Despite the discovery of CSF and serum diagnostic autoantibodies in autoimmune encephalitis, there are still very limited CSF biomarkers for diagnostic and monitoring purposes in children with inflammatory or autoimmune brain disease. The cause of encephalitis is unknown in up to a third of encephalitis cohorts, and it is important to differentiate infective from autoimmune encephalitis given the therapeutic implications.To study CSF cytokines and chemokines as diagnostic biomarkers of active neuroinflammation, and assess their role in differentiating demyelinating, autoimmune, and viral encephalitis.th centile of control values as cut off. We correlated CSF cytokine/chemokines with disease severity and follow up outcome based on modified Rankin scale. One-way hierarchical correlational cluster analysis of molecules was performed in different encephalitis and outcome groups.We measured and compared 32 cytokine/chemokines using multiplex immunoassay and APRIL and BAFF using ELISA in CSF collected prior to commencing treatment from paediatric patients with confirmed acute disseminated encephalomyelitis , anti-NMDAR encephalitis , and enteroviral encephalitis . We generated normative data using CSF from 20 non-inflammatory neurological controls. The sensitivity of CSF cytokine/chemokines to diagnose encephalitis cases was calculated using 95In descending order, CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 had the best sensitivity (>79.1%) when all encephalitis patients were included. The combination of IL-6 and IFN-α was most predictive of inflammation on multiple logistic regression with area under the ROC curve 0.99 (CI 0.97–1.00). There were no differences in CSF cytokine concentrations between EVE and anti-NMDAR E, whereas ADEM showed more pronounced elevation of Th17 related  and Th2  related cytokine/chemokines. Unlike EVE, heat map analysis showed similar clustering of cytokine/chemokine molecules in immune mediated encephalitis (ADEM and anti-NMDAR E). Th1 and B cell (CXCL13 and CXCL10) molecules clustered together in patients with severe encephalopathy at admission and worse disability at follow up in all encephalitis. There was no correlation between CSF neopterin and IFN-γ or IFN-α.A combination panel of cytokine/chemokines consisting of CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 measured using multiplex immunoassay may be used to diagnose and monitor intrathecal inflammation in the brain. Given their association with worse outcome, certain key chemokines  could represent potential therapeutic targets in encephalitis. Paediatric neuroinflammatory disorders represent a heterogeneous group of disorders including encephalitis, demyelinating disorders and other immune-mediated autoinflammatory CNS disorders . It is iCytokines and chemokines are key intercellular mediators in inflammation and have been shown to be elevated in a number of inflammatory disorders of the brain : Table CIn order to improve our CSF diagnostic repertoire, we studied the use of CSF cytokines and chemokines in well-defined encephalitis syndromes, and examined the ability of these molecules to differentiate demyelinating, autoimmune and viral encephalitis etiologies.As part of the ethically approved protocol, we wrote to all parents and gained written consent to use the stored CSF for this study. This study was approved by HREC of Sydney Children’s Hospital network (LNRSSA/14/SCHN/283).We included patients with encephalitis who had stored frozen acute CSF available, using neurology and neuroimmunology clinical databases at the Children's Hospital at Westmead, Sydney, Australia between 2006 and 2014 . The casTwenty non-inflammatory neurological controls  were useThirty-two cytokines were measured by multiplexed fluorescent bead-based immunoassay detection  according to the manufacturer’s instructions, using a combination of 23-plex (MPHCYTOMAG60K23), 6 plex (MPHCYP2MAG62K06), and 3-plex (MPHCYP3MAG63K03) Millipore Human Cytokine panel kits. The 23-plex kit contained antibody-conjugated beads for following cytokines and chemokines: IL-1ra, GM-CSF, IL-1b, TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17A, IFN-γ, CCL2/MCP-1, CCL5/RANTES, CXCL1/GRO, CXCL10/IP-10, CCL3/MIP-1a, CCL4/MIP-1b, IL-12 (p40) and IL-12 (p70), IFN-α, G-CSF and CCL11/Eotaxin. The 6-plex kit was used to detect IL-21, IL-23, CXCL13/BCA-1, CCL17/TARC, CCL21/6Ckine and CXCL12/SDF-1. The 3 Plex kit contained antibody-conjugated beads for CXCL9/MIG, CXCL11/I-TAC, and CCL19/MIP-3b. All CSF samples were primarily obtained for routine diagnostic work-up prior to commencing treatment, and samples were frozen at -40°C till use for the analysis. The CSF samples were thawed overnight for analysis. For each assay, the curve was derived from various concentrations of the cytokine standards assayed in the same manner as patient samples. All samples were measured undiluted.APRIL (sensitivity >2pg/ml) and BAFF (sensitivity >0.4ng/ml) were performed using ELISA kits as per the manufacturer’s instructions . All samples were analyzed undiluted except 9 samples, where volume was insufficient for BAFF analysis. In these cases, results were multiplied by the appropriate dilution factor.http://www.merckmillipore.com. The detectability of cytokine/chemokines was variable in control groups and up to 40% of cytokines and chemokines (n = 14/34) were above detection limit in ≥50% controls as shown in Table A in The methodological details including assay method, lower detection limits and coefficient of variance are available at the manufacturer’s website, R: A language and environment for statistical computing programme v 3.2.2. The graphs were composed using Graph Pad Prism software version 6. In order to assess the clinical utility of the CSF cytokine/chemokines as biomarkers to diagnose all encephalitis patients , we calculated sensitivity using 95th centile of control value as the upper limit of normal range. CSF cytokine/chemokines were also transformed according to log10 /2) and tested in univariable logistic regression models to predict inflammation in encephalitis versus controls. The cytokine/chemokines with area under the receiver operating characteristic (ROC) curve (AUC) of at least 0.8 were then tested in a multivariable model with backward selection.Statistical analysis was performed using SAS version 9.3 and Statistical analyses of nonparametric continuous data  were performed using Kruskal-Wallis test for multiple groups with Bonferroni correction and 2-tailed P values were calculated. We also compared cytokine/chemokines concentrations in all encephalitis patients categorised based on severity of disease at nadir  or presence of disability at final follow-up  using the Mann-Whitney U test.R. The cytokine/chemokine molecules are listed in the same order for each of the three encephalitis groups to allow a visual comparison of the pattern between the groups. The heat maps colors correspond to correlations grading from -1 , no correlation (white) to 1 . Similarly heat maps were also generated for all encephalitis patients based on severity of encephalitis at nadir, and final follow up outcome using MRS score as described earlier.The Spearman rank correlation coefficient was used for analyses of correlations between cytokine/chemokines, and between cytokine levels and clinical parameters in the encephalitis groups. One-way Hierarchical clustering was performed using Spearman’s correlation coefficient as a proximity distance matrix, which was then plotted using a dendrogram. Heat map was created for each of the diagnostic groups using the “heatmap.2” function in The children with ADEM and anti-NMDAR-E were older compared to EVE groups (p = 0.01). The CSF timing from onset of neurological symptoms was shorter in ADEM and EVE group compared to anti-NMDAR-E . The sensitivity of CSF pleocytosis (68–81%), neopterin (81–100%) and oligoclonal bands  (10–36%) to detect inflammation in individual encephalitis groups is shown in th centile as cut off (specificity 95%). These cytokine/chemokines are presented according to Th or B cell status in We observed that TNF-α, IL-10, IFN-α, IL-6, CXCL10 and CXCL13 demonstrated >79.1% sensitivity in patients with encephalitis using control 95A heat map was generated to demonstrate statistically significantly elevated cytokine/chemokines and their frequency of elevation in different encephalitis groups compared to controls . A broadThere were no significant differences in cytokine/chemokine concentrations between EVE and anti-NMDAR E groups. Th1 , Th2 , Th17  and B cell  related cytokine/chemokines showed statistically significant elevation in ADEM compared to EVE and/or anti-NMDAR E . CXCL12 We performed comparisons between most sensitive CSF cytokine/chemokines  and CSF protein, CSF pleocytosis, timing of CSF sample from the onset of neurological symptoms, and age at encephalitis presentation using spearman correlation tests . The majority of the correlations between CSF protein and CSF cytokines were not significant except for moderate correlation with CXCL13 , IL-10  and TNF-α  in ADEM, and IL-10 in EVE . Only CSF CXCL13 showed a positive correlation with CSF cell count in all encephalitis patients . CSF cytokines/chemokine levels generally declined with time of CSF sampling although the correlations were not significant in EVE, Anti-NMDAR E, ADEM and all encephalitis groups. There was no correlation between age and CSF cytokine and chemokine levels in different encephalitis groups. There was no evidence of correlations between CSF cytokine/chemokines and severity of disease at presentation or disability outcome using modified Rankin scale (data not shown). There was no evidence of differences in cyto/chemokine concentrations in all encephalitis patients grouped according to severity of the disease or presence of disability.Hierarchical clustering was used to identify sets of cytokines whose expression levels among patients within individual disease category are correlated. The results are represented in heat maps to facilitate visualization of interaction between cytokine/chemokines among a large set of variables in individual disease groups and are unaffected by heterogeneity in overall cytokine levels within each group. The cytokines and chemokine formed two major clusters of cytokines and chemokines, which correlated within each group. First cluster (Cluster A) included Th1 , Th2 , Treg (IL-10) and Th17  and other molecules . The second cluster (Cluster B) molecules included Th1 , B cell related chemokines  and other molecules . The clustering pattern showed some similarities in both anti-NMDAR E and ADEM groups and showed positive correlations within cluster A molecules. However, in contrast to ADEM, there were significant negative correlations between cluster B  and cluster A molecules (shown in red) in anti-NMDAR E. The correlations between the molecules were weaker in EVE compared to anti-NMDAR E and ADEM. As shown in The aim of the present study was to describe the CSF cytokine/chemokine concentrations in patients in well-defined encephalitis groups and discuss their utility as biomarkers of inflammation. This is based on the hypothesis that CSF cytokines may correspond to the intrathecal activation of immunoactive cells, and the evaluation of these cytokines might be a good indicator of the type of immune activation, the severity of inflammation and disease activity in neuroinflammatory disorders of the brain. In our cohort, among currently available investigations, the sensitivity of pleocytosis, oligoclonal bands and neopterin were similar to previous studies , 18, 19.In our cohort of encephalitis, the Th1 (CXCL10 & TNF-α), T reg (IL-10), B cell (CXCL13) related cytokine/chemokines, and cytokines with broad spectrum of activities including IL-6 and IFN-α, were elevated in more than 75% of the cases. The combination of IL-6 and IFN-α predicted inflammation on multiple regression analysis. In our study, we used non-inflammatory neurological controls to generate a reference range. The ‘normal cut off’ for CXCL13, CXCL10 and IFN-α in our study was similar to normal reference