N--carbamoyl] (DC-chol) (DOTAP/DC-chol liposomes). However, the mechanism(s) underlying the mucosal adjuvant effects exerted by the cationic liposomes have been unclear. In this study, we investigated the role of granulocyte–macrophage colony-stimulating factor (GM-CSF), which was reported to act as a mucosal adjuvant, on the mucosal adjuvant activities of DOTAP/DC-chol liposomes when administered intranasally to mice.Infectious diseases remain a threat to human life. Vaccination against pathogenic microbes is a primary method of treatment as well as prevention of infectious diseases. Particularly mucosal vaccination is a promising approach to fight against most infectious diseases, because mucosal surfaces are a major point of entry for most pathogens. We recently developed an effective mucosal adjuvant of cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[Here, we show that, although intranasal vaccination with cationic liposomes in combination with antigenic protein elicited GM-CSF expression at the site of administration, blocking GM-CSF function by using an anti-GM-CSF neutralizing antibody did not alter antigen-specific antibody production induced by DOTAP/DC-chol liposomes, indicating that GM-CSF may not contribute to the mucosal adjuvant activity of the cationic liposomes when administered intranasally.The online version of this article (10.1186/s13104-018-3591-3) contains supplementary material, which is available to authorized users. Vaccines have been a great public health success in past decades. However, the development of additional safe and efficient vaccines against various infectious diseases is still a challenge –3. AmongN--carbamoyl] (DC-chol) (DOTAP/DC-chol liposomes) potently induced both mucosal and systemic immune responses to the antigen in mice  (DC-chol) were purchased from Avanti Polar Lipids . Recombinant mouse GM-CSF, anti-GM-CSF neutralizing antibody, and rat IgG2a κ isotype control antibody were all purchased from BioLegend . Low endotoxin  egg white ovalbumin (OVA) was obtained from Wako Pure Chemical Industries .Female BALB/cCrSlc mice (7–10 weeks old) were purchased from Japan SLC . Animals were housed in a specific pathogen-free environment and all animal experiments were approved by the institution’s committee for laboratory animal experiments of the Tokyo University of Pharmacy and Life Sciences . 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[DOTAP/DC-chol liposomes were prepared as follow . 10 μmolMice were immunized twice intranasally once a week (days 0 and 7). Mice were divided into three groups as follows: (1) vehicle (PBS), (2) OVA alone (5 µg/mouse), or (3) OVA (5 µg/mouse) plus liposomes (400 nmol/mouse) or recombinant mouse GM-CSF (4 µg/mouse described previously ). After A 96-well plate was coated with OVA in a 0.1 M carbonate buffer (pH 9.5). The plate was washed and then blocked with 1% bovine serum albumin  containing PBST (BPBST) for 60 min at 37 °C. After washing, the plate was incubated with samples  for 60 min at 37 °C. To detect OVA-specific IgG antibody, plates were washed with PBST, treated with peroxidase-conjugated anti-mouse IgG  in BPBST. The plate was washed, and combined with TMB substrate  and then further incubated for color development. To detect OVA-specific IgA, IgG1, and IgG2a, plates were treated with biotin-conjugated anti-mouse IgA, IgG1, or IgG2a (BioLegend) in BPBST, and then avidin-horseradish peroxidase (BioLegend) in PBST was added. Plates were incubated with TMB substrate system (KPL). The reaction was terminated with 1 N phosphoric acid, and optical densities were measured at 450 nm/650 nm –24. Endp6 cells/well in 0.5 mL of culture medium in 48-well flat-bottomed plates (IWAKI) and re-stimulated with OVA  for the indicated time at 37 °C in a 5% CO2.Splenocytes were prepared as described earlier , 27. BriThe cytokine concentrations were measured using ELISA MAX Standard Sets (BioLegend) according to the manufacturer’s instructions. The data were expressed as the mean ± standard deviation. At least three independent experiments were conducted.Δ-threshold cycle method using β2-microglobulin as a reference gene. GM-CSF expression is presented as the fold change relative to expression in the control sample.BALB/c female mice were euthanized by sodium pentobarbital administration . Their nasal tissues and spleens were then excised, and the total RNA was extracted from these samples using a FavorPrep Tissue Total RNA Mini Kit , followed by DNase I  treatment. cDNA was synthesized from total RNA using a ReverTra Ace qPCR RT Master Mix . Then, qPCR was carried out according to the manufacturer’s instructions using a THUNDERBIRD SYBR qPCR Mix (Toyobo). The primers used for PCR were the following: GM-CSF, forward, 5′-TGGGCATTGTGGTCTACAGC-3′, and reverse, 5′-GCGGGTCTGCACACATGTTA-3′; β2-microglobulin, forward, 5′-TTCTGGTGCTTGTCTCACTGA-3′, and reverse, 5′-CAGTATGTTCGGCTTCCCATTC-3′. The expression of GM-CSF was determined using the comparative BALB/c female mice were pre-treated intraperitoneally with anti-GM-CSF neutralizing antibody (100 µg/mouse) 2 days before (day-2) and 1 h before immunization (days 0 and 7) as reported previously –31. Micet-test with Welch’s correction and the Kruskal–Wallis test with Dunn’s post hoc test for cytokine and antibody production, respectively. Differences with p values of < 0.05 were considered significant.Statistical differences were calculated with unpaired First, we evaluated the production of OVA-specific antibodies after intranasal immunization of OVA in combination with DOTAP/DC-chol liposomes in BALB/c female mice. As expected, intranasal