CSF and serum factors as biomarkers that may aid in distinguishing ALS patients from control subjects and predicting ALS progression as well as prognosis.The aim of this study was to identify CSF samples from 105 patients with ALS and 56 control subjects were analyzed for 13 factors using ELISA. The revised ALS functional rating scale (ALSFRS‐r) was used to evaluate the overall functional status of ALS patients, and we also followed up with ALS patients either by phone or with clinic visits for five years after enrollment in this study. Finally, we examined the correlations between factor levels and various clinical parameters and evaluated the predictive value for prognosis through a multivariate statistic model.Serum and CSF, and twelve markers were increased in serum. In the correlation analyses, there were trends toward higher bFGF, VEGF, MIP‐1α levels in ALS with a longer disease duration and slower disease progression in both CSF and serum. Higher MCP‐1 levels were associated with worse disease severity and faster progression, and the IFN‐γ levels were positively associated with disease progression in either CSF or serum. Finally, a better prognosis was observed with higher levels bFGF in CSF and VEGF in CSF and serum; conversely, patients with higher levels of IFN‐γ in the CSF had shorter overall survival.A total of eight factors were obviously elevated in ALS patients is distinct from control subjects and may be useful in clinical practice and therapeutic trials.We demonstrated that a factor profile of  However, we excluded controls with any life‐threatening disease or who were taking any medicine. The demographic features, including age, gender, and body mass index (BMI), did not show significant diversity between the ALS patients and control subjects (p > .05). The demographic and clinical characteristics are summarized in Table This study was approved by the Clinical Experimentation Committee of Human of Tianjin First Center Hospital 2014022S). All the participants in the study provided written informed consent prior to enrollment. A total of 105 patients with sporadic ALS and 56 control subjects evaluated in the neurological wards of Tianjin First Center Hospital between April 2006 and June 2015 were enrolled in the study. Eligible patients were diagnosed by experienced neurologists according to revised E1 Escorial criteria , IL‐6 , IL‐10 , IL‐15 , IL‐17 , G‐CSF , GM‐CSF , bFGF , VEGF , MIP‐1α , MIP‐1β , MCP‐1 , and IFN‐γ . Every factor was assayed using ELISA kits and conducted per the manufacturer's protocols. All the marker measurements were performed by blinded independent investigators to avoid subjective bias.2.4t‐test or Mann–Whitney U‐test was used to test the differences in factor levels between the groups. Correlations between the factor levels and various clinical parameters were assessed by either Pearson or Spearman correlation analysis. Univariate and multivariate survival analyses were performed using Cox proportional hazard models to evaluate the effects of several independent variables on survival, including demographic and clinical data. The univariate survival analysis selected covariates into the multivariate Cox proportional hazard model using a low threshold of p < .2 to include variables with potential or possible effects. Risks were expressed as the hazard ratio (HR) and 95% CI. The significant level was set at p < .05.Analyses were conducted using GraphPad Prism Version 5.0 (RRID:SCR_002798) and SPSS Statistics version 17.0 (RRID:SCR_002865). Continuous demographic and clinical data were described as the means ± standard deviation (SD) in the tables. Either a two sample 33.1First, to identify factors that are obviously different between ALS patients and control subjects, we detected 13 factors in serum and CSF samples using ELISA. The analytical results of the factors are detailed in Table 3.2To further assess whether the changes in these factors had diagnostic value, we performed a series of correlation analyses between the factors and clinical parameters, including disease duration, ALSFRS‐r scores and DPR. We found that bFGF, VEGF, and MIP‐1α levels in the CSF and serum were positively correlated with disease duration Table . The lev3.3p < .2 to select covariates for the multivariate Cox proportional hazard model to adjust for the effects of the multivariate analysis. A better prognosis was observed with higher levels of bFGF in the CSF and VEGF in either the CSF or serum. Interestingly, survival analysis showed that patients with higher levels of IFN‐γ in the CSF had a shorter overall survival. A summary of the survival analysis is shown in Table After the aforementioned analysis, several factors have exhibited obvious connections with disease severity and progression. Thus, we asked whether these factors could predict survival using univariate and multivariate survival analyses. Among the 81 recruited ALS patients who provided both CSF and serum, 44 patients were deceased, and 37 (follow‐up period from 3 months to 60 months) were still alive at the time of last visit. In the univariate analysis, we analyzed the following factors to determine which of these were related to disease prognosis: gender; age; site of onset; BMI; duration; ALSFRS‐r; DPR and levels of bFGF, VEGF, MIP‐1α, MCP‐1, IFN‐γ in the CSF and serum. Subsequently, we used 4In this study, we utilized ELISAs to identify a panel of serum and CSF factors for ALS, with five significant findings as follows: (i) A total of eight factors were obviously elevated in the CSF, and twelve factors were increased in serum; (ii) VEGF levels in either serum or the CSF positively correlated with disease duration, negatively correlated with DPR and could be a potential predictor for prognosis; (iii) Similar correlations were found with bFGF, except that only bFGF levels in the CSF showed significance in the survival analysis; (iv) MIP‐1α levels were positively associated with duration and negatively correlated with DPR, whereas MCP‐1 levels in both serum and the CSF were negatively correlated with ALDFRS‐r scores and had opposing relevance with DPR; and 5) A positive relationship with DPR was found with IFN‐γ, and a poor prognosis was associated with higher levels of IFN‐γ in the CSF. These factors, including cytokines, chemokines, and growth factors (which are involved with neuroinflammation and impaired neurotrophic support) are proposed to influence the pathogenesis of ALS.Neuroinflammation plays an important role in disease progression of ALS. At regions of motor neuron injury, inflammation is a prominent pathological change and is indicated by microglial activation, astrogliosis, infiltration of monocytes and T cells, and the release of proinflammatory versus anti‐inflammatory cytokines (Zhao, Beers, & Appel, The increase in serum G‐CSF and serum GM‐CSF discovered in our research also weakly points to changes of neuroinflammation in ALS. G‐CSF is a traditional hematopoietic growth factor that was approved for granulocytopenia therapy. In addition to affecting the hematopoietic system, G‐CSF can exert anti‐inflammatory effects as well as promote neurogenesis in the CNS (Pitzer et al., Compared to unobvious alterations of the G‐CSF and GM‐CSF, three chemokines in our study emerged with strong differences between ALS patients and control subjects. Chemokines are a group of secreted proteins whose primary function is to induce inflammatory cell migration (Ramesh, MacLean, & Philipp, IFN‐γ is also critical to the neuroinflammatory process and has always been considered as a proinflammatory cytokine to promote the proliferation of cytotoxic microglia (Evans, Couch, Sibson, & Turner, Apart from neuroinflammation, impaired neurotrophic support has also been long recognized as a hallmark of ALS. Trophic factors guide the generation and location of motor neurons; furthermore, they are involved in axon guidance and synapse formation (Tovar‐y‐Romo, Ramirez‐Jarquin, Lazo‐Gomez, & Tapia, Currently, a diagnosis of ALS is primarily based on medical history, clinical examination, electrophysiological results, and the exclusion of similar disorders. Therefore, finding reliable biomarkers that could provide accurate information regarding the onset and progression of ALS in clinical practice and trials is urgently necessary. The past year has seen progress in a range of areas, multiple candidate biomarkers have emerged, specifically from neurochemical analysis of biofluids (Chen et al., 5In conclusion, we identified a panel of promising biomarker candidates of ALS that may be useful in clinical practice and therapeutic trials and that offer diagnostic, prognostic or monitoring potential to help elucidate the pathogenic mechanism of ALS. Moreover, CSF is more appropriate for biomarker discovery than serum because of its proximity to the affected regions exhibiting ALS‐induced motor neuron death. More large‐scale studies with detailed longitudinal follow‐up will be necessary to further evaluate the value of these biomarkers.The authors declare no financial or other conflict of interests.