values reported in the literature . The ‘noCSF CXCL13, CXCL10, IL-6 and IFN-α are elevated in a number of infectious and immune disorders. The cellular source, cellular target, role in neuroinflammation, and association with neuroinflammatory disorders and other neuroinflammatory markers are presented in the When we compared the cytokine/chemokine profile in different encephalitis groups, patients with ADEM showed predominant elevation of Th1 , Th2 , Th17 , B cell  and other cytokines  molecules, which supports the hypothesis that both cell mediated and humoral effector mechanisms may play a role in this condition, similar to experimental allergic encephalomyelitis (EAE) model in mouse , 30. In The elevation of cytokine/chemokines in EVE is consistent with previously reported studies and the elevation of CXCL13, BAFF and APRIL highlight humoral mechanisms involved in protection against viral infections, possibly as a consequence of induction of these molecules by interferons –41. The Another distinctive feature in chemokine profile was lower levels of CSF CXCL12 in EVE and anti-NMDAR encephalitis compared to elevated levels in ADEM. The normal polarized expression of CXCL12 on the basolateral surface of the microvasculature was reported to shift to the luminal side during inflammation leading to low levels in CSF , 49]. H. H49]. Hin vivo [There was no evidence of differences in cytokine/chemokine concentration between EVE and anti-NMDAR E suggesting shared immunological pathways involving T cell, B cell and macrophages. Similar to our study, Michael et al showed no major differences  in cytokine, chemokine and other inflammatory mediator concentration between heterogeneous groups of infectious and autoimmune encephalitis in adults . Despitein vivo . The obsin vivo . Previouin vivo –58.In our study, the correlations of CSF cytokine/chemokines with severity at admission and disability at follow up were not statistically significant unlike previous studies , 59, 60.The important limitation of the study is that even though we measured a broad array CSF cytokines and chemokines in all encephalitis groups, we could not perform comparisons between serum and CSF samples to due to the lack of availability of stored frozen serum samples. Previous cytokine/chemokines studies on neuroinflammatory disorders of brain showed higher CSF to serum ratios of these molecules and some of these molecules have low transfer rate across blood brain barrier  24, 39,–68. The To conclude, we presented cytokine/chemokine profile from relatively well-defined encephalitis groups. Our study suggests that combining several biomarkers will enhance our ability to diagnose and monitor CNS inflammation. Despite similarities between CSF cytokine/chemokine concentrations in EVE and anti NMDAR E, we identified differential cytokine/chemokine interactions on cluster analysis, which may play a role in autoimmune pathogenesis (ADEM and anti NMDAR E). Some of these molecules (CXCL13 and CXCL10) may represent potential therapeutic targets in view of their association with severity of encephalopathy at admission and worse disability at follow up in all encephalitis cases.Future clinical studies are needed to determine the utility of combination cytokine panels in diagnosing the presence of active intrathecal inflammation and monitoring disease activity in guiding therapeutic decisions. In addition to characterising the shared cytokine/chemokine functional pathways, other mechanisms need further investigation such as possible disturbances of immune active and immune regulatory cells, their signalling pathways and other molecules involved in regulating chemotaxis and degranulation which may be responsible for predisposition to autoimmunity following viral encephalitis. The potential role of monoclonal antibodies against CXCL10, CXCL13 as therapeutic targets in encephalitis needs further investigation.S1 Fig#MOG antibody positive demyelination is shaded blue and MOG negative is shaded black in ADEM group.(TIFF)Click here for additional data file.S1 FileTable A. The detectability of CSF cytokine/chemokines in controls. Table B. Comparison between median CSF cytokine and chemokine concentrations in ADEM, anti NMDAR E and EVE groups according to T and B cell effector groups.Table C. Key Cytokine/chemokines, their cellular source, cellular target, role in neuroinflammation, and association with neuroinflammatory disorders and other neuroinflammatory markers.(DOCX)Click here for additional data file.


The lack of clinically-reliable biomarkers makes impossible to predict sperm retrieval outcomes at testicular sperm extraction (TESE) in men with non-obstructive azoospermia (NOA), resulting in up to 50% of unnecessary surgical interventions. Clinical data, hormonal profile and histological classification of testis parenchyma from 47 white-Caucasian idiopathic NOA (iNOA) men submitted to microdissection TESE (microTESE) were analyzed. Logistic regression analyses tested potential clinical predictors of positive sperm retrieval. The predictive accuracy of all variables was evaluated using the receiver operating characteristic-derived area under the curve, and the clinical net benefit estimated by a decision-curve analysis (DCA). Overall, 23 (49%) and 24 (51%) patients were classified as positive and negative sperm retrievals at microTESE. While circulating hormones associated to a condition of primary hypogonadism did not predict sperm retrieval, levels of anti-Mullerian hormone (AMH) and the ratio AMH-to-total Testosterone (AMH/tT) achieved independent predictor status for sperm retrieval at microTESE, with a predictive accuracy of 93% and 95%. Using cutoff values of <4.62 ng/ml for AMH and <1.02 for AMH/tT, positive sperm retrieval was predicted in all individuals, with 19 men out of 47 potentially spared from surgery. DCA findings demonstrated clinical net benefit using AMH and AMH/tT for patient selection at microTESE. Azoospermia affects about 1% among all men and 10–15% of infertile men5. With no sperm found at multiple semen analyses, non-obstructive azoospermia (NOA) is the most severe form of infertility5. Despite genetic causes have been associated with male infertility7, genetic defects are found only in 17–20% of NOA individuals10. Except for a portion of patients with central endocrine disorders, the remaining 80% of NOA men having negative results on genetic testing are classified as idiopathic NOA (iNOA)11.The relevance of male infertility has progressively grown in Western societies, with significant medical, psychological, and socio-economic implications. Seven out of 100 men are infertile, with up to 40% of infertile conditions still of unexplained or idiopathic originin vitro fertilization programs; in this context, microdissection TESE (microTESE) has been advocated as the gold-standard technique to improve sperm yield with minimal tissue excision12. However, the lack of useful predictive biomarkers suggestive for successful sperm retrieval at microTESE in NOA men still represents a relevant gap with a very negative return for the patient. Indeed, no significant association has been found between microTESE sperm retrieval outcomes and (i) preoperative testicular volume, (ii) baseline follicular stimulating hormone (FSH) levels, (iii) basal level of Testosterone (T) or increased T level following treatments with aromatase inhibitors, clomiphene citrate or human chorionic gonadotropin14. Thereof, we sought to identify novel and user-friendly prognostic factors reliably predicting surgical outcomes in iNOA men in the real-life setting. Among other variables, we considered serum levels of testis-derived hormones than might be representative of the primary testicular failure, such as the Anti-Müllerian Hormone (AMH), which is suggestive for a Sertoli cells’ immature phenotype15, and T. Of biological relevance, over the adult life AMH expression is under the control of FSH and the inhibitory action of T16. Likewise, a paracrine effect of AMH was reported on Leydig cells, with an inhibition of steroidogenesis17. For the specific purpose of this study, we considered the ratio of AMH-to-total T (AMH/tT) as a potential effective biomarker to predict the severity of the primary failure of the testis parenchyma.In patients with clinical evidence of NOA, testicular sperm extraction (TESE) is the technique of choice, which rationally has to be planned as a part of Complete data  from the last 47 white-Caucasian men with iNOA submitted to microTESE at two tertiary-referral centers  were analyzed in a retrospective study.18. Primary infertility was defined as when a couple had never been able to conceive18. Men with iNOA were included in the study when having no spermatozoa because of non-obstructive causes in at least two consecutive semen analyses according to the WHO criteria5. Idiopathic NOA was defined after comprehensive diagnostic evaluations of all know causes for non-obstructive azoospermia. Thereof, patients with the following clinical features were excluded from the study: azoospermic patients with (i) testicular factors previously associated with infertility ; (ii) genetic abnormalities previously associated to azoospermia, thus considering mutations of the cystic fibrosis conductance regulator gene (CFTR) associated with congenital bilateral absence of the vas deferens such as CFTR F508del, CFTR F508del heterozygosis, CFTR 5 T/7 T, CFTR 7 T/7 T, and CFTR poly 7 T/9 T, homo and heterozygosis 1298 A > C for the MTHFR gene; microdeletions on the Y chrosomome such as AZFa/b/c; Klinefelter or Kallman syndromes; (iii) known hypothalamic/pituitary defects; (iv) either pituitary or testicular surgery and/or previous vasectomy; (v) previous tumors, including testicular tumors; (vi) testosterone replacement therapy; and, (vii) any other known reason for genital tract obstruction. Conversely, inclusion criteria were (i) a clinical diagnosis of iNOA associated with primary couple’s infertility; (ii) age ≤ 45 years; (iii) white-Caucasian race; (iv) freedom from any known viral and bacterial infections and antibiotic therapies at the time of surgery and throughout the preceding 6 months; and, (v) a comprehensive blood set of analyses over the 12 months before surgery. Weight and height were measured for each participant, and body mass index (BMI), defined as weight in kilograms by height in square meters, was calculated. Waist circumference was measured for every patient19. Testes volume was assessed through a Prader orchidometer, calculating the mean value between the two sides.According to the World Health Organisation (WHO) criteria, infertility was defined as not conceiving a pregnancy after at least 12 months of unprotected intercourse regardless of whether or not a pregnancy ultimately occurredPatients were then dichotomized according to the surgical outcome .20 at two high volume centers for Reproductive Medicine. At the time of testicular microdissection, the parenchyma was immediately placed in 5 ml of Quinn’s™ Sperm Washing Medium  and minced mechanically with sterile slides. The sample was then transferred into a Falcon tube and centrifuged at 600 g for 10 minutes. The pellet was suspended in a minimum volume of 0.5 ml Quinn’s™ Sperm Washing Medium. Sperm retrieval was checked under an inverted microscope at ×400 magnifications. Sperm were counted, and sperm retrieval was expressed as the number of sperm/high power field (HPF) and then eventually cryopreserved. For the freezing procedure, an equal volume of Quinns Advantage™ Sperm Freezing Medium  was added to the suspended sperm pellet, and then homogenized and placed at room temperature for 20 minutes. After the room temperature incubation, the sample was heat sealed in a variable number of paillettes CBS™  and placed at 4 °C for 5 minutes. As a final step, all the paillettes were placed in liquid nitrogen vapor for 15 minutes and then stored in liquid nitrogen. For thawing, the paillettes were warmed to 37 °C and then opened to transfer the sample into a 10 ml Falcon tube. Sample was then washed again with Quinn’s™ Sperm Washing Medium and centrifuged at 600 g