assist clinical evaluation of elevated troponin I test in non-CAD patients. Other than CAD, increased oxygen demand of cardiomyocytes or decreased myocardial blood flow can also cause elevated troponin I, also known as type II myocardial infarction. The causes of demand ischemia have been reported as tachycardia, anemia, hypertensive emergency, congestive heart failure, kidney disease, sepsis, and pulmonary embolism (PE).,4 Individual analysis of each underlying diagnosis has been shown in multiple studies. Troponin I elevation has been documented in sepsis patients without CAD on autopsy. Multiple reports identified troponin I elevations in patients with congestive heart failure (CHF).,7 It has been reported that the heart rate can be a primary dependent factor in patients with paroxysmal supraventricular tachycardia (PSVT) without CAD. Elevated troponin I levels were documented in patients with atrial fibrillation (Afib). Anemia in sickle cell crisis and acute gastrointestinal bleeding have been identified as causes of troponin I elevation ,11 as was hypertensive crisis. Patients with chronic kidney diseases tended to have baseline troponin I elevation. Half of patients diagnosed with PE had similar laboratory findings. However, there were only a few reports that excluded patients with CAD, and no study has summarized the proportion of these factors. The aim of this retrospective study was designed to investigate alternate diagnoses other than CAD that could contribute to troponin I elevations.Troponin I is one of the most commonly tested biochemical markers in the emergency room (ER) and hospital setting. Elevated troponin I is diagnostic for myocardial injury.22.1Patients tested for troponin I in a local community hospital in Scranton, PA, USA, between November 1, 2016, and December 15, 2016, were included. Exclusion criteria included patients with known CAD or coronary etiologies, positive stress test or evidence of CAD based on cardiac catheterization results during hospitalization, and patients who were discharged (death or transfer) without proper evaluation. The study was approved by the Institutional Review Board.2.2 The assay of troponin I utilized was the high sensitivity troponin I assay. Elevation of troponin I was defined according to the parameter of the hospital and was equal or greater than 0.03 ng/mL. If there were multiple troponin level measured during one hospital visit, only the highest one was documented. Among multiple Hb level, HR, SBP records, only the closest one to the troponin I test was documented. The clinical data were collected only once for the patients with multiple admissions.Analytic data were collected including age, sex, hemoglobin level (Hb), heart rate (HR), systolic blood pressure (SBP), and glomerular filtration rate (GFR) by the Cockcroft Gault equation and underlying conditions, including CHF, sepsis, PE, and Afib.2.3U test to examine continuous outcome between patients with and without elevated troponin I levels. The chi-square test and 2-tailed Fisher exact test were used to evaluate categorical data. The association between elevated troponin I level and clinical variables by binary logistic regression was analyzed. Potential predictors of elevated troponin I included age, gender, hemoglobin level, heart rate, systolic blood pressure, glomerular filtration rate, presence of atrial fibrillation, congestive heart failure, pulmonary embolism, and sepsis. These were comprehensively included in the univariate logistic regression. Factors with a P value less than 0.1 in the univariate model were introduced in to the multivariate logistic regression to estimate the adjusted odds ratio (OR) and 95% confidence interval (CI) by forward selection. A simple clinical score was created to predict elevated troponin I based on the results of multivariate logistic regression. The discriminatory ability of the clinical score was evaluated using concordance statistics (c-statistics). Statistical analysis was conducted with SPSS for Windows version 20.0 . Statistical significance was set at a 2-tailed P value less than 0.05.Normality assumption was not met, so the authors employed the Mann–Whitney 3A total of 1368 cases were tested for troponin I during an ER visit or hospitalization. Five hundred and eight patients were omitted due to readmission. Two hundred and forty-seven patients had known CAD or abnormal stress test/cardiac catheterization results during admission. Twenty-seven patients were discharged (died or being transferred) without proper evaluation. A total of 586 patients were included in this study . Patients with elevated troponin I also had higher HR and were more likely to have Afib, CHF, and sepsis . In univariate logistic regression, age, HB, HR, GFR, Afib, CHF, and sepsis were associated with higher odds of having elevated troponin I  on day 1 of hospitalization was reported to have no prognostic value in sepsis patients. It is not an uncommon practice for septic patients to have troponin I levels checked in the ER on admission. The authors conclude that