We found that intravenous administration of acacetin prodrug (10 mg/kg) decreased the ventricular arrhythmia score and duration, reduced ventricular fibrillation and infarct size, and improved the impaired heart function induced by myocardial ischemia/reperfusion injury in anesthetized rats. The cardioprotective effects were further confirmed with the parent compound acacetin in an ex vivo rat regional ischemia/reperfusion heart model. Molecular mechanism analysis revealed that acacetin prevented the ischemia/reperfusion-induced reduction of the anti-oxidative proteins SOD-2 and thioredoxin, suppressed the release of inflammation cytokines TLR4, IL-6 and TNFα, and decreased myocyte apoptosis induced by ischemia/reperfusion. Our results demonstrate the novel evidence that acacetin prodrug confer significant in vivo cardioprotective effect against ischemia/reperfusion injury by preventing the reduction of endogenous anti-oxidants and the release of inflammatory cytokines, thereby inhibiting cardiomyocytes apoptosis, which suggests that the water-soluble acacetin prodrug is likely useful in the future as a new drug candidate for treating patients with acute coronary syndrome.The morbidity and mortality of patients with ischemic cardiomyopathy resulted from ischemia/reperfusion injury are very high. The present study investigates whether our previously synthesized water-soluble phosphate prodrug of acacetin was cardioprotective against ischemia/reperfusion injury in an  Ischemic cardiomyopathy resulted from coronary artery disease is the most common cause of mortality worldwide1346Earlier studies on ischemia/reperfusion injury focusing on preconditioning and post-conditioning cardioprotection375Tianshanxuelian inhibits atrial IKur, IKACh, and Ito1213ex vivo rat hearts revealed that acacetin prevented ischemia/reperfusion reduction of mitochondrial anti-oxidative kinases SOD-2 and thioredoxin as well as reduced inflammatory cytokines TLR4, IL-6 and TNFα, thereby inhibited myocyte apoptosis induced by ischemia/reperfusion.In the previous reports, we found that natural flavone acacetin from the Chinese medicinal herb in vivo beagle dogsin vivo conversion was also confirmed in anesthetized rats. in vivo into acacetin as reported previously in beagle dogsOur recent study has demonstrated that the custom-synthesized acacetin prodrug can be converted to the parent compound acacetin in The effect of acacetin prodrug on ventricular arrhythmias induced by ischemia/reperfusion injury was determined in anesthetized rats. Ventricular arrhythmias including premature ventricular beats, ventricular tachycardia, and ventricular fibrillation were induced by ligating the left anterior descending (LAD) artery for 10 min, followed by a 10 min reperfusion as described previouslyP < 0.05 or P < 0.01 vs. vehicle). Importantly, incidence of ventricular fibrillation was reduced by 26%, 67%, and 78% with bolus acacetin prodrug of 5, 10 and 20 mg/kg (P < 0.05 for 10 mg/kg or 20 mg/kg vs. vehicle).Ventricular fibrillation was frequently observed during the early reperfusion in control animals treated with intravenous vehicle , but notP < 0.001 vs. vehicle).The anti-infarct effect of acacetin prodrug was determined in anesthetized rats with 30 min LAD artery ligation, followed by 120 min reperfusion. Acacetin prodrug was administered by intravenous injection (10 mg/kg) before 10 min of the LAD artery ligation. Ventricular infarct size was remarkably reduced by 68.7% in rats treated with acacetin prodrug (P < 0.01 or P < 0.001 vs. vehicle), developed pressure (P < 0.01 or P < 0.001 vs. vehicle), and +dP/dT (P < 0.01 or P < 0.001 vs. vehicle), induced by ischemia/reperfusion injury. These results show that acacetin prodrug is very effective in inhibiting ventricular arrhythmias, reducing infarct size, and preserving heart function in rat in vivo ischemia/reperfusion model. These results also indicate that acacetin prodrug is converted to the parent compound acacetin and exerts significant cardioprotective action against ischemia/reperfusion injury.Moreover, the prodrug prevented the reduction of the mean femoral artery blood pressure  and the P < 0.05 or P < 0.001 vs. vehicle). Significant reduction of the infarct size was observed at concentration as low as 0.3 μM acacetin. In addition, the heart function was also evaluated in isolated rat hearts subjected to ischemia/reperfusion injury (P < 0.05 or P < 0.001 vs. vehicle).To determine the potential molecular mechanisms underlying cardioprotection of acacetin prodrug, the parent compound acacetin was used to test the effect on myocardial ischemia/reperfusion injury in isolated rat hearts. The perfused hearts were subjected to LAD artery ligation for 30 min followed by 120 min reperfusion. The hearts were treated with different concentrations of acacetin before (10 min) and during the regional ischemia . The infn injury . LVP andIt is well documented that myocardial ischemia/reperfusion injury induces intracellular oxidative stress, triggers inflammation, and initiates apoptosis signals to mediate the subsequent myocardial death67P < 0.05 or P < 0.01 vs. vehicle), indicating that cardioprotective effects of acacetin involve the retention of endogenous anti-oxidants.Previous studies demonstrated that the anti-oxidative proteins SOD (superoxide dismutase) and/or thioredoxin are decreased in myocardial ischemia/reperfusion injury or other oxidation stress18ex vivo rat hearts subjected to ischemia/reperfusion injury , and acacetin decreased the inflammatory responses in a concentration-dependent manner .The release of inflammation mediators  has been found to play an important role in mediating and exacerbating myocardial ischemia/reperfusion injury7ex vivo rat hearts subjected to ischemia/reperfusion insult. The pro-apoptotic proteins cleaved caspase-3 and Bax (Bcl-2-associated X), as well as the anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) were examined in hearts treated with vehicle, 0.3, 1, or 3 μM acacetin. P < 0.01 vs. sham). Acacetin significantly antagonized the decrease of anti-apoptotic molecule (Bcl-2) and the increase of pro-apoptotic molecules (Bax and caspase-3) induced by ischemia/reperfusion injury . The reduced Bcl-2/Bax ratio was reversed by acacetin treatment in a concentration-dependent manner (P < 0.05 or P < 0.01 vs. vehicle). These results indicate that acacetin also has a secondary anti-apoptosis effect in addition to its anti-oxidation and anti-inflammation cardioprotective properties.The potential involvement of molecular mechanism of anti-apoptosis by acacetin was further determined in P < 0.01 vs. vehicle), indicating that acacetin inhibits myocytes apoptosis thereby decreases myocardial infarct size in rat hearts subjected to ischemia/reperfusion injury. The histological damage induced by ischemia/reperfusion was also attenuated in hearts treated with 3 μM acacetin  blockade and anti-atrial fibrillation effect1213Acacetin is a small molecule flavone  universally present in various plants and responsible for many of the colours in nature2 reduction to produce H2O2. In patients and animals with myocardial ischemia/reperfusion, tumour necrosis factor-alpha (TNFα) and interleukin-6 (IL-6) were increased with other inflammatory cytokinesIt has been well documented that myocardial ischemia/reperfusion (oxidative stress) stimulates the generation of reactive oxygen species (ROS) involved in myocardial injury242628It should be noted that TNFα has an ambivalent role in myocardial ischemia/reperfusion injury. Both local ischemic conditioning and remote ischemic postconditioning are related to the decrease of the endogenous TNFα induced by myocardial ischemia/reperfusion in rodents323234It is well recognized that the overall preservation of cardiac structure and function is critical for cardioprotection against ischemia/reperfusion injury by inhibiting oxidative stress and inflammation and therefore decreasing apoptosis17363840413It is generally recognized that cardiomyocytes suffer irreversible injury primarily during ischemia/reperfusion, the window time is important for clinical use of cardioprotective drugs to minimize additional cardiomyocyte loss during reperfusion. A large number of studies have recommended introducing protective drugs at the very beginning of reperfusion to reduce infarct size44545Although the present study indicates that acacetin prodrug is likely a promising drug candidate to rescue the myocardial injury on reperfusion, the limitation is that all the results were obtained in rodents. Further experimental study is required in large animals (e.g. swine and/or canine) to gain the preclinical data which can be referred for clinical trials in the near future.The present study demonstrates for the first time that acacetin prodrug confers significant cardioprotective effect against ischemia/reperfusion injury after converting into the parent compound acacetin by preventing ischemia/reperfusion-induced decrease of anti-oxidants, thereby reducing inflammatory response and inhibiting myocardial apoptosis and infarct size, which suggests that acacetin prodrug may improve the long term outcomes of patients undergoing thrombolytic therapy or PPCI.Sprague Dawley (SD) rats were employed in the present study following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , and the experimental procedure was approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong.Adult male SD rats (250–300 g) was anesthetized with pentobarbital , and the jugular vein and femoral vein were cannulated for drug administration and blood sample collection respectively. The water-soluble acacetin prodrug  with purThe simple liquid-liquid extraction with methanol was used for sample preparation. Pentamethylquercetin was selected as internal standard. The analysis was carried out on a Waters HPLC system  equipped with an Alltech column . The mobile phase consisted of methanol-water-phosphoric acid (70:30:0.15), and the flow rate was 1.0 mL/min. UV absorption was monitored at 260 nm.Ischemia-reperfusion model in anesthetized rats was established as described previously10PVCs) + 2 (Log10 episodes of VT) + 2 (Log10 episodes of VF) + ]48After 10-min stabilization, acacetin prodrug  or equivolume vehicle (5% glucose solution) was intravenously administered for 5 min before LAD artery was ligated for 10 min, followed by a 10-min reperfusion. Cardiac injury was characterized by a high incidence of ventricular arrhythmias during reperfusion in this model. Definitions of arrhythmias were based on the criteria of the Lambeth Conventions, including premature ventricular contractions (PVCs), ventricular tachycardia (VT), and ventricular fibrillation (VF)To evaluate the ischemia/reperfusion-induced myocardial infarct size, rats were anesthetized and subjected to 30 min LAD artery ligation and followed by 120 min reperfusion. Acacetin prodrug  was continuously administered for 10 min before LAD artery ligation and during 30 min ischemia. Lead II ECG, blood pressure, and left ventricular pressure were monitored and recorded on an IBM compatible PC computer using a data acquisition system  for offline analysis.2PO4 1.18, MgSO4 1.18, NaHCO3 25, glucose 11.1, CaCl2 2.5) gassed with 95% O2 and 5% CO2. Polyethylene tubing with a cling film balloon tip filled with water and connected to a pressure transducer was inserted into the left ventricle through a small incision in the left atrium to measure left ventricular pressure. ECG was recorded with two electrodes placed on heart surface and the physiological signals were saved on an IBM compatible PC computer using the data acquisition system (Chengdu Instrument Ltd).Adult male SD rats were anesthetized, their hearts were isolated and retrogradely perfused on a Langendorff heart perfusion apparatus at 37 °C with modified Krebs-Henseleit solution (in mM): NaCl 118.5, KCl 4.7, KHRegional ischemia was achieved by ligating LAD artery using a 5–0 silk suture with a section of silica gel tubing. After 20-min equilibration, the hearts were treated by vehicle (DMSO) or different concentrations of acacetin  for 10 min, and during LAD artery ligation (30-min), followed by 2 hr reperfusion. Myocardial ischemia was confirmed by a decrease in left ventricular developed pressure (LVDP) and specific ST-segment elevation on ECG. The hearts were used to perform histological and molecular analysis at end of 2 hr reperfusion.Data were discarded if one of the following criteria appeared: 1) LVDP was below 80 mmHg during equilibration, 2) lack of evidence of successful LAD occlusion, and 3) hearts with irreversible arrhythmia during equilibration or after ischemia.At end of reperfusion, LAD artery was re-occluded at the same location. 0.5% solution of Evans blue dye was infused via aorta to differentiate the area at risk from the non-ischemic zone. The heart was then rapidly excised and frozen at −20 °C for 2–3 hr, and cut into 2-mm transverse slices from apex to base. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in sodium phosphate buffer (pH 7.4) for 20 min at 37 °C and fixed in 10% formalin.After Evans blue/TTC staining, myocardial ischemic risk area was identified as the myocardial tissue without blue staining and presented as a percentage of the total left ventricular area. Viable tissue in the risk area (deep red) was stained by TTC, while infarcted area was clearly visible as TTC-negative  and expressed as a percentage of total myocardial risk area. Images of the heart slices were taken and size of risk area and infarct area was measured by Image J software.After the reperfusion, the heart was fixed with 10% buffered formalin, embedded in paraffin, and then sectioned into 5 μm slices. The tissue slices were stained with haematoxylin-eosin, and analysed by light microscopy.in situ cell death detection kit, POD  following the manufacturer’s instruction. Samples were counterstained with haematoxylin prior to analysis by light microscope. Quantitative analysis of TUNEL-positive cells was performed in 10 random fields at 100× magnification on three different sections from the peri-infarction zone in each animal as reported previously50TUNEL staining of myocardial slices was performed to detect myocyte apoptosis in myocardium using an The related proteins were determined with Western blotting analysis as described previouslypost hoc test. Quantitative data were analyzed using Student’s t-test for paired or unpaired observations to evaluate significant differences between two group means, and ANOVA for multiple groups. A value of P < 0.05 was taken to indicate statistical significance.Group data are expressed as mean ± S.E.M. The incidence of arrhythmias was analyzed using the Fisher exact test. Arrhythmia scores were analyzed using one-way ANOVA followed by a Tukey How to cite this article: Liu, H. et al. Water-soluble acacetin prodrug confers significant cardioprotection against ischemia/reperfusion injury. Sci. Rep.6, 36435; doi: 10.1038/srep36435 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Cryptococcal meningitis (CM) has been known to lead to significant morbidity and mortality. The relative contribution of the complement system in protection and pathogenesis during CM remains largely unknown. The purpose of this study was to evaluate the baseline complement component profiles in human cerebrospinal fluid (CSF) and plasma from non-HIV patients with CM, and therefore to provide insights of possible roles of the complement system in CM.CSF and blood samples from forty two CM patients not infected with HIV and thirteen non-CM control patients (Ctrl) were retrospectively selected and evaluated from the patients admitted to the hospital with a suspected diagnosis of CM. CSF and blood samples were collected at the admission. Enzyme-linked immunosorbent assay (ELISA) for complement components, cytokine IL-12 and western blot for C3 activation were performed on CSF and plasma samples. The levels of complement C1q, factor B (FB), mannose binding lectin (MBL), C2, C3, C4, C5, C4 binding protein (C4BP), Factor I (FI), Factor H (FH), sC5b-9 in CSF and plasma samples were compared. Pearson’s correlation coefficients were calculated on variables between complement components and the levels of total protein in the CSF samples.Our data demonstrated that the CSF levels of complement components of C1q, FB, MBL as well as complement pathway factors sC5b-9 and complement regulator FH were all elevated in patients with CM as compared to the controls, CSF C3 breakdown products iC3b were found in both CSF and plasma samples of the CM patients. A positive correlation was found between the levels of CSF protein and MBL, C1q or FB.The activity of the complement system in CSF was increased in non-HIV patients with CM. C1q, MBL and FB are the important participants in the complement activation in CM. The relative contribution of each of the specific complement pathways and complement cascades in protection and inflammation resolution against CM warrant further investigation. C. neoformans, resulting in pulmonary cryptococcosis that has a predilection to disseminate to the central nervous system (CNS) leading to life threatening meningoencephalitis [Cryptococcal meningitis (CM) is one of the most common diseases in HIV patients and other immune-compromised patients . Over 1 phalitis .The complement system is well known as a major component of the host innate immune defense system against infection. Activation of the complement system in response to invading pathogens is initiated through the classical (CP), alternative (AP) and lectin (LP) pathways. Activation results in C3 cleavage, releasing anaphylatoxins C3a and C5a, and formation of a membrane attack complex to lyse the target cells. Cleavage of C3 generates the key opsonins, C3b and iC3b. These proteins tag the pathogens for phagocytosis . The comIt has been suggested that the complement system is of central importance in host defense against bacterial meningitis. Mice deficient in C1q and C3 demonstrated a decreased survival and enhanced growth of pneumococci in cerebrospinal fluid (CSF) after induction of meningitis . ComplemC.neoformans [C.neoformans from extraneural sites [C5−/− mice were more susceptible to intravenously injected C.neoformans, and more easily developed acute, fatal cryptococcal pneumonia [C.neoformans by neutrophils [C.neoformans, resulting in optimal phagocytosis and subsequent killing of the organisms [Cryptococcus.In contrast to bacterial meningitis, most fungal microorganisms cause a chronic instead of an acute or subacute meningitis . Investioformans . Guinea al sites . C5−/− neumonia . Patientneumonia . Brain sneumonia . Moreovetrophils . In vitrrganisms . The comrganisms , 19. TheTo our knowledge, no study has been reported which examines the complement system activity in CSF and plasma from CM patients. The purpose of this study was to investigate whether the complement system is activated systemically and/or intrathecally during CM. The levels of the complement components in CSF and plasma samples from CM patients were determined and compared with that in the patient control group who were diagnosed with pulmonary cryptococcosis without CNS infection and inflammation involvement.This study was carried out from November 2014 to December 2015 at Huashan Hospital, Shanghai China. Both lumbar CSF and plasma samples were simultaneously obtained from patients with a suspected CM diagnosis at admission for routine chemistry, cytology and culture analysis. Venous blood was collected from individuals for plasma and routine blood cell and culture analysis. The remaining CSF samples were centrifuged and single use aliquots of these samples were stored at −80 °C.C. neoformans or positive India ink smear of CSF. Possible cases were patient with positive cryptococcal antigen titer in undiluted CSF and met at least one of the following criteria : (1) abnormal laboratory tests or an increased open pressure (≥200 mmH2O) of CSF, (2) abnormalities of cranial imaging (Computerized Tomography or Magnetic Resonance Imaging) which could not be explained by other factors. Cryptococcal antigen in CSF was determined with the Latex-Cryptococcus antigen detection system (Immuno-Mycologics) [CM group and patient control group were retrospectively selected from the collected samples. CM group consisted of 42 patients who were ≥18 year old and not infected with HIV with a first episode of CM as diagnosed according to the criteria reported previously . Confirmologics) .Control CSF or blood samples were obtained from patients who presented to the hospital with suspicion of CM but subsequently tested negative for CM or any other pathogen in CSF with normal CSF leukocyte count and protein concentration. The control patients were diagnosed as pulmonary cryptococcosis based on histologic presence of the organism in lung biopsy specimens, or positive result of the serum cryptococcal antigen test with radiographic characteristics of pulmonary disease. . The ethHuman Complement C1q, FB, MBL, C2, C3, C4, C5, C4BP, FH, FI, sC5b-9, and cytokine Interleukin (IL) -12 were measured with commercial ELISA kits . Experiment protocols were followed according to the manufacturer’s instructions.For comparison of complement C3 activation in CSF and plasma samples, 20 μl of CSF and 2 μl of plasma samples were loaded on an SDS-PAGE gel. Samples were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and then incubated with mouse anti-human C3/C3b/iC3b/C3dg monoclonal antibody  in TBST  containing 5% dry milk at 4 °C overnight. Blots were then washed and incubated with goat anti-mouse IgG  diluted in TBST containing 2% dry milk. The membranes were developed with an Odyssey system (Li-COR Bioscience) according to the manufacturer’s protocol.t tests or Mann–Whitney tests (when the variances were significantly different). Nominal data were analyzed with Fisher exact test or Chi χ2 test. Pearson’s correlation coefficients were calculated on the variability between the levels of CSF complement components and CSF total protein. In all cases, a p value of <0.05 was set as the measure of significance.Results were graphed and analyzed using Prism 5.0 (GraphPad). Comparisons between the CM and control groups were made using unpaired The study groups consisted of 42 CM patients and 13 patients diagnosed with pulmonary cryptococcosis without CNS infection and inflammation served as Ctrl group. As shown in Table p-value =0.0479, r = 0.7061 for MBL, p-value = 0.0003, and r = 0.6716, p-value =0.009). However, There were no statistical significances of the plasma concentrations of C1q, MBL and FB between the CM and Ctrl groups , the lectin pathway (MBL) and the alternative pathway (FB) in plasma and CSF samples from CM and Ctrl group. The concentrations of C1q, MBL and FB in CSF samples were significantly higher in CM group than that in Ctrl group Fig. . The meaups Fig. , suggestWe further examined the concentrations of C2, C3, C4 and C5 in the plasma and CSF samples. There were no statistical significances of the concentrations of these complement factors in both plasma and CSF samples between the two groups Fig. .Fig. 2ThThe complement system activation is mediated through the CP, AP and LP pathways. Each pathway converges at the C3 convertase level resulting in C3 cleavage. To further assess whether the C3 activation is involved in CM, levels of C3 breakdown products in CSF and those in simultaneously collected blood plasma samples were evaluated by Western blot. C3 cleavage fragments iC3b was detected in CM group from both plasma and CSF samples  p = 0.0303)  Fig. .Fig. 5Thp = 0.0121)  Fig. .Fig. 6ThIn this study, we showed elevated CSF concentrations of C1q, MBL, FB, sC5b-9 and FH in CM patients compared to that in the control group and the common complement pathway factors iC3b in both CSF and plasma samples of CM patients. Our data suggest that the increased CSF complement system activity might indicate its important roles in host defense and pathogenesis in CM.C. neoformans infection is well established, it is not clear how the complement system responds to pathogens in CM clinically. The brain may represent its own characteristics not like other organ when infected with Cryptococcus. There are few publications in this field. Our data demonstrated that complement components including the initiators of three complement pathways , other complement factors  and regulators  were present in CSF in CM. Moreover, CSF levels of C1q, MBL, and FB were significantly higher compared with that in control group. In this study, we showed C3 and the complement terminal pathways were activated in CSF during CM. Cleavage of C3 generates the key opsonins, C3b and iC3b for phagocytosis [Though the importance of the complement system to ocytosis . Activatocytosis , which docytosis , 26. SigThe differences of complement components between the CM and Ctrl groups were only detected in CSF samples, not in the plasma samples. This indicates that local inflammatory responses impact significantly on activity of the complement system in CSF. The brain as a well-developed organ of CNS is shielded by the blood–brain-barrier (BBB) with tight junction formations. BBB is a structural and functional barrier formed by the microvascular endothelium, astrocytes and pericytes. The complement system has taken part in various developmental and regenerative processes, and is integrated with multiple biological systems and pathways in many researches . We thinHowever, complement has been described as a double-edged sword since activation of the complement system significantly contributes to the pathogenesis of various acute and chronic inflammatory diseases . AdequatC. neoformans infections. Beenhouwer and his colleagues found that the relative inherent susceptibilities of the mouse strains to C. neoformans were as follows: IL-12−/− > IL-6−/− > C57BL/6 > > IL-10−/− [It is well known that a Th1-driven cell-mediated response is necessary for the control of L-10−/− . In the In conclusion, our data revealed the presence of the complement components and the complement activation in CSF in non-HIV patients with CM, and suggested an important role of complement factors C1q, MBL, FB, FH and sC5b-9 in the pathogenesis of CM. Further studies are warranted to confirm the pathophysiological role of these complement factors in CM and to stimulate the development of new therapeutic strategies for CM by directing treatment to specific pathways within the complement cascade.