for 10 minutes. The supernatant was discarded and samples suspended in variable amounts medium.All men underwent microTESE according to the original surgical technique21. Histological classification of human spermatogenesis was carried out according to the criteria suggested by either Johnsen22 or McLachlan21. Morphological evaluation of Leydig cell compartment was also performed, and reported as (i) normal Leydig cells, (ii) Leydig celle hyperplasia  and (iii) Leydig cell tumour .A formal histological examination of the testicular specimen was also performed on Bouin and formalin-fixed specimens, followed by hematoxylin-eosin staining. Records were then discussed according to the reported indications2; range of linearity = 5–3000 pg/ml, coefficient of variability = 3.9%) were measured using a heterogeneous competitive magnetic separation assay . An enzyme-linked immunosorbent assay was used to measure Inhibin B  and AMH . Electrochemiluminescence immunoassay was used for measuring total T levels  and Prolactin . Sex hormone–binding globulin (SHBG) levels were measured via a solid-phase chemiluminescent immunometric assay on the Immulite 2000 . The same laboratory was used for all patients.Venous blood samples were drawn from each patient (7–11 AM) after an overnight fast. Follicle-stimulating hormone , luteinizing hormone , thyroid-stimulating hormone , and 17β-estradiol . Mann-Whitney test was used for comparing continuous variables, and Chi23. Then, AMH and AMH/tT values were dichotomized according to the most informative cut-off value capable of maximizing sensitivity when predicting positive sperm retrieval. Finally, we used decision-curve analysis (DCA) to evaluate the clinical net-benefit of the two predictive markers24. Statistical analyses were performed using GraphPad Prism 5.9; DCA was computed using R version 3.3.0 . All tests were two-sided, with a significance level set at 0.05.The statistical analyses consisted of several steps. First, logistic regression model estimated odds ratios (OR) and 95% confidence intervals (CIs) for the univariable (UVA) and multivariable (MVA) association between AMH, AMH/tT and a positive sperm retrieval at microTESE, after adjusting for covariates previously reported to be associated with primary testicular failure in NOA men . The Receiver Operating Characteristic (ROC) curve was obtained to quantify the predictive accuracy  of AMH and the AMH/tTData collection followed the principles outlined in the Declaration of Helsinki; all patients signed an informed consent agreeing to provide their own anonymous information and tissue specimens for future studies. The study was approved by the Institutional Review Board .2 and tT  in 18 out 24 (75%) individuals, complete germ cell arrest in 5 out of 24 (21%), and hypo-spermatogenesis in 1 out of 24 (4%); conversely, the testis parenchyma of positive sperm retrieval patients was classified as SCOS in 3 out of 23 (13%), germ cell arrest in 5 out of 23 (22%), hypospermatogenesis in 13 out of 23 (56.5%) and normal spermatogenesis in 2 out of 23 (8.5%) Fig. . Normal Table To quantify discrimination of the outcome of microTESE based on the blood level of AMH Fig.  and AMH/The most informative cut-off values for AMH and AMT/tT were then calculated; in this context, circulating AMH levels below 4.62 ng/ml resulted in 100% sensitivity and 82% specificity, with a positive predictive value of 79% and a negative predictive value of 100% for positive sperm retrieval; likewise, AMH/tT values below 1.02 achieved same performances as AMH levels <4.62 ng/ml Table .Table 3SDecision-curve analysis showed the net benefit of using both AMH and AMH/tT to identify patients in terms of sperm retrieval outcomes, with a slight advantage in favor of AMH/tT values toward AMH levels Fig. .in vitro fertilization via intracytoplasmic sperm injection was already attempted in 12 out of 23 couples (52.2%). Of all, 7 embryos were transferred into the uterus, thus resulting in 5 biochemical pregnancies, 5 clinical pregnancies, and 3 newborns.In the testis parenchyma of positive sperm retrievals, a median of 0.038 sperm/high power field (IQR = 0.0033–0.48) were found. At the time of preparation of this manuscript in vitro fertilization programs. Major achievement of this study is the novel identification of two potential user-friendly biomarkers with high predictive accuracy toward positive sperm retrieval at microTESE. Indeed, we found that circulating AMH levels and AMH/tT values allowed dichotomizing a homogenous cohort of white-Caucasian primary infertile men with iNOA into positive vs. negative sperm retrievals at microdissection testicular surgery, with >93% accuracy. In this context, the adoption of AMH below 4.62 ng/ml or an AMH/tT ratio cutoff value below 1.02 resulted in 100% sensitivity for positive sperm retrieval, at the clinical cost of including 5 out of 28 (18%) false positive but reliably sparing unnecessary microTESE in 19 out of 47 men (40.4%).Still there is a disarming lack of clinically useful predictive biomarkers of sperm retrieval outcomes at testicular surgery in NOA men undergoing 25. Similarly, regardless of sperm retrieval outcomes at microTESE, all iNOA men considered in our cohort showed reduced testicular volume. Despite testicular hypotrophy has been advocated to be associated with spermatogenic dysfunction14, current findings confirmed previous data showing that testis volume per se is not a reliable predictor of sperm retrieval outcomes at surgery26. To this must be added that primary hypogonadism in infertile men has been associated with low tT serum levels and high circulating FSH levels28. Data from the European Male Aging Study (EMAS) showed that serum levels of tT <3.03 ng/ml and LH >9.4 mU/ml reliably identified primary hypogonadism in aging men29. Recently, the same two markers were applied also to stratify different types of hypogonadism in primary infertile men30. Current findings confirmed that the aforementioned cutoff values used for FSH, LH and tT did not identify patients with a positive vs. a negative sperm retrieval at surgery, thus confirming known data31. More in depth, the circulating levels of FSH, LH and tT did not emerge to adequately define the severity of the primary exocrine testis failure.Strengths of these findings first come from the confirmation of previous observation that age at surgery, testicular volume and circulating FSH levels were not reliable biomarkers of positive vs. negative sperm retrieval in the real-life scenario. Indeed, even in our relatively small but homogenous cohort of iNOA individuals, age at surgery did not achieve independent predictor status for sperm retrieval outcomes34. In agreement, both serum AMH levels and AMH/tT ratio values were not associated with the histological findings. According to the applied mono vs. bilateral microTESE to find sperms, we may rationally infer that a negative outcome upon bilateral surgery was indicative of the absence of germ cells in both testes; thereof, the increase AMH levels and AMH/tT ratio values were faithful markers of a globally impaired exocrine function of both testes. Hence, being hormonal serum levels representative on the one hand of the production of the entire testis, and on the other of the different degree of the disease, circulating tT and AMH might be considered adequate indicators of the endocrine gonadal system, while an increased AMH/tT ratio in retrieval negative individuals might be representative of a spreading of the pathological insult within the testis, with the potential consequent spreading of SCOS to all seminiferous tubules. A second interpretation is that the gonadal endocrine system of sperm retrieval positive and negative iNOA men may be representative of different stages of testicular development/maturation. The blood level of AMH is high from the neonatal to the pubertal stage in males, with a subsequent decrease to 3–4% of the infant levels throughout the adulthood35. In this context, AMH production is under the control of FSH up to the pubertal stage36 without being subject to control by androgens37; in contrast, AMH level is drastically reduced by T during the adult life16. Likewise, a paracrine effect of AMH was reported on Leydig cells, with an inhibition of steroidogenesis17. Thereof, it would seem that the main physiological role of AMH in the adult male is limited to the paracrine control of testicular function. To this regard, AMH/tT ratio appeared to be representative of the balance between the exocrine and the endocrine compartments within the testis; therefore, the high blood levels of AMH and AMH/tT ratio observed in negative sperm retrieval iNOA men, with a testicular histology of SCOS along with germ cell aplasia, is likely representative of an impaired control of the androgen toward the expression of AMH. We could speculate that in iNOA individuals with negative sperm retrieval at microTESE, high blood levels of AMH and AMH/tT ratio might be even expression of a sort of testis regression to a pre-puberal stage, with immature Sertoli cells unresponsive to T action.Further studies are certainly needed to shed light on the mechanisms behind the condition of germ cell aplasia in idiopathic infertile men. To this aim, the finding of these two novel prognostic biomarkers of sperm retrieval in iNOA men might outline at least two pathogenetic mechanisms. A first interpretation is that while retrieval positive testes emerged to be heterogeneous for the presence of both areas positive for sperm along with seminiferous tubules characterized by germ cell arrest and Sertoli cells only, conversely 75% of sperm retrieval negative testes were classified as SCOS and 21% with complete germ cell arrest. The histological classification of the testis parenchyma was previously associated with the severity of clinical diagnosis in infertile men, but the score was not associated with testicular volume, and serum levels of inhB, FSH, LH and tT38, becoming undetectable at menopause39; this makes circulating AMH extremely relevant especially in terms of assisted reproductive technology40. Conversely, for the first time we showed that elevated blood AMH levels and increased AMH/tT values may reflect the depletion of the germ cell reservoir within the testis in iNOA men. In this context, serum levels of AMH and AMH/tT values appeared as more reliable biomarkers representative of the severity of the primary testis failure.Blood level of AMH is considered as a predictive marker of ovarian reserve in women41 or oligozoospermia42, whereas decreased blood levels of AMH were reported in NOA men41. It has been also reported that circulating AMH lacks prognostic significance in terms of sperm retrieval in NOA men submitted to testicular sperm aspiration or TESE43. Despite that genetic background44 and genetic abnormalities45 are known factors modulating the circulating levels of AMH, previously published studies did not report specific inclusion/exclusion criteria regarding the considered infertile population. In this context, we consider a major strength of our study the detailed definition of specific inclusion and exclusion criteria, which at least allow to selecting a homogeneous cohort of idiopathic infertile white-Caucasian men below 45 years of age.Blood levels of AMH were previously considered not diagnostically significant in men with either obstructive azoospermiaOverall, serum levels of AMH and the AMH/tT ratio hold a number of translational outcomes in clinical terms. Of major clinical relevance, further implementation in the clinical practice would reduce the number of unnecessary surgery, while focusing on patients with the highest chance of sperm retrieval.Our study is not devoid of limitations. Above all, the study has considered a relatively small cohort of iNOA patients; therefore larger studies across different centers and populations will be needed to substantiate our findings. Second, the strict selection of only white-Caucasian iNOA men might necessarily pave the way for setting novel cut-offs for AMH and AMH/tT in infertile men of different genetic backgrounds.