checking troponin I levels in septic patients, especially without underlying CAD, has to be done discriminately to try to curtail and additional unnecessary testing.The authors identified sepsis as the strongest independent factor of troponin I elevation in non-CAD patients in this study. In previous reports, sepsis caused increased cytokines secretion, catecholamine surge, and tissue metabolic demand. Increased oxygen demand in peripheral hypoxic tissue can further increase the cardiomyocytes workload and cause relative insufficient coronary blood flow. There are multiple articles that describe elevated troponin I in CHF patients.,7,20 In this study, the authors identified CHF as an independent cause of elevated troponin I level in non-CAD patients. However, CHF can also be a complication of acute coronary syndrome. Thus, definitive decisions concerning the monitoring and interpretation of elevated troponin I levels will depend on clinical judgment during an ER visit or hospitalization.CHF was also identified as an independent cause of elevated troponin I. Congestive heart failure is known to cause sporadic areas of cardiomyocytes death, abnormal structures of the remaining cardiomyocytes, and ultimately progression to fibrosis. Thus, troponin I elevation can be caused by the cell necrosis process. Thus, the authors concluded that age related differences should be taken into consideration when interpreting the troponin I level.Age was also noted in this study to correlate with increased troponin I levels. It has been reported that age greater than 60 years old is a significant factor that can affect a troponin I level. It has been reported that heart rate can be primary dependent factor in patients with supraventricular tachycardia without CAD. This study reconfirmed that HR has a strong impact on troponin I levels in non-CAD patients.Heart rate was also an independent factor that affected the troponin I level. Heart rate has been well known to be a risk factor that causes increased oxygen demand. This can lead to relative insufficient coronary blood flow.,24 Abruptly increased cardiac index in patients with acute anemia has been demonstrated in a previous article. Anemia in sickle cell crisis and acute gastrointestinal bleeding were also reported to be associated with troponin I elevation.,11 In this study, anemia was confirmed to be a single independent factor for troponin I elevation. However, there was no significant difference between acute and chronic anemia regarding troponin I levels. There were only 6 patients who were diagnosed with acute anemia included in this study. Due to the number of cases, this cohort will need to be further studied.Anemia was another diagnosis that was associated with elevated troponin I in this study. Anemia can cause a hyperdynamic status by increasing heart rate and contractility, which can further increase the oxygen demand of cardiomyocytes which then results in insufficient coronary blood flow.The authors generated a simple clinical scoring system with 1 score given to patients with Hb < 10 g/dL, age ≥ 60 and HR ≥ 100 beats per minute. This was applied to patients without known or clinical suspicion of CAD. Given that sepsis and CHF were the 2 strongest independent factors, the scoring system was initially done on all patients with a second analysis done on those patients excluding sepsis and CHF. With the lowest score, the chance of the patient having an elevated troponin I was 4% versus 2%; with the highest score, the chance of the patient having an elevated troponin I level was 50% versus 43%. The score showed moderate discriminatory ability and provided a tool that could be applied in clinical practice to try to identify subsets of patients who might have elevated troponin I levels due to the factors identified above and who do not generate a high clinical suspicion of CAD. However, hypotension was also listed as a factor for elevated troponin I. The underlying pathophysiology may be due to reduced coronary perfusion during a hypotensive episode. Further investigation is needed regarding hypertension and hypotension as causes of elevated troponin I levels.In this report, SBP showed significant results in the Mann–Whitney test but showed insignificant results in logistic regression. The data were further analyzed. It was identified that within SBP 140 to 180 mm Hg, the percentage of abnormal troponin I was around 7% but increased to 16% to 29% in the rest of the SBP intervals. The nonlinear correlation explained the statistic difference. Hypertensive crisis has been well described as a cause of cardiomyocytes ischemia. The underlying pathophysiology could be due to impaired blood pumping from the heart due to increased afterload. Thus, the association between Afib and elevated troponin I levels needs to be further investigated.Atrial fibrillation (Afib) was shown to have a significant effect on troponin I value in chi-Square and univariate logistic regression in this study. However, Afib failed to show independent significant impact in multivariate logistic