Flavivirus infection, with the outcome strongly dependent on secondary immunopathology. Neutrophils are present in cerebrospinal fluid (CSF) of TBE patients, but their pathogenetic role remains unknown. In animal models, neutrophils contributed both to the Flavivirus entry into central nervous system (CNS) and to the control of the encephalitis, which we attempted to evaluate in human TBE.Tick-borne encephalitis (TBE) is a clinically variable but potentially severe n = 110), meningoencephalitis (n = 114) or meningoencephalomyelitis (n = 16) assessing CSF neutrophil count on admission and at follow-up 2 weeks later, and their associations with other laboratory and clinical parameters. We measured serum and CSF concentrations of Th17-type cytokines  and chemokines attracting neutrophils  in patients with TBE , with non-TBE aseptic meningitis (n = 6) and in non-meningitis controls (n = 7), using commercial ELISA assays. The results were analyzed with non-parametric tests with p < 0.05 considered as significant.We analyzed records of 240 patients with TBE presenting as meningitis  of total pleocytosis, but on follow-up, they were absent in most of patients (58%) and scarce (< 10%) in 36%. CSF neutrophil count did not correlate with lymphocyte count and blood-brain barrier integrity, did not differ between meningitis and meningoencephalitis, but was higher in meningoencephalomyelitis patients. Prolonged presence of neutrophils in follow-up CSF was associated with encephalitis and neurologic sequelae. All the studied cytokines were expressed intrathecally, with IL-8 having the highest CSF concentration index. Additionally, IL-17A concentration was significantly increased in serum. IL-17F and CXCL1 CSF concentrations correlated with neutrophil count and CXCL1 concentration was higher in patients with encephalitis.The neutrophil CNS infiltrate does not correlate directly with TBE severity, but is associated with clinical features like myelitis, possibly being involved in its pathogenesis. Th17 cytokine response is present in TBE, especially intrathecally, and contributes to the CNS neutrophilic inflammation. IL-8 and CXCL1 may be chemokines directly responsible for the neutrophil migration.The online version of this article (10.1186/s12974-018-1138-0) contains supplementary material, which is available to authorized users. Flavivirus species ) transmitted by Ixodes ticks. The European TBEV subtype frequently causes a mild flu-like disease, but in some cases, it is able to penetrate the blood/brain barrier (BBB) causing the second, neurologic phase of the disease, which may take a severe course and leave permanent neurologic sequelae [5]. Both the animal models and the results of human studies point to the involvement of the host immune/inflammatory response in the pathogenesis. TBEV entry into the central nervous system (CNS) probably occurs at least partially via transcellular pathway [Flavivirus encephalitis suggest it may be facilitated initially by the disruption of BBB by the excessive systemic inflammation [Tick-borne encephalitis (TBE) is caused by a sequelae –5. Both  pathway , but aniammation , 8 and sammation . The furammation  and of aammation –15. Inteammation .Flavivirus species, West Nile virus (WNV) and Japanese encephalitis virus (JEV) [The cerebrospinal fluid (CSF) of TBE patients is characterized by the increased albumin concentration and albumin CSF/serum index persisting during the convalescent period, indicating protracted BBB disruption, elevated IgG index pointing to the intensive intrathecal humoral response , and a tus (JEV) , 20. ThiFlavivirus infections are few and conflicting. For example, the activation of neutrophils in contact with JEV and an effective degradation of viral proteins and RNA has been observed in vitro suggesting a protective role [Neutrophils are involved in the early unspecific response and elimination of pathogens, which is typically accompanied by a significant local tissue damage , 22. Thrive role , but intive role . Intrathive role , 14, 30,The Th17-type specific response, essential for the development of a sustained neutrophil-dominated inflammation, is driven by a subset of immune cells  able to secrete a set of cytokines including interleukin-17A (IL-17A), IL-17F, and IL-22 , 31–36. Flavivirus neuroinfections, the involvement of Th17 lymphocytes and related cytokines might explain both the presence of the substantial neutrophil infiltrate and the significant immunopathology. The study by Wang et al. on WNV-infected mice confirmed the intrathecal expression and pathogenetic role of IL-22 and CXCL1 [Although originally linked to the response against extracellular pathogens in skin and mucous membranes, Th17 cells may contribute to the inflammation and immunopathology within CNS. IL-17 and IL-22 act on BBB endothelial cells, increasing secretion of IL-6, CCL2, CXCL1, CXCL2, and IL-8; increasing BBB permeability; and facilitating transmigration of CD4+ cells , 43. In nd CXCL1 . Pietikänd CXCL1 . IL-8 wand CXCL1 , 47.We have attempted to gather more data on the role of neutrophils in the pathogenesis of TBE in humans and on the expression of the cytokines which might contribute to their recruitment into CNS. We have analyzed retrospectively the correlations between the CSF neutrophil count and other laboratory and clinical parameters, including the disease manifestation and severity, in TBE patients hospitalized in six consecutive years in a reference center. We have also analyzed concentrations of chemokines for neutrophils IL-8, CXCL1, and CXCL2, as well as Th17 cytokines IL-17A, IL-17F, and IL-22 in CSF and serum in prospectively recruited TBE patients, to assess their peripheral and intrathecal expression, concentration gradients, and relation to the CSF inflammatory parameters.We have studied TBE patients hospitalized in the Department of the Infectious Diseases and Neuroinfections of the Medical University of Białystok. The initial diagnosis was based on the history of a recent tick bite or exposition to ticks in TBE endemic areas, clinical symptoms, and CSF pleocytosis and confirmed with the detection of the specific anti-TBEV IgM antibodies in serum and/or CSF on admission or by the time of discharge from the hospital. Most of the patients underwent two lumbar punctures: on admission to hospital and before the discharge 10–16 days later. Patients with residual clinical symptoms or protracted inflammatory changes in CSF had additional control lumbar punctures later in the convalescent period, typically 4–8 weeks after admission.The data from the records of patients hospitalized between 2009 and 2014 were analyzed in the retrospective part of the study. Only patients with CSF pleocytosis ≥ 15 cells/μl on admission were included, which excluded patients with peripheral TBEV infection without CNS involvement. Patients in whom the microscopic CSF evaluation on admission was not performed, because of low CSF cytosis or for technical reasons, in whom the original CSF examination was done outside of our department or with a delay of ≥ 3 days after admission, were not included either. This left 240 patients  in whom we have analyzed associations of the peripheral neutrophilia and CSF neutrophil count with the clinical course, manifestation, severity, outcome, presence of an altered mental status, and paresis, as well as with basic laboratory parameters: C-reactive protein (CRP) concentration, blood leukocytosis and lymphocytosis, CSF pleocytosis, lymphocytosis, total protein, and albumin concentration.TBE with a classical biphasic course was defined as a disease with a flu-like peripheral phase with transient improvement followed by a neurologic phase, as opposed to a monophasic onset of a neurologic disease. Meningoencephalitis (ME) was diagnosed based on the altered mental status and/or focal neurologic deficits, meningoencephalomyelitis (MEM) based on sensory or motor symptoms from the spinal nerves, and patients not fulfilling the ME or MEM criteria were diagnosed with meningitis (M). The disease severity was scored in a pre-defined scale from 1 to 6, in which 1 corresponded to an evidently uncomplicated meningitis; 2 very mild or dubious CNS involvement with no altered mental status nor paresis ; 3 mild encephalitis with lethargy, drowsiness, emotional lability, monofocal paresis, gait disorders, but patients remaining in a logical contact and able to walk; 4 moderately severe encephalitis with disorientation, abnormal behavior, multifocal and/or severe neurologic symptoms, and generalized seizures; 5 severe encephalitis with loss of consciousness; 6 coma or death. The mental status was scored from 0  through 1 , 2  to 3 (lack of consciousness). The outcome in surviving patients  was scored from 0 to 3 . The division of the patient cohort according to the above criteria is shown in Table Borrelia burgdorferi after previous symptomatic or asymptomatic infection, which makes the diagnosis of an active disease challenging. In the group co-infected with TBEV, it is especially problematic as the assessment of intrathecal synthesis of the specific antibodies against Borrelia burgdorferi is complicated by the acute BBB disruption. Neither the clinical presentation nor laboratory parameters differed systematically in these patients from the rest of the group, which led us not to exclude them from the study.Six patients were diagnosed by a treating physician with a probable or confirmed neuroborreliosis coexisting with TBE, based on a positive serology and intrathecal synthesis of the specific antibodies. The patients came from the area endemic for Lyme borreliosis and could be seropositive against In the prospective pilot study, IL-8 serum concentration was measured on admission to hospital in 36 patients with TBE hospitalized in 2014 and 2015, to confirm previous findings on IL-8 expression in TBE and study its specificity for TBE versus other viral meningitides . Of thesThe main prospective study group consisted of 15 patients hospitalized in 2015 and 2016, 11 with M and 4 with ME, of whom 3 had mild presentation with mainly cerebellar symptoms and 1 moderately severe presentation with an altered mental status. Eight patients had biphasic and 7 monophasic disease. The data of this group are presented in Table As the control samples, we used sera from 7 healthy blood donors and CSF from 7 patients in whom neuroinfection was ruled out by means of a lumbar puncture. Additionally, concentrations of IL-8, CXCL2, IL-17A, and IL-17F were studied in paired serum and CSF samples obtained on admission from 6 patients with non-TBE lymphocytic meningitis hospitalized during Echovirus 30 meningitis outbreak.The subjects included in the prospective study gave informed consent for the entry, and the study was accepted by the Bioethics Committee of the Medical University of Białystok .The blood and CSF samples for the prospective study were obtained simultaneously, together with the materials for clinically indicated laboratory examinations: examination I on admission to hospital, examination II before discharge, and examination III during the later control visits in the convalescent period.In the IL-8 group, only 5 patients (the same in whom the CSF was studied) were studied at the examination II time point. In the main study group, the examination I samples were available in all TBE patients and examination II samples in 13 patients (only IL-22 and CXCL1 were measured in one of them at examination II time point because of the limited CSF volume). In 5 patients, the later convalescent period (examination III) paired serum and CSF