To the Editor,The combined use of plerixafor and granulocyte-colony stimulating factor (G-CSF) improves mobilization in poor mobilizers. However, there are limited data available on the use of plerixafor in healthy donors ,2. The eth dose of G-CSF, the leukocyte count was 45,000/µL, of which 0.33% (148/µL) were CD34+ cells. Peripheral stem cell apheresis was performed using the Donor Spectra Optia Apheresis System . A total of 15.20x108 nuclear cells/kg were collected. The product contained 3.92x106 CD34+ cells/kg, 14.91x107 CD3+ cells/kg, 17.36x107 CD19+ cells/kg, and 7.17x107 CD56+ cells/kg. G-CSF was discontinued after an adequate number of stem cells had been collected, but the fever persisted. Oseltamivir at 150 mg twice daily was then prescribed for the donor because the respiratory tract virus panel examination revealed influenza A infection. The fever became controlled 24 h after oseltamivir administration. The plerixafor procedure was considered to have permitted “sufficient mobilization” in a healthy donor who could not be mobilized with G-CSF probably because of his influenza infection.A 46-year-old male was selected as an HLA-matched donor for a patient diagnosed with acute myeloid leukemia (AML). Donor assessment was performed in accordance with the standard operating procedure prepared for JACIE . The donor was given 10 mg/kg/day G-CSF. He developed a dry persistent cough, chills, fever of 39 °C, fatigue, and flu-like symptoms on day 3 of G-CSF administration. The donor was considered to have an upper respiratory tract infection, which could not be attributed to only G-CSF administration. The family members of the donor were found to have similar symptoms. Thus, blood and urine cultures were obtained and he was started on levofloxacin in addition to paracetamol; G-CSF was continued. A respiratory tract virus panel was performed on a nasal smear using a PCR-based technique. The peripheral blood leukocyte count was 22,000/µL but CD34+ cells represented just 0.07% of all cells (11/µL) on day 5 of G-CSF administration; this was considered to reflect “poor mobilization”. Therefore, 0.24 mg/kg plerixafor was administered “just in time,” in addition to G-CSF, on night 5, after the donor had been given all necessary information and informed consent had been obtained. Two hours after the 11Many factors including age, sex, body mass index, baseline leukocyte count, and comorbid conditions may compromise mobilization . AlthougThis report indicates that influenza A may suppress the hematopoietic system, negatively affecting stem cell mobilization. The problem may be overcome by plerixafor administration.


The diagnosis of tuberculous meningitis (TBM) can be extremely difficult in the absence of culture confirmation. Cerebrospinal fluid (CSF) adenosine deaminase (ADA) can potentially assist in this regard, although its current value remains unclear. The literature on the usefulness of CSF ADA in TBM diagnosis is inconsistent, especially from an analytical point of view.A retrospective analysis of clinical and laboratory data relating to all CSF ADA requests during 2009 and 2010 in a South African quaternary healthcare setting was performed. A CSF ADA cut-off for TBM diagnosis was calculated using receiver operating characteristic curve analysis. The performance of CSF ADA in different infective and non-infective categories was assessed.p-value < 0.01; sensitivity of 85.9% (95% CI of 77.0 – 92.3) and specificity of 77.7% (95% CI of 75.4 – 79.8%); positive likelihood ratio = 3.85 and negative likelihood ratio = 0.18). At this cut-off 13 TBM cases were missed.In total, 3548 CSF ADA requests were considered over the 2-year period. Of these, 1490 were for patients for whom both a CSF ADA and a mycobacterial culture were requested. The optimal cut-off was calculated at 2.0 U/L (AUC = 0.86; 95% CI = 0.82 – 0.89; An optimal cut-off for routine use could not be established as too many TBM cases were missed. Specimen integrity, lack of ADA assay standardisation and overlap in performance of the assay in different diagnostic categories affect interpretation. The diagnosis of tuberculous meningitis (TBM) in general can be extremely difficult in the absence of culture confirmation. A non-definitive test such as cerebrospinal fluid (CSF) adenosine deaminase (ADA) could potentially assist in the regard, although its current value for the diagnosis of TBM remains unclear.South Africa is a low to middle income country with a heavy burden of infectious diseases, particularly Human Immunodeficiency virus (HIV) and tuberculosis. As the diagnosis of TBM based on culture confirmation can take weeks, clinicians rely heavily on CSF ADA as a rule-in test, because of its fast turn-around time. However, as indicated in Table In view of the limitations described above, the objective of this study was to determine an appropriate CSF ADA cut-off level in a quaternary healthcare setting in South Africa to assist in the diagnosis of TBM.A retrospective study was conducted over a period of two years (2009 and 2010) in which 3495 medical records were reviewed. As some patients had more than one admission the final number of records eligible for analysis was 3548  presence of a positive rapid test as well as a positive enzyme-linked immunosorbent assay (ELISA), (2) presence of a positive initial and confirmatory ELISA, and (3) presence of a positive polymerase chain reaction (PCR) for HIV . In addi6/L was used to further classify patients according to the 2010 World Health Organisation (WHO) guidelines regarding treatment for HIV [A CD4 absolute cell count of 350 x 10 for HIV .confirmed infective categories) according to definitive evidence . For a specific breakdown of the cases considered for the ‘Confirmed TBM’ category, please see Table suspected infective categories) according to the evidence as documented by clinicians in patient files. In cases when CSF ADA was requested as part of a diagnostic work-up, but a non-infectious diagnosis was ultimately made by the clinician, a review of clinical, radiological and histological evidence was performed to exclude the possibility of TBM. The ‘Other’ category includes pathologies not involving CSF or intracranial anatomy. The ‘No diagnosis made’ category includes patients where no clinical, radiological or laboratory evidence was found to assign patients to any of the other categories. CSF ADA performance was assessed in all these categories  for the measurement of total ADA was used and performed on a Pentra analyser  with colorimetric detection. The clinical decision limit for TBM used at this healthcare facility was 6 U/L as determined in a South African study by Blake and Berman [The median, interquartile range (IQR), the 95% distribution of values and outliers for the CSF ADA results for each of the categories defined for this study were determined.Mycobacterium tuberculosis complex (TB) on CSF was either not requested or negative, i.e. on the total population for this study (n = 3548). Using the Mann–Whitney test, CSF ADA results in the confirmed infective categories were compared between ‘Confirmed viral meningitis/encephalitis’, ‘Confirmed ventriculitis’, ‘Confirmed bacterial meningitis’ and ‘Confirmed TBM’ groups.All CSF ADA test results for the demographic categories were compared using the Mann–Whitney test for non-parametric data . Similarp-value of < 0.05 was considered statistically significant.Receiver operating characteristic (ROC) curves and the Youden index were generated to establish the optimal cut-off point for ADA interpretation in patients for whom a CSF specimen was submitted for both mycobacterial culture and ADA determination . Using tn = 1490) was arbitrarily split into a training and validation cohort after random assignment of values to each of the CSF ADA results to assess for overfitting of the model. The ROC curve and Youden index was generated for the training set. The CSF ADA cut-off point determined was applied to the validation set. The same diagnostic accuracy parameters were determined for the validation cohort as above.The dataset  was used to compare the individual ROC curves .CSF sample integrity as determined by its appearance  was used to re-categorise CSF ADA results and these categories were compared by establishing the minimum, median, and maximum values as well as the interquartile ranges (IQRs).A uniform case definition for diagnosis of TBM as determined by Marais and colleagues was used to re-categorise patients and CSF ADA results were assessed in each of the categories . The CSFStatistical analyses were performed on Analyse-it .n = 3170, 89.3%), of which 2009 individuals (56.6%) were between the ages of 20 and 40 years. Male and female patients were roughly equal in number . Most patients were HIV-positive . Of the total population, CD4 cell counts for 1456 (41.0%) patients measured ≤ 350 x 106/L. Table n = 3548).The majority of the patients in this study were adults (n = 3548) follow a non-parametric distribution. For the majority of results (n = 3415) CSF ADA values were less than 10 U/L. The CSF ADA results in Fig. The performance of CSF ADA measurement in each of the infective and non-infective categories is shown in Fig. p < 0.02), higher in male than female patients (p < 0.01), higher in HIV-positive than HIV-negative patients (p < 0.01) and higher in patients with CD4 cell counts ≤ 350 × 106/L as opposed to patients with CD4 cell counts > 350 × 106/L (p < 0.01).In all comparisons of CSF ADA results were higher in adult than paediatric patients (n = 92) are compared to all CSF ADA results for patients where TB culture on CSF was either absent or negative (n = 3456). Similar statistically significant results (p < 0.01) were obtained from the sub-set analysis of medical records (n = 1490) where both culture and ADA was requested on a CSF specimen.In Table p < 0.01). This however was not the case in the comparison between the ‘Confirmed bacterial meningitis’ and ‘Confirmed TBM’ categories (p = 0.06) and the ‘Confirmed ventriculitis’ and ‘Confirmed TBM’ categories (p = 0.42).Individually all CSF ADA results in the ‘Confirmed cryptococcal meningitis’, ‘Confirmed bacterial meningitis’, ‘Confirmed viral meningitis/encephalitis’ and ‘Confirmed ventriculitis’ categories were compared with the ‘Confirmed TBM’ category (see Table n = 1490) and an AUC of 0.86  was obtained. This analysis together with the Youden index indicates that the optimal cut-off of CSF ADA in suspected TBM patients is 2.0 U/L with a sensitivity of 85.9% (95% CI of 77.0 – 92.3) and specificity of 77.7% (95% CI of 75.4 – 79.8%). The positive and negative likelihood ratios were 3.85 and 0.18. It is important to note that, even at this cut-off, 13 of 92 ‘Confirmed TBM’ cases (14.1%) were missed . For this curve the Youden index determined the appropriate CSF ADA cut-off to be 2.20 U/L. When this cut-off was applied to the validation cohort, the sensitivity, specificity, positive and negative likelihood ratios were 87.5% (95% CI of 74.8 – 95.3%), 77.9% (95% CI of 74.6 – 80.9%), 3.96 and 0.16 respectively.Training and validation cohorts where determined for the dataset In sub-set ROC analyses performed for each of the 8 categories listed in Table n = 3548) were re-categorised according to their integrity and are plotted in Fig. n = 3003, 84.6%). Of note is the considerable spread of CSF ADA results for the ‘Ungraded xanthochromia’ category compared to the ‘Clear and colourless’ category, both in terms of outliers and 95% distribution of values. When excluding all specimens that were not clear and colourless , no change was noted in the ROC curve analysis and the same cut-off of 2.0 U/L using the Youden index was determined. Furthermore the overlap in CSF ADA results for the different categories remained.All CSF ADA requests n = 358 were ren = 1024) of cases that had both a CSF ADA and a mycobacterial culture requested, fell in the ‘Not TBM’ category. Of these cases, 20.2% (n = 207) had CSF ADA results ≥ 2 U/L and 6.2% (n = 63) had CSF ADA results ≥ 6 U/L  and may have skewed the ROC curve analysis. Furthermore, pre-analytical and analytical factors may also have contributed. Similar results were obtained when arbitrarily splitting the dataset (n = 1490) in a training cohort and a validation cohort. Overfitting of the model is unlikely to have occurred.An unusually low cut-off for CSF ADA of 2.0 U/L was determined. This contrasts with the first South African study on the use of CSF ADA that determined a cut-off of 6.0 U/L to indicate probable TBM [It is important to also note than when confirmed TBM ADA results were compared with all ADA results for patients where TB culture on CSF was either not requested or negative, a statistically significant difference was determined Table . HoweverIn clinical practice CSF ADA is used for rule-in purposes. If CSF ADA is used for rule-in/screening purposes the sensitivity