regression. In this study, 87 patients were diagnosed with Afib. Out of the 87, 79 patients were greater than age 60, and 20 patients had HR greater than 100 beats per minute (bpm). This implies that age and HR can be confounding factors in patient diagnosed with Afib. Parwani et al analyzed 354 patients with Afib and concluded troponin I release can be induced by Afib. However, the study included CAD patients, and there was no control group of patients without Afib.2 are age greater than 60 years old. This implies that age can also be a confounding factor in GFR in this study. It has been reported that troponin I elevation is associated with chronic kidney disease and decrease GFR. Ziebig et al enrolled 24 patients after heart surgery with measurement of both serum and urine troponin I and classified the patients based on urine GFR. This suggested that impaired kidney function affected the troponin I removal. However, 8 out of 24 patients had myocardial infarction as a complication of CAD in the study. Thus, a further prospective study with quantified GFR levels in non-CAD patients may provide more information.In this study, GFR showed no significant effect on troponin I. The method used to calculate the GFR was based on the Cockcroft Gault equation. None of the patients had quantified standard GFR urine test. Age was a numerator on the Cockcroft Gault equation and 193 out of 217 patients with GFR < 60 mL/min/1.73 m In this study, PE associated with increased troponin I levels was not statistically significant. This may be due to the insufficient sample size (only 7 patients with PE in this study).It has been reported that half of the patients with PE have elevated troponin I. However, both SBP and sex failed to show statistical significance in this study. Based on real world clinical practice, the mean age of the patient in this study was age 65 years and many of the patients had comorbid ailments as listed.There is 1 recently published study that revealed that sex, age, and SBP are strong factors of elevated troponin I in healthy adults with mean age 37 years old.Current research regarding troponin I focuses on single factors as causative etiologies of elevated levels and rarely excludes CAD patients. In this study, patients with underlying CAD were excluded. This gave the authors an independent assessment of the other factors that led to increased troponin I levels.There were several limitations in this study. This was a retrospective study in a single center. Not all the patients had definite diagnostic tests such as cardiac catheterization, computed tomography angiogram, or magnetic resonance imaging to rule out CAD. However, all the medical records were reviewed and the patients enrolled were evaluated by hospitalists and cardiologists based on clinical judgment of symptoms and risk stratifications. All the patients with known CAD, abnormal stress test/cardiac catheterization results and patients without proper cardiac evaluation were excluded. Before doing this, the highest troponin I level was greater than 80 ng/mL; after excluding the CAD patients, the highest troponin I level decreased to 1.79 ng/mL. This implies that the patients left after omitting CAD patients had milder cardiomyocytes damage. A larger prospective study is needed for further investigation. Finally, although the score showed moderate discriminatory ability within the cohort, external validation is still needed to confirm the results.5Troponin I is one of the commonly tested biochemical markers reflecting underlying cardiomyocytes damage. In this study, sepsis was identified as the strongest independent cause of elevated troponin I levels in non-CAD patients. Thus, the authors concluded that troponin I levels should be discriminately checked on septic patients, especially without underlying CAD. The scoring system composed of age, Hb, HR provided an additional tool to assist in predict potential troponin I abnormalities.


The KT Ratio is considered an index that represents IDO. The participants were examined with the Beck Depression Inventory II (BDI-II), the Montgomery Aasberg Depression Rating Scale (MADRS), and a neuropsychological test battery. We found no significant differences between the two study groups with respect to TRP, KYN, or KT Ratio in serum or CSF. Differences in neuropsychological performance between the two patient groups could be seen in the following tests: Animal Fluency, Digit Symbol, the DKEFS Color-Interference Test (Naming Part), Trail Making Test A and B, and the Grooved Pegboard Non-dominant Hand. KYN in serum correlated highly with KYN in CSF. KYN in serum correlated significantly with both age and gender. When analyzing males and females separately, we found that women had a lower level of TRP in both serum  and CSF . Women had a lower level of KYN in serum (p = 0.029) than men did. Age was positively associated with KYN. KYN in CSF correlated only with age, however; there were no gender differences. No significant relationship was seen between BDI-II and MADRS on the one hand, and KYN and TRP on