samples were additionally studied for the concentration of IL-22 and CXCL1, to assess the long-term dynamics of the pro-neutrophilic cytokine response. Two patients  were studied with standard laboratory techniques. Anti-TBEV IgM antibodies in serum and CSF were detected with Enzygnost Anti-TBE/FSME IgM kit from Siemens , following the standard procedure.Concentrations of cytokines were measured with commercial ELISA kits, strictly following the manufacturer’s instructions: CXCL1 and IL-8 with a Quantikine® ELISA from R&D Systems , IL-22 with IDELISA™ from ID Labs Inc. , IL-17A, IL-17F, and CXCL2 from Shanghai Sunred Biological Technology Co. (China). The sensitivity of the assays according to the manufacturers’ data was 10 pg/ml for CXCL1, 5 pg/ml for CXCL2, 3.5 pg/ml for IL-8, 0.5 pg/ml for IL-17A, 0.5 pg/ml for IL-17F, and 10 pg/ml for IL-22. Any read-out falling below that level was considered 0 in the analysis.Qalb) was calculated as albuminCSF/albuminserum. Because of the lacking data on serum albumin concentrations at the day of the first lumbar puncture, it could be calculated in only 36 patients (15%) of the retrospective cohort and in 14 out of 15 in the prospective cohort. The indexes of cytokine concentrations in CSF were calculated as I = / in 14 prospective cohort patients in whom the serum albumin concentration was known.CSF albumin quotient , Kruskal-Wallis non-parametric ANOVA for comparisons of multiple groups , R Spearman test for the analysis of correlations, and chi-square test for the analysis of the distribution of the scored variables. The intra-group comparisons  were performed with Wilcoxon pair test.The data were analyzed with Statistica 10 software with the use of non-parametric tests and In the retrospective study, patients with the most severe presentation or sequelae were too few to be included in the formal statistical analysis. Also, not all data were available for all the patients , decreasing the case numbers in particular analyses.R = − 0.28) but significant (p < 0.005) decrease of the peripheral blood neutrophil count with the time since the onset of fever  tick bite; (2) onset of symptoms; and (3) onset of fever, to the lumbar puncture, whenever there were sufficient data in the medical records and checked for the association with the admission laboratory parameters. There was no correlation between any of the analyzed intervals and any of the studied CSF parameters, blood leukocyte count, and CRP concentration, meaning they were not critically dependent on the duration of the disease. There was only a weak  higher neutrophil fraction, neutrophil count, total protein, and albumin concentration. The CSF neutrophil count did not differ between patients with monophasic and biphasic course of the disease and did not correlate with the score of the disease severity, consciousness level, or outcome (not shown). However, it was significantly higher in patients with paresis (p < 0.05) and especially in patients with a spinal paresis in comparison with the rest of the cohort (p < 0.03)  Fig. b.Table 4R = 0.32, p < 0.001)   were excluded (00/μl werp < 0.05), while the protein and albumin concentrations remained higher in MEM   (Table p < 0.01), resembling trend observed for CSF albumin (p < 0.05) and total protein (p < 0.05), but contrasting with the pattern for total and lymphocytic pleocytosis , and this association of the residual neutrophil infiltrate with persistent neurologic deficits was significant  and manyfold higher than in serum (p < 0.05)  of TBE sera on admission and in none of the convalescent or control serum samples. It was detectable in all admission TBE CSF studied, in one convalescent CSF sample and in none of control CSF. IL-8 median concentration in admission CSF was significantly increased (05) Fig. .Fig. 4Thp < 0.05), in TBE patients (p < 0.05), and in serum samples (p < 0.05)  of non-TBE meningitis sera and in all CSF samples. Its concentration in non-TBE meningitis CSF was significantly higher than in controls (3% of nonp < 0.0001 for both) but not in serum. The CXCL1 median concentration in CSF was 5-fold and significantly higher than in serum (p < 0.01) while CXCL2 did not create a consistent concentration gradient towards CSF. The CXCL2 CSF/serum quotient was variable, in individual patients ranging from 0.16 to 1.62 (above 1.0 in four). The CXCL1 and CXCL2 CSF concentrations decreased in examination II (p < 0.01 for both), but CXCL2 remained elevated (p < 0.05) compared to controls. In patients examined during later follow-up (examination III), CXCL1 was undetectable in 3 out of 5 , IL-17F (p < 0.01), and IL-22 (p < 0.01) in CSF of TBE patients, and IL-17A was significantly upregulated in serum as well (p < 0.0001). Their concentrations did not differ significantly between serum and CSF. All the concentrations decreased in examination II (p < 0.01), but IL-17A in CSF remained elevated in comparison with controls (p < 0.01). IL-22 in examination III was still detectable in 4 of 5 sera and (at low concentration) in 2 out of 5 CSF samples  and was the highest in a patient with the most severe clinical symptoms and altered consciousness. There was an analogous non-significant trend for IL-22, but not for IL-17A, IL-17F, and CXCL2  and not different from the TBE group. Concentration of IL-17A was higher in serum and CSF of non-TBE patients than in controls (p < 0.01) and TBE (p < 0.05). IL-17F was not upregulated significantly in non-TBE meningitis, and its concentration in admission CSF tended to be lower than in TBE (p = 0.066)  and CSF (p < 0.05), and all the other cytokines were significantly upregulated in CSF. There was a tendency for higher Th17 cytokine concentrations than during examination I, with a trend being most notable for IL-17A and IL-17F in both serum and CSF . Two patients examined in the peripheral phase tended to have higher serum concentrations of the studied cytokines than the 2 examined later.The cytokine concentrations measured in 4 patients in the peripheral phase or at the onset of the neurologic phase of TBE were comparable to the examination I values  higher in ME than in M, with almost no overlap between the groups . Additionally, serum IL-17F and CXCL2 correlated significantly with CSF neutrophil count (p < 0.05) and CXCL2 with total pleocytosis (p < 0.01)  and IL-17F (p < 0.05)  Fig. . There w05) Fig. . Analyzip < 0.05, Fig. p < 0.005 for the correlation of the admission CXCL2 in serum with follow-up pleocytosis, p < 0.05 for other) . Interestingly, there was a tendency for a negative correlation of the initial cytokine concentrations with the residual follow-up neutrophil counts, significant for IL-17A in CSF (er) Fig. .Fig. 9ThThe results of the retrospective study are in accordance with previous data and also give additional insights into the mechanism and consequences of the neutrophil CSF infiltration. The CSF neutrophil fraction was similar to previously described by Mickiené et al. in TBE, and somewhat lower than that reported in WNV-infected patient groups, which was 41% in meningitis and 45% in encephalitis according to Tyler et al., and 50% decreasing to 37% over 3 days since onset according to Rawal et al. , 48. In Flavivirus encephalitis come mainly from animal models and remain conflicting. In the mouse model of viral encephalitis studied by Zhou et al., neutrophils constitute the first population of cells infiltrating CNS and their depletion leads to an increased virus replication and impaired migration of mononuclear cells. The authors hypothesized that neutrophils initiate the protective accumulation of mononuclear cells within CNS by first contributing to BBB disruption and then by secreting chemokines including CCL3, CCL4, CXCL9, CXCL10, and CXCL11 [Flavivirus encephalitis, which could be mediated either by an increased viral load entering CNS with migrating neutrophils or by their direct contribution to the intrathecal immunopathology. In mice infected with Murray Valley encephalitis virus, both the depletion of the neutrophils dominating in the brain infiltrate and the inhibition of the inducible nitric oxide synthase (iNOS) prolong the survival, suggesting an immunopathology dependent on the neutrophil-generated NO [The data on the pathogenetic role of the neutrophilic CNS infiltration in d CXCL11 , some ofd CXCL11 , 49. On rated NO . In the rated NO . In micerated NO . The Bairated NO . Howeverrated NO , 52.Flavivirus encephalitis remains unclear. In WNV patients described by Tyler et al., the CSF parameters did not differentiate meningitis from encephalitis and although a higher pleocytosis, neutrophil fraction, and CSF protein concentration correlated with a worse outcome, the prognostic value of individual factors was weak [The significance of these observations for the pathogenesis of human was weak . In the was weak . The senwas weak , 27, butwas weak .To identify mediators of the neutrophilic response in TBE, we have first verified the synthesis of IL-8 described in previous studies. IL-8 was previously reported in CSF of patients with aseptic meningitis of various etiology , 54, andFlavivirus encephalitis [Following the IL-8 detection, we have studied expression of two additional chemokines for neutrophils (CXCL1 and CXCL2), as well as of Th17 cytokines previously detected in animal models of phalitis , 58, 59.Besides IL-8, there are few data on the expression of Th17 cytokines and chemokines for neutrophils in other human CNS infections to provide context for our TBE observations. According to Pietikäinen et al., the concentration of IL-17 is higher in CSF of TBE patients in comparison with neuroborreliosis, a condition characterized by almost exclusively lymphocytic pleocytosis . Zhang eFlavivirus neuroinfection, particularly in mediating the neutrophil migration. In the mouse model of WNV encephalitis, the lack of IL-22 expression decreased CNS viral load, infiltration by neutrophils, and mortality rate, attenuating all the aspects of the intrathecal infection and inflammation. However, when WNV was injected intrathecally, IL-22 was moderately protective [Flavivirus encephalitis but later contributes to the intrathecal protective response, which resembles the sequential role of neutrophils in WNV infection as described by Bai et al. [Both our data and previous animal results point to an important role of IL-22 in the pathogenesis of otective . These ri et al. , 27. Thei et al. . Accordii et al. . In vitri et al. , confirmThese results may have implications for a better understanding of a still debatable route of TBEV entry into CNS, a crucial event differentiating between the mild peripheral and potentially severe neuroinvasive infection. In vitro TBEV is able to cross BBB by infecting and replicating persistently within brain endothelial cells, without directly affecting BBB structure and permeability . This foWe found the concentrations of pro-neutrophilic cytokines to decrease in the early convalescent period of TBE, confirming the association of a Th17 response with its early stages. However, this early response had a lasting influence possible to trace in the convalescent CSF parameters. Especially, the CSF concentration of IL-17A correlated negatively with the residual CSF neutrophil infiltrate, which in the retrospective study associated with a more severe presentation. According to Zhou et al. the protective mononuclear CNS infiltrate is facilitated by neutrophils, but once it is established, it may suppress further neutrophil migration, for example, through effects of interferon gamma (IFN-γ) on the local chemokine synthesis . AccordiNeutrophils