should be optimized, but this may result in the cut-off of 2.0 U/L being dropped even further in the current data set. If ADA is used for rule-out purposes the specificity should be optimized, resulting in an elevation of the cut-off with a considerable loss of sensitivity , the absence of a statistically significant difference between the ‘Confirmed bacterial meningitis’ and ‘Confirmed TBM’ categories concurred with findings of a number of studies , 20, 21.6/L as intuitively it is assumed that cellular responses are impaired in these patients. Co-morbidities may account for this. Information on treatment for HIV was not collected. It is assumed that if cases were on treatment immune reconstitution would be seen, hence elevated values. Although these differences were statistically significant (p < 0.05), in all cases the median CSF ADA values were less than 1.0 U/L.In the 3548 CSF ADA requests reviewed, results for the categories according to demography, HIV status and CD4 cell count yielded a statistically significant difference for all. Statistically significant differences in ADA results in adult and paediatric patients are in keeping with the work by Donald and colleagues . It has Several pre-analytical and analytical factors limit adequate interpretation of the results from this study. No specific specimen collection and handling procedures are noted in the Diazyme kit package insert  regarding CSF samples. There are no guidelines regarding the stability of ADA specifically in CSF samples. It is unclear what the correct specimen volume should be for optimal assay performance. Feres and colleagues mention in their validation of the Diazyme assay for measurement of ADA  in CSF that 22 μL was used . This isIt is important to note the effect of sample integrity on ADA. ‘Ungraded xanthochromia’ accounted for 81 specimens and showed the widest distribution of 95% of their values. Both this category and samples assigned xanthochromia +/− to 2+ (accounting for 52 or 1.5% of samples) showed a wide scatter in their outliers, similar to the category for clear and colourless samples (accounting for 3003 or 84.6% of samples). Ungerer and colleagues demonstrated that erythrocytes contribute 100% of ADA-1 (ADA isoform 1) and polymorphonuclear cells contribute 70% ADA-1 and 30% ADA-1 + CP (two ADA isoform 1 molecules connected by a combining protein) to the total ADA activity in cell extracts . This man = 1024, 68.7%) whom had a CSF ADA and mycobacterial culture requested, fell in the ‘Not TBM’ category. By applying the CSF ADA cut-off of 2.0 U/L, about a fifth of these cases  would have been treated if the decision to treat was based on the CSF ADA alone. This is about three times more than the number of patients that would have been treated using a cut-off of 6.0 U/L . Either way, irrespective of whether a cut-off of 2.0U/L or 6.0 U/L is used, a considerable number of patients would have received unnecessary treatment. The uniform case definition was established after extensive literature review and international expert consensus [Using a uniform case definition for TBM , by weigonsensus . AlthougThe measurement of ADA in general is complicated by poor standardisation . Giusti No consensus exists regarding the linear range of this assay. The calibration curve for the Diazyme assay  lies between 0 – 50 U/L. However, the package insert for the Diazyme assay  states that dilution should occur for samples with results > 200 U/L. Delacour, et al., (2010) performed a linearity study and determined the analytical measurement range to be 0.5 – 120 U/L .Ideally control material should reflect analyte levels at which clinical decisions are made. Currently this is true for pericardial, peritoneal and pleural fluids, but not for CSF.The imprecision of the Diazyme assay  at the proposed cut-off of 2.0 U/L is unknown. Data from our validation studies at the lower control level of 10.6 U/L showed a within-run standard deviation of 0.172 U/L, total standard deviation of 0.601 U/L, and the total coefficient of variation of 5.8%. As the allowable coefficient of variation was 7.0%, this performance was deemed to be acceptable. It is possible that the imprecision of the assay is poor at a cut-off of 2.0 U/L. This would require further investigation.The retrospective analysis of clinical and laboratory data constituted a further limitation of this study. While every effort was made to find all data parameters relevant to the study, this was not always possible due to incomplete data in patient files and on the laboratory information system. This may have affected the categorisation process. Reflex testing of CSF ADA may have occurred. Many of the controls will have had a high pretest clinical probability of other diagnoses. In view of the fact that submission of insufficient CSF volume impacts on the sensitivity of TB culture, it is possible that microbiological confirmation of TBM was missed in some cases, as less than 10% of the 1490 patients who had a culture and ADA requested on a CSF specimen had more than one specimen submitted. As the majority of the population studied were HIV positive and for a quarter the HIV status was not known, these results cannot be extrapolated to non-HIV populations. This requires further investigation.The authors cannot recommend CSF ADA as either a rule-in or a rule-out test. A clinical appropriate cut-off could not be determined, as at a cut-off of 2.0 U/L still 13 out of 92 cases of ‘Confirmed TBM’ were missed. Several pre-analytic and analytical factors affect interpretation of CSF ADA results. No definite conclusions can be reached regarding CSF ADA cut-off levels, without fully understanding these factors. The effect of sample integrity cannot be underestimated and there is a pressing need for standardisation of CSF ADA measurement. CSF culture remains the gold standard for the diagnosis of TBM. Its poor performance overall, however, makes it a problematic gold standard to which CSF ADA is compared to and a replacement is needed. The data from this study show that the CSF ADA result may potentially mislead clinicians in the diagnostic process. As such careful assessment and weighting of all the available evidence should prove more fruitful . A futur


Electromyography systems are widely used within the field of scientific and clinical practices. The reliability of these systems are paramount when conducting research. The reliability of Myon 320 Surface Electromyography System is yet to be determined. This study aims to determine the intra-session and inter-day reliability of the Myon 320 Surface Electromyography System. Muscle activity from fifteen participants was measured at the anterior deltoid muscle during a bilateral front raise exercise, the vastus lateralis muscle during a squat exercise and the extensor carpi radialis brevis (ECRB) muscle during an isometric handgrip task. Intra-session and inter-day reliability was calculated by intraclass correlation coefficient, standard error of measurement and coefficient of variation (CV). The normalized root mean squared (RMS) surface electromyographic signals produced good intra-session and inter-day testing intraclass correlation coefficient values (range: 0.63–0.97) together with low standard error of measurement (range: 1.49–2.32) and CV  measures for the dynamic-and-isometric contractions. The findings indicate that the Myon 320 Surface Electromyography System produces good to fair reliability when examining intra-session and inter-day reliability. Findings of the study provide evidence of the reliability of electromyography between trials which is essential during clinical testing. Electromyography (EMG) is the study of electrical activity produced by skeletal muscles. EMG analysis has become an important tool in many areas of scientific and clinical research   measures .Fifteen healthy male participants , who were physically active, with no history of knee, hip or shoulder surgery or neuromuscular conditions volunteered for this study. Participants were asked to refrain from physical activity 24 h prior to taking part in the experiment in order to avoid the effects of cumulative muscular fatigue. All participants completed a physical readiness questionnaire and consent form before participating in the study. Ethical approval was granted by the University of the West of Scotland, School of Science and Sport Ethics Committee.Participants were required to attend the laboratory on two separate occasions. The length between each of the trials was required to be greater than 2 days but no longer than 10 days. At the first visit to the laboratory the participants were familiarized with the environment and the exercises prior to data collection. All visits were performed at the same time of day to minimize the effects of diurnal variation and any variation of the procedure. Experimental data preparation and collection was performed by the same researcher to eliminate researcher variation. The order in which the exercises were performed was randomized for all testing conditions.The sEMG activity was recorded using surface electrodes  and a set of 6 Surface EMG Transmitters . Prior to the sEMG data collection for the dynamic and isometric contractions, the skin was prepared by hair removal from the tested area, as well as skin abrasion and alcohol cleaning. This skin preparation procedure is essential in order to reduce the impedance of the interface between the skin and electrode. Pairs of sEMG electrodes were attached to the skin no more than 2 cm apart (center to center) over the dominant side of the anterior deltoid (AD) and vastus lateralis (VL) and extensor carpi radialis brevis (ECRB) muscles Figure . To stanFor the dynamic contractions, two separate movement patterns were assessed: one for the upper and one for the lower extremity. For the upper extremity, a bilateral front raise, the lifting of an object in front of the body, exercise was performed with sEMG electrodes placed on the right AD muscle. All participants completed the bilateral front raise exercise with a calibrated 10 kg Taishan bumper plate weight . To execute the exercise, and standardize procedures, participants were instructed to stand with their feet shoulder width apart, holding the bumper plate with both hands around the waist line. From this position, participants raised the arms up in front of the body until the weight was directly above the head, with only a slight bend in the elbows, which was maintained throughout the movement. The shoulder at this stage of the exercise was required to be between 170 and 190° anterior to the body. The weight was then returned to the start position. Three trials of the front raise exercise were performed, with each trial consisting of three repetitions. Each of the three repetitions was performed at a rate of 4 s for the concentric phase and 4 s for the eccentric phase of the exercise, lasting a total of 24 s. This timing sequence was regulated through an interval timer, which enabled participants to move at a constant pace over the three trials, therefore making the movements more reliable. Between each trial, participants rested for 5 min to limit the effect of muscular fatigue. Retro-reflective markers were applied to the shoulder and hip area. This enabled the researchers to identify joint angles required to complete the movement.For the lower extremity, sEMG data was collected from the right VL muscle during the unloaded squat exercise. During the squat, participants were instructed to have their feet shoulder width apart, whilst looking straight ahead. They were then asked to flex their knees between 100° and 80°, before returning to full knee extension, keeping their back as straight as possible. Three trials of the squat exercise were performed, with each trial consisting of three repetitions. The timing sequence as detailed above for the front raise exercise was implemented for the squat exercise, with the 5 min rest period between trials. Retro-reflective markers were applied to the hip, knee and ankle joints to enable the researchers to identify joint angles at the start and end of the exercise.Isometric contractions were performed via three sub-MVC recordings from the right ECRB forearm muscle during a handgrip strength test. Following electrode placement and signals being verified, participants were seated with their right arm firmly strapped into the previously discussed experimental rig. Grip strength was recorded with a handheld dynamometer . Firstly, participants were asked to perform two MVICs in order to normalize the sEMG data. Fifty percent of the greatest MVIC reading for the handheld dynamometer was selected for the three reproducibility trials. Participants had to build up to sub-MVCs in 3 s and then hold it for a further 3 s . Two 5 s MVICs were performed for each of the three muscles tested in the following positions; VL while the back was