the other. KYN in CSF as the KT Ratio in both serum and CSF was associated with neuropsychological performance. Thus, we suggest that KYN and KT Ratio are related more strongly to neuropsychological performance than to affective symptoms in depression.It has been suggested that the metabolic enzyme indoleamine 2,3-dioxygenase (IDO) is a biological mediator of inflammation related to the psychopathology of depression, with a Kynurenine (KYN) increase in the Tryptophan (TRP) metabolic pathway, resulting in reduced Serotonin. In this study, we examined KYN, TRP, and the ratio of KYN to TRP concentrations × 10 Depression is the world’s most widespread mental disorder, affecting over 350 million individuals . The mec3) KT Ratio is a reliable indication of IDO  is a biological mediator of inflammation related to depressive disorders, as seen in animal models and after drug therapy in humans with interferon-α . The effn of IDO . Becausen of IDO . In addin of IDO . 3OH-KYNn of IDO . Both 3On of IDO .Although several studies have examined plasma or serum levels of inflammatory markers in psychopathology of depressive disorders, data on Tryptophan, Kynurenine, Indoleamine 2,3-dioxygenase in both serum and cerebrospinal fluid (CSF) have rarely been studied. A PubMed search with the words Tryptophan, Kynurenine, serum, CSF resulted in 21 studies, none of which were related to depression. We wanted to rectify this omission by examining these variables in a depressive group and a control group of patients with no depression. In addition, we asked: ‘How are serum and CSF levels connected?’We hypothesized that patients with a diagnosis of depression would present with a specific and different serum and CSF, KYN, and TRP profile than would patients with no depression. In particular, we expected to find higher levels of KYN and KT Ratio in both serum and CSF among depressed patients than among non-depressed patients. We therefore examined two patient groups: one with depression and one without. The non-depressed group, which served as a control group, was referred to a neurological clinic because of diffuse  symptoms such as fatigue, but no diagnosis could be established. Because a recent study has demonstrated that IDO is related to depressive symptoms more strongly in women than in men , we analCognitive dysfunction is common in depression . It is n(1)Does the concentration of TRP, KYN, and KT Ratio in serum and CSF differ between patients with depression and a similar patient group without depression? And as a secondary aim, do patients with depression differ in cognitive performance compared to patient group with no depression?(2)Are TRP and/or KYN and KT Ratio concentrations in serum and CSF associated with affective symptoms and/or cognitive performance?TR, mood disorders. For participants in the depression group, it was decided that brain imaging should be conducted if there were any indication or suspicion of cerebral disorder. None of the depressed patients showed any indication or suspicion of such a disorder, so they were not examined with brain imaging.A group of 49 depressed patients were recruited from inpatient wards and outpatient clinics of the Mental Health Unit at Innlandet Hospital Trust, Norway. They were all over 18 years of age and were diagnosed with depression using (a) the International Statistical Classification of Diseases and Related Health Problems, 10th revision [ICD-10], F32–34 spectra; and (b) DSM-IVThirteen of the 49 depressive patients were using a combination of anti-depressive and antipsychotic medication; 22 were medicated with anti-depressives only and 3 with only antipsychotic medication.The control group comprised 31 patients over 18 years of age with neurological symptoms who were referred to the Department of Neurology at the same hospital for a thorough investigation. The inclusion criteria were no disease and no neurological sign of disease. None of the patients had neurological signs of disease; and none of their laboratory tests indicated cerebral disorder. Clinically, they had many similar symptoms seen in depressed patients, including tiredness, fatigue, lack of initiative, and the feeling that the body is not functioning. They had no overt signs of depression, however: mood changes or emotional state. Other than depression, the most common symptom was fatigue, as reported by 45 of the depressed patients and 25 of the controls. Nineteen of the control patients had a neuropsychological assessment and were included in the study. Of these 19 controls, 14 reported fatigue. Lack of funding prevented our giving 12 patients from the control group a neuropsychological examination when they were recruited.All patients were recruited consecutively from either the psychiatric or neurological ward after the psychiatrists or neurologists, who were informed about the study, determined that the patients met the criteria for inclusion in the study.g. CSF samples were obtained by a trained neurologist and centrifuged within 30 min at 2000 g for 10 min. Serum and CSF was aliquoted and stored at -70°C until analyses were performed. All blood samples were taken between 08.30 and 10.00. The spinal tap was done before 12.00 noon, except for two depressed patients for whom the procedure was performed closer to 13.00. The patients were not required to fast before blood or CSF sampling.A medical history was recorded, a routine clinical examination performed, and hematological and biochemical screening tests conducted on all participants. Peripheral blood was collected, allowed to clot for 30 min before 10 min of centrifugation at 2000 A neurologist conducted a detailed examination of the patients, including a routine examination of CSF, in the Department of Neurology.TRP and KYN levels in serum and CSF were determined by high-performance liquid chromatography . In brieAll patients were examined independently with two depression scales. (a) Montgomery Aasberg Depression Rating Scale (MADRS)  yielded • Mini-Mental State Examination (MMSE) is a test for global cognitive function .• Trail Making Test A (simple); Trail Making Test B (complex): the Trail Making Test measures attention, visual searching, mental processing speed, set shifting, and cognitive flexibility .• Grooved Pegboard Test, a test of fine motor control: this test measures response time in seconds with dominant and non-dominant hand .• Hopkins Verbal Learning Test-R (HVLT): immediate total recall and delayed recall , 2001.• Brief Visual Memory Test-R (BVMT) measures immediate and delayed visual recall .• Wechsler Adult Intelligence Scale, 3rd Edition (WAIS-III), the subtests of which are Digit Symbol and Symbol Search measures processing speed, visual perception, attention and concentration, motor and mental speed .• The D-KEFS Color Word Interference Test: Test 1, color naming; Test 2, color name reading; Test 3 inhibition; Test 4 inhibition/switching. The test measures the Stroop effect, attention, cognitive processing, and mental stimulus control .• Controlled Oral Word Association Test (COWAT), which measures the ability to produce words within 1 min: Words , Clothes (number of clothes), Animals  .The study was reviewed and approved by the Regional Committees for Medical and Health Research Ethics, South East A, Reference No. 2009/2196a, and all participants provided a written informed consent form before they were included in the study.U-test or chi-square analyses were performed between the two groups to test group differences in age, education, gender, KYN, TRP, KT Ratio, depressive symptom forms, and neuropsychological test results. Correlations among KYN, TRP, and KT Ratio, age, and gender were assessed with Spearman’s rho. Linear multiple regression was used to study independent predictors. Statistical significance was set at p < 0.05. SPSS Version 24  was used for the analyses.Independent Mann–Whitney Clinical examination, CSF examination, computed tomography, or magnetic resonance imaging revealed no cerebral neuropathology in any of the patients . Controlling for age, we analyzed our data to see if the anti-depressive drugs or the combination of anti-depressive and anti-psychotic drugs had had an impact on the test results in the depression group. No significant results were found (data not shown).Table 1. There were no significant differences with respect to TRP, KYN, or KT Ratio in serum or CSF. When we analyzed men and women separately independent of depression, however, women were found to have a lower level of TRP in both serum  and CSF  than men. Women also had a lower level of KYN in serum (p = 0.029), but not CSF (p = 0.15). No differences were found for KT Ratio (data not shown). When the analyses were broken down for men and women in the depression and the control group separately, the same results were found in the depression group for TRP in serum (p = 0.035) and CSF (p = 0.04) and for the neurological group for TRP in serum (p = 0.018) and CSF (p = 0.006). The KYN serum difference between women and men was not significantly different in either the depression or control group (data not shown).Participant characteristics with comparisons between the two study groups are shown in As expected, the depression and diffuse neurological symptom groups differed significantly on both Beck-II and MADRS. Differences in neuropsychological performance could be seen in Animal fluency, WAIS-III Digit symbol, The D-KEFS color naming, Trail Making Tests A and B, and the Grooved Pegboard non-dominant hand, with best performance in the neurological group. There results were basically the same when controlled for age and gender.Table 2 presents correlations among KYN, TRP, and the KT Ratio in serum and CSF by age and gender. KYN concentrations in serum and CSF correlated significantly with each other; this was not the case with TRP. Age was positively correlated with KYN levels, but KYN in serum correlated positively with age only for men. On the other hand, KYN in CSF correlated positively with age, but there were no gender differences. Regarding TRP, the correlations with age and gender were weaker, but in the same direction as for