migrate into CSF of patients with TBE independently of the lymphocyte influx and BBB disruption. The CSF neutrophil count does not differ between meningitis and meningoencephalitis, but is higher in myelitis, and normalizes with delay in patients with encephalitis and with persistent neurologic complications, suggesting a complex involvement in the intrathecal pathology. The pro-neutrophilic Th17 cytokines and chemokines signaling through CXCR1 and CXCR2 are upregulated intrathecally, and at least some of them (IL-17A) also systemically, starting from the peripheral and continuing into the neurologic phase of TBE, which allows them to exert their influence during the critical events deciding about the onset and severity of the CNS involvement. Of Th17 chemokines, IL-22 is most vividly expressed intrathecally. Of chemokines attracting neutrophils at least IL-8 and CXCL1 create chemotactic gradients towards CSF and may be responsible for neutrophil recruitment, and CXCL1 concentration correlates with CSF neutrophil count and with encephalitic presentation. By analogy with animal models, a signaling axis involving IL-22, its mediator chemokines and CXCR1/CXCR2 receptors may increase the TBEV influx into CNS with infected neutrophils, thus providing an important route of neuroinvasion leading to a more severe CNS involvement. After the encephalitis is established, however, an intrathecal neutrophilic response may be protective, contributing to its control and eradication.Additional file 1:R and significance p of the correlation is given in the upper right corner. (BMP 2655 kb)Correlation between the time since onset of fever and peripheral blood neutrophil count. The time since the onset of fever till the admission to hospital as reported by a patient and recorded in medical documentation (in days) is presented on horizontal axis and the neutrophil count on admission (in cells/μl) on vertical axis. The data from the individual patients are shown with crosses, the linear fit with a continuous line and the 95% confidence interval with dashed lines. The strength Additional file 2:R and statistical significance p of the linear fit are shown in the upper right corner of each panel. NS not significant. (BMP 19542 kb)Correlations between the serum and cerebrospinal fluid concentrations CXCL1, CXCl2, and Th17 cytokines. Concentrations of IL-17A, IL-17F, IL-22, CXCL1, and CXCL2 in serum (S) samples obtained from tick-borne encephalitis patients on admission to hospital are shown horizontal axes and in cerebrospinal fluid (CSF) samples obtained simultaneously on vertical axes (expressed in pg/ml). The data from individual patients are shown with points (patients with meningitis) and circles , the linear fit with a continuous line, and the 95% confidence interval with dashed lines. The strength Additional file 3:The lack of a significant correlation between the cerebrospinal fluid concentrations of IL-17A, IL-22 and CXCL2 and the cerebrospinal fluid pleocytosis and neutrophil count. The plots presenting cerebrospinal fluid (CSF) total pleocytosis (left) and neutrophil count (right) in tick-borne encephalitis patients on admission to hospital on vertical axes (expressed in cells/μl), plotted against CSF concentrations on cytokines: IL-17A (upper row), IL-22 (middle row), and CXCL2 (lower row) presented on horizontal axes (expressed in pg/ml). The data from individual patients are shown with points (patients with meningitis) and circles , the linear fit with a continuous line. The apparent trend for a positive correlation for IL-17A and CXCL2 did not reach the level of the statistical significance. NS non-significant. (BMP 20257 kb)Additional file 4:The lack of a significant correlation of the cerebrospinal fluid concentrations of the studied cytokines with the cerebrospinal fluid lymphocyte count, total protein, and albumin concentration. The results for IL-17F and CXCL1 are shown, representative for all the cytokines studied. The cerebrospinal fluid parameters in tick-borne encephalitis patients on admission to hospital are shown on vertical axes: lymphocyte count expressed in cells/μl (left), total protein concentration expressed in mg/dl (center), albumin concentration in mg/dl (right), plotted against the simultaneous CSF cytokine concentrations presented on horizontal axes, expressed in pg/ml: IL-17F in the upper and CXCL1 in the lower row. The data from individual patients are shown with points (patients with meningitis) and circles , the linear fit with a continuous line. The trend for a positive correlation apparent on some plots did not reach the level of the statistical significance. NS non-significant. (BMP 20761 kb)


In this investigation, we reported the assessment of CSF biomarkers and their correlations with vesicular monoamine transporter 2 (VMAT2) bindings measured with 18F-9-fluoropropyl-(+)-dihydrotetrabenazine (18F-AV133) that is being developed as a biomarker for PD. We test the hypothesis that monoaminergic degeneration was correlated with CSF biomarker levels in untreated PD patients.Cerebrospinal fluid (CSF) biomarkers, such as α-synuclein (α-syn), amyloid beta peptide 1–42 , 18F-AV133 brain PET, and T1 weighted MRIs. Region of interest (ROI) and voxel-wise Pearson correlation between standardized uptake value ratio (SUVR) and CSF biomarkers were calculated.The available online data from the Parkinson’s Progression Markers Initiative study (PPMI) project were collected and analyzed, which include demographic information, clinical evaluations, CSF biomarkers  hypothesis-driven ROI analysis found a significant negative correlation between CSF Aβ1–42 levels and VMAT2 densities in post cingulate, left caudate, left anterior putamen, and left ventral striatum in PDs. CSF t-tau and p-tau levels were significantly negatively related to VMAT2 SUVRs in substantia nigra and left ventral striatum, respectively. Voxel-wise analysis showed that left caudate, parahippocampal gyrus, insula and temporal lobe were negatively correlated with Aβ1–42. In addition, superior frontal gyrus and transverse temporal gyrus were negatively correlated with CSF p-tau levels.Our major findings are: 1) Compared with controls, CSF α-syn and tau levels decreased significantly in PD; 2) α-syn was closely correlated with AβThese results suggest that monoaminergic degeneration in PD is correlated with CSF biomarkers associated with cognitive impairment in neurodegenerative diseases including Alzheimer’s disease. The association between loss of dopamine synaptic function and pathologic protein accumulations in PD indicates an important role of CSF biomarkers in PD development. Althocontrols . Furthercontrols , 8–10, icontrols .18F-AV133, a positron emission tomography (PET) tracer for VMAT2 imaging that is being developed for commercial distribution, has been shown to be a promising tracer for detecting and monitoring the VMAT2 reduction in PD [18F-AV133-PET data is likely to allow us to directly assess the degeneration of monoaminergic neuron in the living PD brain. In fact, a correlation between 18F-AV133 uptake and cognitive impairment has been reported in Lewy body dementia patients [Vesicular monoamine transporter 2 (VMAT2) is the protein responsible for transporting dopamine, serotonin, and norepinephrine into synaptic vesicles . VMAT2 ipatients .1–42, tau and molecular imaging has been widely studied as measures of diagnosis and disease progression in Alzheimer’s disease (AD) in recent years [18F-AV133-PET. The ultimate goal is to improve the understanding of the interactions between pathologic protein accumulation and the loss of synaptic dopamine function in understanding motor and cognitive aspects of PD.The combination of CSF Aβnt years –18. Combnt years . Thus, ihttp://www.ppmi-info.org/data) were downloaded in December 2014. PPMI is sponsored by The Michael J. Fox Foundation and funded by the Foundation in partnership with 16 biotech and pharmaceutical companies. It is a landmark study launched in 2010 to find biomarkers—disease indicators that are critical missing links in the search for better Parkinson’s disease (PD) treatments. The PPMI data were collected from over 33 clinical sites in 11 countries and the PPMI study was approved by the local Institutional Review Boards (IRBs) of all participating sites. Study subjects and if applicable, their legal representatives, gave written informed consent at the time of enrollment for imaging data, genetic sample collection and clinical questionnaires. Because PPMI is an observational study, research volunteers do not take any experimental drug or placebo, but agree to contribute data and samples for up to five years. The detailed information and complete list of PPMI sites’ IRBs could be found at http://www.ppmi-info.org/.The anonymized and de-identified data from the Parkinson’s Progression Markers initiative (PPMI) database , 18F-AV133 PET scans and T1 weighted structural MRI scans [18F-AV133 images acquired at 80.8 (± 2.8 SD) min post tracer injection were used in the study. As described below in more detail, the downloaded 18F-AV133 PET images were then processed and analyzed at both regions of interest and voxelwise levels. The relationships among 18F-AV133 brain uptake, CSF biomarkers, and PD severity and/or progression, approximated by the UPDRS  motor scores and MoCA  scores were examined. The study was approved by the medical ethical committees at the respective centers, and written informed consent was obtained from all participants.Four hundred and fifteen PD patients and 190 controls with CSF analysis from baseline were included in the current study. In addition, six PD patients and two controls were excluded due to missing p-tau or t-tau values, resulting in 409 PD and 188 controls for further analysis. All participants in the study completed an extensive evaluation battery, including clinical assessments, CSF biomarkers . Because blood contamination might influence CSF α-syn concentrations, hemoglobin (Hgb) was measured in each CSF sample to monitor CSF contamination by red blood cell [All CSF samples were obtained by lumbar puncture as described previously . More deood cell , 5. For ood cell . Based oood cell , 22.18F-AV133 VMAT2 binding in PD progression, the PET and MRI images of the PD patients were reoriented so that the striatum contralateral to the symptomatic side was always on the left of the brain [18F-AV133 [http://www.ppmi-info.org/: AV-133 PET Image Processing Methods for Calculation of Striatal Binding Ratio (SBR). SUVR images were calculated as PET (images)/PET  in the standard space . ROI SUVRs were then obtained by applying ROIs to SUVR images. A 3D spatial Gaussian filter of 8 mm full width at half maximum in x, y, z direction was applied to SUVR images for voxel-wise statistical analysis using SPM8.All PET and MRI images were processed using Statistical Parametric Mapping software  and MATLAB (The MathWorks Inc.). The MRI images were normalized to standard Montreal Neurologic Institute (MNI) space using SPM8 with a high resolution MRI template provided by VBM8 toolbox , and thehe brain , 25. The8F-AV133 , 27. TheAnalyses were performed with Statistical Package for the Social Sciences (SPSS) statistics . Severity of motor signs in patients with Parkinson’s disease was evaluated by using the Unified Parkinson’s Disease Rating Scale motor subscale III (UPDRS-III) , 28. The1–42 was only slightly decreased in PDs (p = 0.475). In contrast to the findings in AD [Four hundred and nine PD patients and 188 healthy controls with baseline CSF biomarkers levels from PPMI were included in this study. The