against the wall with 90° of knee flexion, AD while holding a 10 kg weight anterior to the body and shoulder flexed at 90°, and ECRB while seated with the right arm firmly strapped into a previously validated rig (unpublished data). In accordance with Hashemi Oskouei et al. , the rigAll sEMG data was sampled at 1,000 Hz. During the processing procedures, all sEMG data was digitally filtered (20–400 Hz) in order to reduce transients and instrumentational noise and root mean squared (RMS) values calculated. For MVIC recordings, the maximum 1 s value across the 2 MVIC recordings for all muscles was identified and selected in order to normalize the bilateral front raise, squat and handgrip exercises. For the dynamic contractions, an RMS time window of 50 ms was employed. For the bilateral front raise exercises, the total duration of the movement was averaged and analyzed for reproducibility between the three trials. The identical procedure was also carried out for analysis of the squat exercise. In order for the researcher to analyse the dynamic exercises, kinematic data was recorded through the Vicon Bonita Motion System , sampling at a rate of 250 Hz. For the sub-MVIC handgrip test, an RMS time window of 100 ms was used and the 3 s 50% contraction was averaged to determine reproducibility of the three trials.A two way random effects model with single and average ICC measures, with a 95% confidence interval, was used to measure the repeatability of the average normalized RMS sEMG signal during the intra-session testing. Inter-session reliability  was determined by comparing the average normalized RMS sEMG muscle activity for the three trials for each exercise of both testing sessions. ICC, CV and SEM were obtained using the Statistical Package of Social Sciences (SPSS V 22.0). ICC was categorized as follows: good reliability: 0.80–1.00; fair reliability: 0.60–0.79; poor reliability: <0.60  of their MVIC value.The average normalized RMS sEMG data between participants from the AD muscle over the three sub-MVC trials of the bilateral front raise exercise displayed good within-day reliability [ICC  = 0.97] and an acceptable CV of 4.73% (95% CI = 1.35–9.79). The average muscle activation between participants was 66.05% ± 20.15 for the sub-MVC bilateral front raise exercise. SEM between participants was 2.06. Inter-day reliability for the average normalized RMS sEMG for the AD during the bilateral front raise exercise produced good reliability [ICC  = 0.94] and an acceptable CV of 3.86% (95% CI = 0.82–7.46). The average muscle activation for inter-day testing between participants was 65.85% ± 18.51 for the sub-MVC front raise exercise. The SEM between participants during inter-day testing was 1.49.For the squat exercise, the average normalized RMS sEMG data from the VL muscle over the three sub-MVC trials displayed good within-day reliability [ICC  = 0.95] and an acceptable CV of 5.73% (95% CI = 1.48–8.94). The average muscle activation during intra-day testing between participants was 67.87% ± 21.25 for the sub-MVC squat exercise. SEM between participants was 2.32. Inter-day reliability for the average normalized RMS sEMG from the squat exercise produced good reliability [ICC  = 0.93] and an acceptable CV of 4.77% (95% CI = 1.62–7.52). The average muscle activation for inter-day testing between participants was 67.10% ± 20.63 for the sub-MVC squat exercise. The SEM between participants during inter-day testing was 1.84.For the isometric handgrip test the average normalized RMS sEMG data from the ECRB forearm muscle over the three trials displayed good within-day reliability [ICC  = 0.87] and an acceptable CV of 5.89% (95% CI = 0.36–12.36). The average muscle activation between participants was 45.98% ± 8.82 for the handgrip test. SEM between participants was 1.57. On the other hand, inter-day relative reliability was fair during single isometric contractions [ICC  = 0.63]. CV also increased to 7.18% (95% CI = 3.40–12.71). The average muscle activation for inter-day testing between participants was 45.91% ± 8.09 for the sub-MVIC handgrip test. The SEM between participants during inter-day testing for the isometric contraction was 1.93.This is the first study to assess the reliability of the Myon 320 sEMG system during low velocity controlled movements, such as those routinely used in rehabilitation. The researchers investigated intra-session and inter-day reliability during sub-maximal dynamic and isometric contractions while recording sEMG measurements using the Myon 320 sEMG System. The main findings were that the Myon 320 sEMG System displayed good reliability associated with normalized RMS sEMG measures (ICC > 0.80) for intra-session and inter-day testing during dynamic sub-MVC. During 50% MVIC contractions the Myon 320 sEMG System produced good intra-session repeated measures (ICC > 0.80) and fair inter-day measures (ICC 0.60–0.79). The normalized RMS sEMG within the group of participants in the study displayed a strong correlation with the 50% MVIC during the intra (45.98%) and inter-day (45.91%) testing.The high intra-session ICC for the normalized RMS sEMG signal during the bilateral front raise and squat exercises presented in the current study is consistent with previously published literature . The literature for inter-session reliability is somewhat contrasting to the findings of the current study. Worrell et al.  reportedWith regards to isometric contractions, the good ICC (0.87) values for the normalized sEMG RMS data during intra-session testing in the current study is consistent with previously published research (Dankaerts et al., With regards to inter-session reliability during isometric contractions in this study, it would appear that reapplying the electrodes on a subsequent day reduces the repeatability of the normalized RMS sEMG signal. These findings are in agreement with previous published literature (Hashemi Oskouei et al., When using the Myon 320 sEMG System, the present study shows that it is possible to obtain good reliability for normalized RMS sEMG during intra-session and inter-day testing during dynamic sub-MVC, when exercises are performed at low velocities. This study also highlights the fair reproducibility of the normalized RMS sEMG from the extensor muscles of the forearm during a handgrip task during inter-session testing, which is in agreement with previously published literature. Therefore, the current study demonstrates that the Myon 320 sEMG System is a reliable sEMG measurement tool, for low velocity controlled movements.All authors contributed to the development of this manuscript. UU, GS, and CE were involved in the experimental design. GS, MW, DB, and AG were involved in the data collection. UU, GS, MW, DB, and AG were involved with the data processing and analyses. UU, GS, CE, MW, JB, JSB, NG were involved in the writing and proof reading of the manuscript.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.


In this cohort, NT-proBNP changes could not be demonstrated to be related to anthracycline or trastuzumab treatment. Pharmacodynamic models for troponin T and LVEF were successfully developed, identifying maximum troponin T concentration after anthracycline treatment as a significant determinant for trastuzumab-induced LVEF decline. These models can help identify patients at risk of drug-induced cardiotoxicity and optimize cardiac monitoring strategies.Trastuzumab is associated with cardiotoxicity, manifesting as a decrease of the left-ventricular ejection fraction (LVEF). Administration of anthracyclines prior to trastuzumab increases risk of cardiotoxicity. High-sensitive troponin T and N-terminal-pro-brain natriuretic peptide (NT-proBNP) are molecular markers that may allow earlier detection of drug-induced cardiotoxicity. In this analysis we aimed to quantify the kinetics and exposure–response relationships of LVEF, troponin T and NT-proBNP measurements, in patients receiving anthracycline and trastuzumab. Repeated measurements of LVEF, troponin T and NT-proBNP and dosing records of anthracyclines and trastuzumab were available from a previously published clinical trial. This trial included 206 evaluable patients with early breast cancer. Exposure to anthracycline and trastuzumab was simulated based on available dosing records and by using a kinetic-pharmacodynamic (K-PD) and a fixed pharmacokinetic (PK) model from literature, respectively. The change from baseline troponin T was described with a direct effect model, affected by simulated anthracycline concentrations, representing myocyte damage. The relationship between trastuzumab and LVEF was described by an indirect effect compartment model. The ECThe online version of this article (10.1007/s10928-018-9579-8) contains supplementary material, which is available to authorized users. The final model parameter estimates are summarized in Table Aant is the amount of anthracyclines, Ke the elimination rate constant, TRP is troponin T, TRP0 is troponin T at baseline, before starting anthracycline treatment, SLOPE is the parameter that describes the proportional increase of troponin T from baseline. The goodness of fit plots . The BSV for both EC50 and recovery could not be identified, therefore the BSV on recovery was not included in the final model. The BSV in baseline LVEF was moderately positively correlated with the EC50 parameter (r = 0.585), indicating that patients with a low baseline LVEF tend to have a higher sensitivity to trastuzumab-induced LVEF decline (lower EC50). The model described the data adequately, however a slight underprediction was seen for the lower LVEF values , leading to similar results. However, high relative increases of NT-proBNP, approximately 27 weeks after the lowest LVEF value, were observed in 4 patients with a significant decline in LVEF and in 3 patients with no significant decline in LVEF . NT-proBNP increment could, therefore, be a delayed effect of previous LVEF decline, a sign of other underlying cardiac disease or a forecast of development of congestive heart failure, since these patients did not report an increase in heart failure symptoms (NYHA) at the time of the peak. Therefore, NT-proBNP was not included in the LVEF model.SLOPE parameter (anthracycline-troponin T model) and on the EC50 and recovery half-life parameter (trastuzumab-LVEF model). No covariates were identified that significantly improved model fit of the anthracycline-troponin T model, except for type of anthracycline on the SLOPE parameter. The SLOPE-parameter, was significantly affected by the type of anthracycline administered, as illustrated by twofold lower estimate for epirubicin compared to doxorubicin (anthracycline-type effect estimate of 0.524 (relative standard error (RSE) 17.5%). This means that, for example, administration of one dose of 100 mg doxorubicine leads to an increase in troponin T from 3 to 5.6 ng/L, where an equivalent dose of epirubicin would increase troponin T from 3 to 4.3 ng/L. Considering the LVEF-trastuzumab model, the sensitivity for LVEF decline (EC50 parameter) was significantly affected by the predicted maximum concentration of troponin T after anthracycline treatment. Sensitivity for LVEF decline was higher (decreased EC50 parameter) for patients with a high maximum concentration of troponin T, resulting in a more pronounced decline in LVEF during trastuzumab treatment . The maximum troponin T concentration reduced the between subject variability in sensitivity for LVEF decline from 98.0% in the base model  to 82.9% in the covariate model. The sensitivity analysis demonstrated that the predicted concentration of troponin T at 21 days after the last anthracycline dose was an equally significant covariate on the EC50 parameter . The other tested covariates did not significantly improve the model.The covariates were evaluated on the The pharmacodynamics of troponin T and LVEF changes during anthracycline and trastuzumab treatment, respectively, were successfully described by the reported models. The maximum concentration of troponin T was a significant determinant of sensitivity to trastuzumab-induced cardiotoxicity, defined as a decline in LVEF values.SLOPE parameter, showing that epirubicin had an approximately twofold lower proportional effect on baseline troponin T concentrations compared to doxorubicin. This finding is expected, since at equivalent doses, epirubicin demonstrates less cardiotoxicity than doxorubicin. Moreover, the cumulative lifetime anthracycline dose threshold, associated with increased incidence of heart failure, is also almost a twofold higher for epirubicin (950 mg/m2) compared to doxorubicin (550 mg/m2) [Baseline troponin T concentrations were directly affected by anthracycline concentrations. The type of anthracycline significantly affected the linear irubicin 50 mg/m2 0 mg/m2) . The ant0 mg/m2) . In addi0 mg/m2) , 27.50 parameter, indicating that patients with a low LVEF