KYN. TRP in serum was significant only for gender, whereas TRP in CSF was related to both age and gender. KT Ratio in CSF and serum correlated highly with each other, and showed also positive correlations with age but not with gender.Table 3 presents correlations of TRP, KYN, and the KT Ratio to predict scores on BDI-II, MADRS, and neuropsychological tests in linear regressions, controlling for age, gender, and group status . Because of their distributions, Trail Making Test A and B, Grooved Pegboard Test, and inhibition of word and color in the D-KEFS inhibition were log transformed. No significant correlations were found between BDI-II/MADRS on the one hand and KYN, TRP or KT Ratio on the other.Table 3, KYN in CSF correlated with neuropsychological performance when age, gender, and group membership were controlled. Further associations were seen when the KT Ratios were examined. No such correlations were found for TRP, and no significant associations were found between BDI-II, MADRS on the one hand and KYN, TRP, or KT Ratio on the other. When the same tests were performed without controlling for group status, however, the partial correlations between KYN in CSF and KT Ratios relative to the neuropsychological test performance were stronger.As can be seen in p = 0.013; Grooved Pegboard dominant hand , partial correlation = 0.34, p = 0.007; Grooved Pegboard non-dominant hand , partial correlation = 0.31, p = 0.017; Digit Symbol WAIS-III, partial correlation = -0.30, p = 0.017, D-KEFS color naming, partial correlation = 0.31, p = 0.016.• KYN in CSF: MMSE, partial correlation -0.32, p = 0.022; HVLT-delayed recall, partial correlation -0.301, p = 0.018; Log Trail Making Test Part A, partial correlation 0.34, p = 0.008; D-KEFS color word inhibition/switching, partial correlation = 0.32, p = 0.012.• KT Ratio in serum: HVLT-learning, partial correlation -0.294, p = 0.029, DKEFS inhibition/switching, partial correlation = 0.37, p = 0.003.• KT Ratio in CSF: D-KEFS color naming, partial correlation = 0.28, We are unaware of any other study that compared a depression group to a similar patient group, as we have done in this study with regard to KYN, TRP, and KT Ratio in serum and CSF.p = 0.03), even after adjustment for baseline depression scores. In a study of young adult (24–39 years) Finns, it was found that the IDO enzyme is involved in immune regulation of early atherosclerosis, particularly among females, and could constitute a marker of immune activation for atherosclerosis in females  in serum and CSF correlated significantly with each other. TRP in serum and CSF showed no significant correlation. Theoretically, Interferon-γ will enhance TRP metabolism by inducing IDO and thereby catalyze the first step of TRP degradation, forming the KYN. Our results could not support Interferon-γ as a specific trait in depression, however. In addition, when we studied cytokines in serum and CSF, as reported in another paper , we founAlthough the two groups were similar with respect to clinical symptoms, there was a clear trend toward differences in cognitive performance between the depression and control groups. Depression in itself, therefore, seems to be responsible for the depression group’s inferior cognitive performance.KYN in CSF was related to some degree to neuropsychological performance. Poorer performance on MMSE, Grooved Pegboard, Digit Symbol WAIS-III, and D-KEFS Color all correlated with KYN in CSF. The partial correlations for these tests were modest—all below 0.4. More associations were seen with the KT Ratio, however. HVLT learning and delayed recall and the Trail Making Test Part A were all significantly associated with KT Ratio in serum, and this finding seems to be partly independent of depression. Higher levels of the KT Ratio indicated poorer performance on the neuropsychological tests. The KT Ratio in CSF showed significant associations with D-KEFS color naming and D-KEFS inhibiting/switching. These findings could indicate support for the theory that too much KYN at the expense of Serotonin results in metabolites that influence cognitive performance . Thus KYNo significant differences were found between a group of depressed patients and controls regarding KYN, TRP, KT Ratios in serum and CSF. Women seem to have a lower level of TRP in both serum and CSF than men do. There was a clear trend toward inferior neuropsychological performance in the depression group compared to the controls. KYN, TRP, and KT Ratio are strongly related to age and gender in the two samples, independent of depression. In the original studies of KYN and TRP related to medication with Interferon-α, the vegetative or sickness symptoms emerged weeks before depression ,c, 2004.KH designed the study and did the writing of the manuscript. KE took part in the planning of the study and contributed to the writing process. JW took part in the planning of the study and contributed to the writing process. PF took part in the planning of the study and contributed to the writing process.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.