means and standard deviations of demographics and clinical assessments for the PD and control groups are listed in  p = 0.47. In contPrevious studies demonstrated that contamination of blood in CSF could have an influence on the level of some proteins , 3, 5, s1–42 in PD participants . CSF α-syn and p-tau, t-tau levels also displayed strong positive correlations  . These results were not altered appreciably with or without controlling for baseline UPDRS motor scale and MoCA scores. As previously reported [We analyzed the correlations among the CSF biomarkers within the PDs at baseline (BL). There was a significant correlation between CSF α-syn and Aβp<0.001) . However1–42 levels, were found between the early- and late-onset counterparts in the BL analysis . The mean age of this groups of patients was 64.51 yr (range: 33.7–77.3 yr) and the mean disease duration was 18.19 (range: 2–23) months. The median MoCA score and motor scale of these patients were 25.50 (range: 17–30) and 22 (range: 10–46). One limitation of the AV133-PET data in PPMI was that not all the AV133-PET was applied at the same visit, i.e. the PET images might be collected at different visits. As the objective is to analyze the correlations between CSF biomarkers and VMAT2, so the CSF data collected at the same visits as AV133-PET were used. Finally, 11 sets of data collected at baseline, 10 sets of data collected at visit 04 (visits 12 months after BL), and 1 set of data collected at visit 06 (visits 24 months after BL) were used for further analysis.Among the PD patients with CSF biomarkers measurements, 22 of them had 18F-AV133 binding in PD patients were illustrated in The means and standard deviations of ROI SUVRs of 18F-AV133. Results of ROI analysis showed significant negative correlations between Aβ1–42 and 18F-AV133 SUVRs. Correlations were -0.478 for posterior cingulate, -0.628 for left caudate, -0.513 for left anterior putamen, and -0.612 for left ventral striatum  that included the parahippocampal gyrus, insula and temporal lobe  mainly involving the left caudate was negatively correlated with CSF Aβ42 level . SPM mapral lobe . In addiral lobe .18F-AV133 SUVRs in brain regions known for cognitive function, we speculated that these proteins might be related to the cognitive function in PD patients. Subsequently we analyzed the relationship of these proteins with cognitive deficits that indexed by MoCA scores in PD [1–42 level in PD-D when comparing with PD-ND, although not statistically significant, was noted . Analysis of tau levels also showed slightly increased tau levels in cognitive deficit PDs  CSF α-syn positively correlated with Aβ1–42 and tau levels in PD participants, and the correlation was more greater in the early-onset PDs; (iii) CSF Aβ1–42 and tau levels were inversely correlated with VMAT2 SUVRs in cortical brain areas associated with cognitive dysfunction.Our main findings were that: (i) as expected, CSF α-syn were significantly lower in PD as compared with controls. AβSeveral studies, including the present one, have shown that CSF α-syn is significantly decreased in patients with PD compared with controls –6, 8, 11In addition to significantly lowered α-syn level in PD patients, we also found levels of tau decreased in PD than in controls. It is suggested that CSF Aβ, tau, and α-syn may interact synergistically to promote the accumulation of each other in the cortex, and thus contribute to the pathological cascade of PD . So the 1–42 and tau levels showed up, while no relation was found in the analysis of the whole group. One potential testable hypothesis could be that the aggregations and deposition secondary to cross-seeding of the pathologic proteins may be less toxic than the oligomers composed of pure tauopathies, or synucleinopathies, as the amyloid oligomers have emerged as the most toxic species of amyloid-β and tau oligomers may be more closely related to tau neurotoxicity than the presence of the tangles themselves [The direct interactions among CSF biomarkers in Parkinson’s disease have been repeatedly reported , 40. In emselves , 45.1–42 levels and VMAT2 SUVRs in brain areas known for emotion and cognitive function , indicating that the cognitive status might be inversely correlated with CSF Aβ1–42 in PD patients. Although not statistically significant, our observation noted a tendency for an increased CSF Aβ1–42 level with the worsening of the cognitive status in PD. This is counterintuitive because, as previously reported, the CSF levels of Aβ1–42 were lower in patients with PD compared to healthy controls in most studies [1–42 in PD may be caused by the progressive deposition of Aβ1–42 in the brain, or by a decreased production of Aβ1–42 by neurons [1–42. However, our study showed an inverse relationship between neuronal dysfunction and CSF Aβ1–42 levels. Actually, similar findings had been reported by Bouwman et al. They discovered a significant increase in CSF Aβ1–42 over baseline levels in patients with probable AD [1–42 is reduced compared to controls, this may represent a compensatory response and levels might increase with greater cognitive impairment to suggest AD diagnosis [1–42 concentrations [1–42 increase accompanied by a decrease of neuron glucose metabolism might be better explained by the progressive loss of living neurons which causing constitutively producing and secreting β-amyloid protein into CSF.A relationship between β-amyloidopathy and cognitive functions in PD has been reported by in vivo imaging and postmortem studies . The mai studies . The dec neurons , 48. Thubable AD . They coiagnosis , 50. Oneiagnosis . Arlt etThe levels of CSF tau showed an inverse correlation with VMAT2 SUVRs in substantia nigra and left ventral striatum . This is quite similar to the results from the analysis of glucose metabolism and CSF markers , 53. Cer1–42 and tau levels negatively correlated with VMAT2 SUVRs in brain regions associated with cognitive dysfunction in PD, indicating a relationship between these pathologic proteins and dopamine degeneration in PD. Considering the high heterogeneity in PD development, we realized one main limitation of the study is that the CSF biomarker and VMAT2 correlation study was based on only 22 patients. If supported in larger studies the VMAT2 PET measures could relate to subsequent cognitive dysfunction especially in those early onset PD subjects under 50 years. The role of this relationship in PD progression, especially in patients with cognitive decline, can be studied longitudinally with the ongoing PPMI project to identify the earliest neurobiological changes associated with cognitive decline in PD.This pilot study provides imaging evidence that CSF AβS1 FigCSF α-syn and CSF Hgb concentrations were significantly correlated in Parkinson’s disease subjects with Hgb levels above the 200 ng/ml .(TIF)Click here for additional data file.S2 Fig1–42) (A), α-syn and total tau (t-tau), phosphorylated tau 181P (p-tau) (B) in healthy controls evaluated at baseline (n = 188). No significant correlations were found among these indicators. Because interpretation of α-syn might be confounded by blood contamination of CSF samples, 33 subjects with Hgb levels above the 200 ng/ml were excluded from α-syn analysis. Lines indicate trends within each group as determined by linear regression.Correlations between cerebrospinal fluid (CSF) α-syn (α-synuclein) and amyloid beta 1–42 (Aβ(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The knowledge of cerebrospinal fluid (CSF) physiology is mainly presented in hypotheses. Today there are two relevant hypotheses on CSF physiology; the classic one, known as the third circulation ,2, and tSeven years ago, after more than 30 years of our experimental work in the field of CSF physiology, we were the first research group that completely abandoned the classic CSF hypothesis and proposed a new one explaining the relationships between CSF, interstitial fluid (ISF), surrounding tissue of CSF system and blood, based on experimental data, clinic evidence, and relevant scientific publications . AccordiThe regulation of intracranial pressure (ICP) was explained by the classic hypothesis of CSF physiology. It is believed that ICP is regulated by the relationship between CSF secretion, circulation, and absorption. During permanent active CSF formation (secretion), ICP depends on the resistance to CSF flow (circulation) throughout the CSF system and resistance to CSF absorption into the blood. This means that a higher resistance should result in higher ICP. ICP alterations during the changes of body position could also be interpreted in the same way. Furthermore, ICP varies with body position -11, but form = the rate of CSF secretion; Rout = the CSF outflow resistance, and Pdural = the pressure in the dural veins. The Equation 1 for ICP calculation is based on the classic hypothesis because it states that the ICP mainly depends on CSF secretion and absorption by the dural sinuses. Furthermore, Equation 1 shows that the change of body position cannot directly influence the ICP, which means that the observed change of ICP after verticalization can only result from the changes in CSF secretion or absorption rates and/or dural pressure  changes. Hence, if someone would like to study the influence of body posture on ICP, they should determine which of the mentioned parameters are affected. In Holmlund et al’s article , and, consequently, ICP. Finally, the authors explained the experimental results and the mechanism of ICP regulation during changes of body position exclusively in accordance with the classic CSF hypothesis.where I article ,a partiaWe believe that the ICP regulation cannot be explained using the proposed Equation 1 in which those parameters are crucial for ICP calculation. Our new approach to CSF physiology has also required re-evaluation of traditional hypothesis of ICP regulation.2O), but after verticalization becomes sub-atmospheric (about -4.0 cm H2O) inside the cranium and highly positive (about +30.0 cm H2O) in thw lumbar subarachnoid space (2O and +30.0 cm H2O) in the vertical position produces a constant hydrostatic pressure gradient, also observed in humans (2O) in horizontal position, while negative ICP values were measured inside the cranium (about -4.0 cm H2O) and highly positive ones (about +30.0 cm H2O) inside the lumbar space in an upright position (2O to +30.0 cm H2O) both in the phantom experiment and in cats. These values increase in linear fashion with each distance unit from the measuring point inside the cranium toward the lumbar region  and phantom experiment (“plastic-rubber” model) -11. It wid space -11. Thisn humans -8. The sWe did not observe the difference in behavior of CSFP between experimental animals and phantom after verticalization. In phantom, there were no biological and physiological influences, and “CSFP” was defined only by the laws of fluid mechanics. We concluded that CSFP in experimental animals also depended on the laws of fluid mechanics and anatomical characteristics of the cranial and spinal CSF spaces  rather tThe presented hypothesis of the CSFP regulation takes into account the CSF pressure inside the entire CSF system  as a unique department in which the changes of pressure in one part of the system are inextricably linked to the changes in the other . The hypOur research -11 is veAccording to our results -11, the Hypotheses are only theories, verified by more or less numerous facts. Those verified by the most facts are the best, but even then they are never final, never to be absolutely believed.” In other words, the hypotheses, including the classic one, should last so long as experimental and clinical data fit into their framework.Finally, we should not lose sight of what the father of experimental medicine, Claude Bernard said: “