baseline tend to be more sensitive to trastuzumab-induced LVEF decrease.The trastuzumab-LVEF model demonstrated a decline in LVEF values that improved after treatment cessation, demonstrated by a recovery of LVEF towards baseline. This analysis prospectively validated the model for LVEF developed in a previously published PK-PD analysis . A high Repeated NT-proBNP measurements were not integrated in the model, since NT-proBNP showed high variability. However, some patients experienced high relative increases of NT-proBNP, approximately 27 weeks after their nadir of the LVEF value. An increment in NT-proBNP could therefore possibly be a delayed effect of a prior decline in LVEF, which could be a sign of other cardiac comorbidities or a forecast of development of congestive heart failure. However, early time course data for NT-proBNP, during anthracycline treatment, was lacking for most patients, which could be a reason why NT-proBNP was not identified as an early cardiac biomarker in this cohort. Additionally, NT-proBNP baseline concentrations were not significantly related to anthracycline-induced troponin T increase nor to LVEF decline during trastuzumab treatment.50 and found not significantly different. In addition, no differences in parameter estimates or model fit were seen when the trastuzumab-LVEF model was evaluated for each treatment group separately.Age, cardiac history and interval between anthracycline treatment and initiation of trastuzumab treatment have been previously reported as risk factors for trastuzumab-induced cardiotoxicity . In addiThe biomarker models were not estimated simultaneously. However, anthracyclines induce troponin T release by damaging cardiac cells, where trastuzumab is hypothesized to cause functional changes in contractile proteins, not associated with cell damage or troponin T changes –7. ThereThe established models can help evaluate the feasibility of using a cardiac biomarker (e.g. troponin T) to identify patients at risk of trastuzumab-induced LVEF decline. However, clinical applicability is challenged by unstandardized analytical assays for determination of cardiac biomarkers and algorithms to calculate LVEF, definition of the optimal sampling time point, identification of a cut-off value and subsequently determination of a proper strategy in case of identification of an abnormal cardiac value. In addition, subclinical LV dysfunction can be estimated using alternative methods, such as determination of LV diastolic dysfunction and myocardial strain. These parameters give insight in early changes in LV remodeling and have been recommended to monitor patients treated with cardio-toxic anti-cancer drugs .Although prospective validation is warranted, the developed models can be applied to evaluate cardiac monitoring strategies, such as previously described for LVEF . SimulatIn conclusion, to our knowledge this is the first PK-PD analysis that integrated longitudinal data of two cardiac biomarkers during anthracycline and trastuzumab treatment. In this cohort, changes in NT-proBNP could not be demonstrated to be related or predictive of anthracycline- or trastuzumab-induced cardiotoxicity. The analysis identified maximum troponin T concentration after anthracycline treatment as a significant determinant of subsequent trastuzumab-induced LVEF decrease.Supplementary material 1 (DOCX 578 kb)Below is the link to the electronic supplementary material.


Drosophila melanogaster small nuclear heat shock protein DmHsp27 was investigated by mutagenesis using size exclusion chromatography and native gel electrophoresis. Mutation of two sites of phosphorylation in the N-terminal region, S58 and S75, did not affect the oligomerization equilibrium or the intracellular localization of DmHsp27 when transfected into mammalian cells. Deletion or mutation of specific residues within the NTR region delineated a motif (FGFG) important for the oligomeric structure and chaperone-like activity of this sHsp. While deletion of the full N-terminal region, resulted in total loss of chaperone-like activity, removal of the (FGFG) at position 29 to 32 or single mutation of F29A/Y, G30R and G32R enhanced oligomerization and chaperoning capacity under non-heat shock conditions in the insulin assay suggesting the importance of this site for chaperone activity. Unlike mammalian sHsps DmHsp27 heat activation leads to enhanced association of oligomers to form large structures of approximately 1100 kDa. A new mechanism of thermal activation for DmHsp27 is presented.The importance of the N-terminal region (NTR) in the oligomerization and chaperone-like activity of the  The small heat shock proteins (sHsps) are a ubiquitous family of ATP-independent stress proteins found in all domains of life –5. They Small Hsps contain a tripartite architecture composed of a conserved α-crystallin domain (ACD) flanked by variable N-terminal region (NTR) and C-terminal extension (CTE) –13. The Drosophila melanogaster DmHsp27, DmHsp26, CG14207, maize Hsp22 and yeast Hsp26 have been described [Phosphorylation of human sHsps has been reported to regulate their structure and function –27. Thesescribed –32. Howeescribed .Drosophila melanogaster Hsp27 (DmHsp27) is a nuclear-localized sHsp [zed sHsp –35. In azed sHsp . We prevIn addition to phosphorylation sites, the NTR contains some sequences that are conserved. Deletion of a conserved FGFG motif (position F29 to G32) results in formation of large oligomers and an increase in chaperone-like activity at non-stress (non-activating) temperature using insulin as a chaperone assay. We further determined the effects of single point mutations within the FGFG motif on oligomeric structure and chaperone-like activity. Mutation of phenylalanine 29 to an alanine or tyrosine (F29A or F29Y) and glycine 30 or 32 to arginine (G30R or G32R) affects oligomerization and display a better chaperone-like activity than the wild-type protein in non-heat shock condition using insulin. Surprisingly, heat activation of DmHsp27 leads to enhanced association to form large size oligomers of approximately 1100 kDa suggest a new mechanism of thermal activation for DmHsp27.The NTR of vertebrate and plant sHsps has previously been described as a determinant of chaperoning activity and substrate specificity , 18. At DmHsp27 is present in up to four isoforms according to the tissue and developmental stage . It has It has been reported that phosphorylation of human HspB1 and HspB5 is a mechanism for nuclear localization in unstressed cells . The rolMethanococcus sHsps shows high conservation of two residues (F29 and G30 in DmHsp27)  .We next examined the effect of these conserved residues within the NTR on the oligomeric structure and localization of DmHsp27. A deletion of the first 86 amino acids of the N-Terminal region (Del_Nter), had a striking effect on the oligomeric equilibrium, which showed a wide band (from 480 to 146 kDa) and one extra band in the region of dimer . This muTo further investigate residues that are important for the oligomeric structure and the chaperone-like activity of DmHsp27, we used blast similarity of NTR-DmHsp27. The obtained sequences all belong to insect sHsps. Alignment of these sequences helped to delineate a conserved sequence motif (FGXG) from phenylalanine 29 to glycine 32 in DmHsp27 . AnalysiSince deletion of residues FGFG (29–32) induces formation of a large oligomer, we investigated this region more carefully using single point mutations. Specifically, we focused on F29  and F31 (less conserved), two residues with large side chains. Phenylalanine in both positions was mutated to an alanine, a smaller amino acid (F29A and F31A), or to an amino acid (tyrosine) that mimicked the size of the original amino acid but altered the hydrophobicity (F29Y and F31Y). The highly conserved glycines G30 and G32 were mutated to alanine (G30A and G32A) and to a positively charged larger amino acid arginine (G30R and G32R). Mutations in F31 (F31A and F31Y)  or in G3The size estimation by native gel electrophoresis was confirmed using SEC for DmHsp27, F29A, F29Y, G30R and G32R mutants. As seen in As reported previously DmHsp27WT can prevent Luc, CS, and MDH heat-induced aggregation more efficiently than insulin DTT induced aggregation  and Fig It is possible that DmHsp27 binds more easily to Luc, CS and MDH than to the insulin substrate resulting in a stronger aggregation protection. Alternatively, heat activation of DmHsp27 could lead to a better protection. To test these hypotheses, we performed the insulin chaperone assay at 42°C. DmHsp27 and all mutants showed a high capacity to reduce insulin aggregation at 42°C compared to 20°C . At 20°CThe formation of large oligomers induced by elevated temperature are partially reversible as indicated by native gels  on at least 2 serines in NTR (S58 and S75) –32. HereUsing molecular dynamics simulations Patel et al.  showed tAlthough, NTR has been described as poorly conserved, we delineate a conserved motif phenylalanine 29 to glycine 32 (FGFG) in orthologues of DmHsp27. This motif is equivalent to the described “the phenylalanine-rich region” in vertebrate sHsps . A largePrevious studies on the structure of Hsp16.9 from wheat  and Hsp1Saccharomyces cerevisiae, in which the transition into a state of increased substrate binding affinity and chaperone activity occurs through slight conformational changes without perturbation of the oligomeric state [C. elegans Hsp17 forms large sheet-like super-molecular assemblies (SMA) at high temperatures and only the SMA form exhibits chaperone-like activity in suppressing the aggregation of non-native substrate proteins [There is significant controversy concerning the mode of activation of sHsps. sHsps reveal temperature-dependent chaperone activity in preventing aggregation of substrate proteins , 65. Mosic state . Anotheric state . HspB1 eic state . Hsp22  at KpnI and XhoI sites by PCR as described in [The cDNA of wild type DmHsp27 (DmHsp27WT) was cloned using GIBSON ASSEMBLY (NEB) into bacterial expression vector pETHSUK  and mamribed in . MutatioFollowing the detailed protocol in  pETHSUK The protein purification was accomplished by affinity chromatography using Ni-NTA agarose (Qiagen) column. His-Sumo-tag was digested with Sumo-Hydrolase followed by size exclusion chromatography (SEC) on Superose 6 10/300 column (GE Lifesciences) as described earlier . All mutSEC was used to analyze the quaternary structure of DmHsp27. 300μg of proteins were loaded on Superose 6 10/300 column (GE Lifesciences) equilibrated with 20 mM Tris-HCl pH 8, 150 mM NaCl. SEC was achieved at room temperature and eluted at 0.5 ml/min. For estimating the molecular weight, the column was calibrated with protein markers immunoglobulin M (IGM) from bovine serum (900 kDa) (Sigma), (thyroglobulin (669 kDa), Ferritin (440 kDa), Aldolase (158 kDa), Conalbumin (75 kDa), Ovalbumin (43 kDa) and Blue Dextran 2000 to determine the void volume) (GE Lifesciences) as detailed in .For native gel electrophoresis samples were kept at room temperature (20°C) or heated at 42°C using a water bath during 10 min and loaded on a gel 4–12% gradient native Tris-Glycine gels (Thermo Fisher Scientific). Gels were run at 150 V at room temperature (20°C) or at 42°C (on a water bath) using pre-cast Mini-Cell electrophoresis system . The protein complexes were stained with Coomassie blue immediately after electrophoresis.Luciferase, citrate synthase, L-malate dehydrogenase and insulin were used as substrates to evaluate the chaperone-like activity as as described in Morrow et al 2006  and MoutThe heat-induced aggregation assay was performed using: luciferase  at 42°C, citrate synthase  and L-malate dehydrogenase  at 45°C in the absence or presence of DmHsp27 or its mutants. Altough, insulin  non-Heat-induced aggregation was induced by disulfide bonds reduction at 20°C. All substrats were pre-incubated alone or in the presence of DmHsp27 or its mutants and aggregation was followed by an increase in the optical density at 320 nm on a spectrophotometer with thermostated cells.(+)-DmHsp27:Lipofectamine (Invitrogen) complex (1.5 μl Lipofectamine/1.5 μg DNA), cells were washed with culture medium and incubated for 48 h to express DmHsp27 before immunofluorescence.Hela cells were maintained in MEM Alpha (Gibco) supplemented with 5% FBS. Cells were plated in advance at a confluence of 175 000 cells/well (6 well plate) containing glass coverslip for transfection. Later, cells were incubated for 4 h in OptiMEM (Gibco) containing the plasmid pcDNA3.1Immunofluorescence was performed as described in , 78. BriS1 FigNative gradient (4–12%) polyacrylamide gel electrophoresis of recombinant DmHsp27 and NTR mutants heated at 42°C for 1h and cooled back at 20°C for 30 min (A) or 2h (B). Positions of standard protein markers with known molecular weights are shown on the left.(TIF)Click here for additional data file.


Annual hibernation is an adaptation that helps many animals conserve energy during food shortage in winter. This natural cycle is also accompanied by a remodeling of the intestinal immune system, which is an aspect of host biology that is both influenced by, and can itself influence, the microbiota. In amphibians, the bacteria in the intestinal tract show a drop in bacterial counts. The proportion of pathogenic bacteria is greater in hibernating frogs than that found in nonhibernating frogs. This suggests that some intestinal gut microbes in amphibians can be maintained and may contribute to the functions in this closed ecosystem during hibernation. However, these results were derived from culture-based approaches that only covered a small portion of bacteria in the intestinal tract.In this study, we use a more comprehensive analysis, including bacterial appearance and functional prediction, to reveal the global changes in gut microbiota during artificial hibernation via high-throughput sequencing technology.Polypedates megacephalus) could reduce microbial diversity, and artificially hibernating frogs tend to harbor core operational taxonomic units that are rarely distributed among nonhibernating frogs. In addition, artificial hibernation increased significantly the relative abundance of the red-leg syndrome-related pathogenic genus Citrobacter. Furthermore, functional predictions via PICRUSt and Tax4Fun suggested that artificial hibernation has effects on metabolism, disease, signal transduction, bacterial infection, and primary immunodeficiency.Our results suggest that artificial hibernation in the brown tree frog  contains supplementary material, which is available to authorized users. Lactobacillus bacteria and may diminish the prevalence of allergic disease. In communities in which consumption of fermented foods is high and antibiotics are not used, causes of allergy and asthma are low. These studies suggest that changes in diet and associated changes in the gut microbiota are driving the increasing incidence of inflammatory disease.The gut mucosal immune system forms the largest vertebrate immune compartment . It is wAkkermansia muciniphila, of the phylum Verrucomicrobia, is increased during hibernation in the Syrian hamster and ground squirrel. However, in larger animals, such as free-ranging brown bears, Verrucomicrobia, including Akkermansia muciniphila, are decreased during hibernation. The gut microbiota turnover of hibernators can further modulate metabolic functions. For example, the molar proportion of acetate ions rose during hibernation, which may be due to the activity of mucolytic bacteria, such as Akkermansia muciniphila, that convert mucins to acetate ions [Akkermansia muciniphila has been studied to associate with the mucus layer and is able to grow on mucin as its sole carbon and nitrogen source [Bacteroides fragilis is the predominant bacterium in the microbiota from the hibernating bear, whereas both Streptococcus and TM7 are reduced during hibernation. Furthermore, two studies of calorie-restricted mice reported an increase in Bacteroides fragilis [Streptococcaceae and TM7 [Hibernators, such as the Syrian hamster, ground squirrel, and brown bear, have been shown to restructure gut microbiota during hibernation –9. Slow ate ions . Akkerman source –12. Othefragilis  but decr and TM7 . Togethe and TM7 .Rana pipiens) and bullfrogs also led to a drop in bacterial counts and a change in the composition of gut microbiota [Rana catesbiana) [In amphibians, studies have shown that artificial hibernation of northern leopard frogs  , which oesbiana) , 20. Caresbiana)  also fouesbiana) , 22, sugAmplicon-based sequencing of marker genes is widely used for large-scale studies that involve many different sampling sites or time series. The conventional 16S rRNA gene-based analysis is a powerful tool for assessing microbial composition, but does not provide insight into the metabolic potential in the microbial communities. Therefore, the prediction of the functional capabilities of a microbial community based on marker gene data would be highly beneficial. In this study, we use high-throughput sequencing technology to provide a comprehensive analysis to characterize the shift of gut microbes before and after artificial hibernation. We first distinguish the difference of dominant gut microbes between artificially hibernating frogs (AH frogs) and nonhibernating frogs (NH frogs). In addition, the relative abundances of both potentially pathogenic facultative and anaerobic bacteria were characterized. Furthermore, we predicted the gene content of a microbial community from a marker gene survey via PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states)  and Tax4NH frogs were collected from Wazihwei Nature Reserve  and private botanic gardens  in the wild (snout–vent length (SVL), 2.8–8.3 cm), where the population of brown tree frogs is widely distributed in Taiwan. Individuals were collected in three distinct seasons  were measured Table . Fecal cFor NH frogs, we used barcoding pyrosequencing to determine microbial community composition. The primer set was 515 F/806R, which targets the V4 region of the 16S ribosomal RNA gene, found to be well suited to the phylogenetic analysis of pyrosequencing reads . ConsideFor AH frogs, we used Illumina pair-end sequencing for microbial 16S rRNA gene amplicon. The primer set was 515 F/806R, which was the same as for NH frogs. All sequences were analyzed via the Quantitative Insights Into Microbial Ecology (QIIME) pipeline . SequencBray–Curtis dissimilarity distances were calculated among individual samples to determine the differences in bacterial community composition across individuals . Beta-dit-test was used to compare the abundance changes between NH and AH frogs. Only differences for which p-value < 0.05 are reported.To examine the relationship of gut microbiota between NH and AH frogs, Venn diagrams were created using the R package to visualize the OTUs that were shared between NH and AH frogs. Student’s Bacteroides, Clostridium, Eubacterium, Fusobacterium, Peptococcus, Peptostreptococcus, Propionibacterium, and Ruminococcus, and facultative genera included Azotobacter, Bacillus, Corynebacterium, Enterococcus, Flavobacterium, Lactobacillus, Pseudomonas, and Streptococcus. There are several pathogenic bacteria associated with red-leg syndrome (RLS)–one of the main infectious diseases that affects amphibians and causes high mortality [Aeromonas hydrophila, Citrobacter freundii, Chryseobacterium indolgenes, Edwardsiella tarda, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Streptococcus iniae [To reveal the potential pathogenicity, several bacteria were selected for comparison. For example, facultative and anaerobic bacteria that were found in amphibians as shown in Banas et al. , in brieortality . The etius iniae –43.p-value < 0.05 are reported.We implemented both PICRUSt and Tax4Fun to predict the functional shifts in AH frogs. The PICRUSt approach was proposed to predict KEGG Ortholog (KO) functional profiles of microbial communities using 16S rRNA gene sequences . This alt-test, p-value < 0.05).Our sequencing reads resulted in an average of 21,289 ± 6300 high-quality sequences per sample for the colonic samples in NH and AH frogs. The observed OTUs of all AH frogs are fewer than those in NH frogs . However, Firmicutes were significantly more abundant in NH frogs than in AH frogs . In addition, we compared the compositional similarity between AH and NH frogs by calculating the pairwise distance among OTU abundance  suggested no significant functional differences within NH frogs . However, we found most predicted functional categories in the KEGG pathways are significantly change in AH frogs, compared with NH frogs , bighead carp (53% recovery rate), mud carp, and large-mouth bass [L. hongkongensis was also highly isolated (80% recovery rate) in amphibians by pulsed-field gel electrophoresis [L. hongkongensis was present but rare in brown tree frogs, suggesting that the brown tree frog might be able to resist this pathogen.An interesting case is nteritis –63. L. hong Kong . In the uth bass , 66. In phoresis . This evHibernation is associated with a dramatic remodeling of many intestinal functions, such as energy and intestinal immune system, in both small and large hibernators. For example, an enrichment of Bacteroidetes and lower relative abundance of Firmicutes has previously been observed in the microbiota of hibernators , 68, 69.Akkermansia. However, the functional predictions via PICRUSt and Tax4Fun showed that AH frogs not only significantly increase KEGG pathways in infectious disease, but also significantly decrease signal transduction. Immune changes are often associated with altered host–microbe signaling [In addition, recent studies showed that seasonal reorganization of the microbiota is a major driver of the immune alterations because the immune system is the primary sensor of gut microbes and their metabolites , 73. Mucignaling , 77–79, ignaling . TherefoCitrobacter and Pseudomonas. Functional predictions via PICRUSt and Tax4Fun infer that AH frogs change pathways corresponding to the genes for metabolism, organismal system, information processing, and disease. We infer that artificial hibernation may impose potential effects on bacterial invasion and primary immunodeficiency.Artificial hibernation in the brown tree frog reduced microbial diversity and levels of Firmicutes in the intestinal tracts. AH frogs tend to harbor core OTUs that are rarely distributed among NH frogs. Artificial hibernation also increased the relative abundance of RLS-related pathogens, such as