Muscular targets that are deep or inaccessible to surface electromyography (sEMG) require intrinsic recording using fine-wire electromyography (fEMG). It is unknown if fEMG validly record cortically evoked muscle responses compared to sEMG. The purpose of this investigation was to establish the validity and agreement of fEMG compared to sEMG to quantify typical transcranial magnetic stimulation (TMS) measures pre and post repetitive TMS (rTMS). The hypotheses were that fEMG would demonstrate excellent validity and agreement compared with sEMG.In ten healthy volunteers, paired pulse and cortical silent period (CSP) TMS measures were collected before and after 1200 pulses of 1Hz rTMS to the motor cortex. Data were simultaneously recorded with sEMG and fEMG in the first dorsal interosseous. Concurrent validity (r and rho) and agreement (Tukey mean-difference) were calculated.fEMG quantified corticospinal excitability with good to excellent validity compared to sEMG data at both pretest (r = 0.77–0.97) and posttest (r = 0.83–0.92). Pairwise comparisons indicated no difference between sEMG and fEMG for all outcomes; however, Tukey mean-difference plots display increased variance and questionable agreement for paired pulse outcomes. CSP displayed the highest estimates of validity and agreement. Paired pulse MEP responses recorded with fEMG displayed reduced validity, agreement and less sensitivity to changes in MEP amplitude compared to sEMG. Change scores following rTMS were not significantly different between sEMG and fEMG.fEMG electrodes are a valid means to measure CSP and paired pulse MEP responses. CSP displays the highest validity estimates, while caution is warranted when assessing paired pulse responses with fEMG. Corticospinal excitability and neuromodulatory aftereffects from rTMS may be assessed using fEMG. Corticospinal excitability measured with transcranial magnetic stimulation (TMS) is nearly universally assessed via electromyography (EMG) electrodes placed on the skin’s surface (sEMG). sEMG isIntramuscular or indwelling fine wire electrodes (fEMG) capture activity from a small population of single motor units (SMUs) near to the electrode. Use of fEMG with TMS has been implemented to assess coil orientation effects on the latency of evoked responses,8, recruThere is no literature reporting on the validity and agreement of multiunit fEMG to sEMG to quantify common corticospinal measures such as CSP and paired pulse tests. Further, it is unknown if corticospinal excitability measures using fEMG are sensitive to neuromodulation such as repetitive transcranial magnetic stimulation (rTMS). Therefore, before intramuscular targets can be used to measure corticospinal excitability or capture the effects of non-invasive brain stimulation, the validity of fEMG must be assessed against the commonly used sEMG method. With this purpose, we investigated TMS testing outcomes using simultaneous recording of fEMG and sEMG, before and after a session of inhibitory neuromodulation with rTMS.Eleven healthy, right-handed adults ages 24–35 (Mean ± SD: 25.6±3.2) were recruited and ten  completed the study. One subject failed to complete the experiment due to an equipment failure and could not return to the testing site. This study was approved by the University of Minnesota Institutional Review Board and Clinical Translational Science Institute and conformed to the ethical standards of the institutional research committee and with the 1964 Helsinki declaration. Written informed consent was obtained from all individual participants included in the study.A pretest/posttest design was used to compare responses both before and after one session of inhibitory (1Hz) rTMS. The pre and posttest assessment consisted of primary motor cortex excitability testing similar to a previously established protocol –16.Corticospinal excitability was assessed using both sEMG and fEMG simultaneously collected from the right first dorsal interosseous using a 70-mm figure-of-eight coil connected to a Magstim 200 stimulator . sEMG was recorded with a pair of bi-polar silver-silver chloride electrodes . The active electrode was placed on the muscle belly and the reference on the metacarpophalangeal joint . fEMG ac2 [Subjects were seated in a comfortable, semi-reclined chair during testing. Real time EMG signals were displayed on a screen to insure relaxation of the target muscle. The coil was positioned with the handle directed posterolaterally 45° to the mid-sagittal line of the head over the approximate location of the optimal site to elicit an MEP, which is referred to as the hotspot. To determine the hotspot, single-pulse magnetic stimuli were delivered until a MEP was elicited and then the intensity of stimulation decreased to locate the site in the primary motor cortex with the lowest threshold muscle response. Resting motor threshold (RMT) was established similar to a previously established protocol–16. The Y is the individual SICI, ICF or CSP values of the posttest; Peak-to-peak amplitude of each fEMG and sEMG trace was calculated for single and paired pulse responses for the SICI and ICF tests. The mean value of the 10 single pulses was calculated within each subject and each SICI and ICF value was divided by the mean single pulse value for pretest and posttest respectively. For CSP, the EMG data were first rectified, and then a 10ms moving standard deviation (SD) calculation was applied to the data. The onset of the CSP was the time point of the delivery of the stimulus. The average of the moving SD of the pre-stimulus data (-100ms to -5ms) was used as a threshold to determine the offset of the CSP. Offset was defined as the point that the moving SD value returned to the pre-stimulus level. To compe pretest. No outlA normality test (Kolmogorov-Smirnov) was conducted on all measures with the significance set at 0.05. To determine concurrent validity between fEMG and sEMG Pearson’s (r) or Spearman’s (rho) correlation coefficient was calculated, as appropriate for normal and non-normal distributions respectively. Correlation coefficient interpretation was based on general guidelines suggested by Portney and Watkins 2009) for both Pearson’s and Spearman’s coefficients: good to excellent relationship if r >0.75, moderate to good if r = 0.50 to 0.75, and fair if r = 0.25 to 0.50, and little to no relationship if r = 0.00 to 0.25. To asse09 for boThree of the ten subjects reported mild and short duration headaches following the testing session. No other adverse events were found. fEMG traces contained multiunit data sufficient to produce similar waveform to sEMG .Wilcoxon Signed Rank paired test on individual trials indicated no difference between EMG methods for CSP at pretest (p = 0.33) or posttest (p = 0.63) and no difference for ICF at pretest (p = 0.36) or posttest (p = 0.18). There was a significant difference between EMG methods for SICI at pretest (p = 0.04) but not at posttest (p = 0.15). Tukey’s mean difference matched pair comparisons indicated no difference between EMG methods in SICI at pretest , SICI at posttest , ICF at pretest , ICF at posttest , CSP at pretest  and CSP at posttest .Visual analysis of the relationship between sEMG and fEMG at pretest and posttest revealed a large number of SICI estimates by fEMG clustered near to zero, while sEMG estimates demonstrated a larger range of values .Following rTMS, normalized change scores were not statistically different between electrode types for all outcomes. There was a tendency for fEMG to underestimate change scores of paired pulse responses as evident in the normalized change scores . fEMG reOur results indicate that fine wire electrodes are a valid means of collecting the EMG signal for TMS measures including SICI, ICF and CSP. Among these measures, CSP displayed the highest validity and agreement while paired pulse responses were found to have excellent validity despite variable agreement when compared to sEMG. SICI data should be interpreted with caution when using fEMG, as results may be underestimated.Previous studies in the first dorsal interosseous have shown that fEMG data are sensitive to the conditioning effects of paired inter- and intra- hemispheric TMS using peri-stimulus time histograms,22. Our The clustering effect of data near to zero in correlation graphs, and the lower estimates of validity and graphical agreement for ICF and SICI in fEMG compared to sEMG may be the result of a limited pool of motor units that summate on the indwelling electrode. A small sample of motor units within the recording area may limit the ability for the fEMG trace to vary in amplitude across trials, unlike sEMG that assess a larger sample of motor units. Since ICF and SICI are calculated based on a ratio of MEP amplitude, this finding should be carefully considered in future work. CSP duration would not be influenced as greatly by this, as it is quantified using the timing of EMG activation, not the amplitude of the MEP. SICI responses may be more susceptible to a floor effect, as SICI reflects the ratio of the paired pulse inhibited MEP amplitude to the single pulse MEP amplitude. If only few motor units are within the recording area, a paired pulse SICI test may result in full suppression of all motor units and hence a very small MEP amplitude or no response could be recorded in the fEMG trace. Thus, the outlined SICI data points near zero fEMG  may reflB medicated intracortical inhibition, as CSP displayed the highest levels of validity and agreement for both fEMG and sEMG. However, it has been reported that SMU based analyses, such as a peristimulus frequencygram, estimate a longer CSP compared to probability based analyses and standard sEMG[Our results support the use of CSP to assess the effects of rTMS on corticospinal excitability, specifically GABAdard sEMG. Todd andard sEMG. Accordidard sEMG. Yet, usThere are limitations to this study. One limitation is that the number of SMUs recorded in each subject were not assessed. If too few SMUs lie within the recording area, there may be insufficient summation of motor units for amplitude modulation in response to paired pulse protocols. This may be one reason fEMG is less sensitive to changes in SICI, as too few SMUs would not allow amplitude modulation of the EMG trace. It is unknown how many MUs are required to generate reliable measures of corticospinal excitability. Additionally, the RMT for the TMS intensity was determined using the sEMG traces, which may not translate to the same threshold with fEMG. This concern is lessened because threshold values are nearly the same  using sEMG based on amplitude and fEMG data based on SMU firing probability. Lastly,Our results indicate that CSP and paired pulse outcomes display excellent validity while agreement varied between testing outcomes. This methodology may be used in other venues of neurophysiology research that seeks to perform comprehensive TMS testing and delineate neuromodulatory effects in musculature that is inaccessible to sEMG. Future applications may aim to enhance neuromotor control to pelvic floor muscles, the diaphragm or laryngeal musculature.S1 AppendixData for outcomes at both pretest and posttest are provided for sEMG and fEMG. Tabs include separate outcomes: Short-interval intracortical inhibition (SICI), intracortical facilitation (ICF), cortical silent period (CSP).(XLSX)Click here for additional data file.