vaccination induced the production of OVA-specific nasal IgA in nasal fluid and IgG in the serum compartment. In contrast, intranasal immunization with PBS (vehicle) or OVA alone did not exhibit significant OVA-specific antibody production in either mucosal or systemic under these experimental conditions  intranasal administration with DOTAP/DC-chol liposomes induced the expression of GM-CSF at the site of injections; (2) recombinant GM-CSF showed the mucosal adjuvant effect when nasally administered in mice; and (3) GM-CSF expression in nasal area induced by DOTAP/DC-chol liposomes was not required for the mucosal adjuvant activities.The development of safe and efficient mucosal adjuvants is needed to prevent fatal infectious diseases. To accomplish this, understanding the mechanism(s) underlying mucosal adjuvant induction of immune responses to antigenic proteins is essential. Generally, adjuvants show their activities through the depot effect, with the gradual release of antigen at the site of infection and increase in antigen uptake by APCs. Resent research has focused on the role of APCs in activating innate immunity . In partHerein, we demonstrated that nasal administration of DOTAP/DC-chol liposomes induced the gene expression of GM-CSF at the site of administration; however, the protein level of GM-CSF in the nasal area after the nasal immunization of DOTAP/DC-chol liposomes has not been evaluated. The major limitation of this study is that though the protocol for studying the biological activities of GM-CSF neutralization at the mucosal compartments by intraperitoneal injection of anti-GM-CSF neutralizing antibody has been reported in literatures –31, we hAdditional file 1: Figure S1. DOTAP/DC-chol liposomes potentiate both mucosal and systemic OVA-specific antibody responses. The data show the OVA-specific nasal IgA and serum IgGs for each immunized group . The data were obtained from three independent experiments. The statistically significant value (*p < 0.0001) shown were calculated from the Kruskal–Wallis test with Dunn’s post hoc test.


To the Editor—We thank Dr Dian and co-authors for their interest in our recent publication [lication . One of Our prognostic tool did not perform well on the Indonesian cohort, although differences in the tool’s performance between the 2 cohorts are not altogether surprising. As indicated by the authors, patients in the Indonesian cohort had more severe disease at the start of treatment than the Vietnam cohort; but baseline Medical Research Council disease severity grade were included in our prognostic models and thus would be unlikely to explain the tool’s poor performance in the Indonesian cohort.Mycobacterium tuberculosis from CSF in Vietnam [Instead, recent publications allow comparison of measures and determinants of cerebrospinal fluid (CSF) inflammation and outcomes between the 2 populations and suggest there may be biological differences between them that may lead to different prognostic variables . For exa Vietnam , but unl Vietnam . Studies Vietnam , 7. ThesThe authors suggest that CSF culture and neutrophil counts might be more reliable predictors of death than those we modeled. CSF cultures were not included in our models because the results are not available before the start of treatment and therefore are not of clinical utility when defining prognosis at diagnosis. CSF neutrophil count is strongly correlated with CSF lymphocyte count and, for this reason, was not included in the model; although as described above, neutrophils do appear important in TBM pathophysiology. However, we thank the authors for exposing a fault in the web-based tool: CSF lymphocyte counts >500/µL cannot currently be entered. We have therefore modified the tool to allow submission of higher counts.


We designed and synthesized a donor-acceptor-donor dye consisting of a 2,7-disubstituted fluorenone with diethylaminophenyl moieties present as strong electron donating groups. Switching between twisted intramolecular charge transfer (TICT) emission and excimer emission was achieved, with no ground state changes, by simply changing the solvent used. In a nonpolar solvent, excimer emission was observed; with increasing polarity, the emission gradually disappeared, and the TICT emission appeared. Twisted intramolecular charge transfer (TICT) with donor-acceptor dyes is a basic phenomenon utilized in various applications, and investigations have been carried out in order to analyze this phenomenon in detail ,2. One oIn the case when a donor-acceptor dye forms an excimer, the ratio of TICT emission decreases, and excimer emission appears  18,19].,19.18,19In this paper, we report a newly synthesized donor-acceptor type dye that can be switched between TICT and excimer emission, without any ground state changes, by simply changing the solvent. We hypothesized that fluorenone was a suitable structure to use in this study owing to its tendency to form excimers . FurtherThe structure of the dye and its synthesis condition are shown in 3)4 and Cs2CO3 catalysts in THF at 60 °C for 2 days.The dye was synthesized via a Suzuki-Miyaura coupling  using 2,We obtained absorption spectra of the compound in various solvents: acetonitrile, DMF, chloroform, THF, and toluene. The dye concentration was set as the concentration at which a maximum absorbance of 0.1 was achieved. −6 to 1.0 × 10−5 mol/L. For the other two peaks, the intensities gradually stopped increasing. This suggested that the 645 nm fluorescence peak was due to excimer emission, and the other two peaks originated from single molecule emissions. From previous reports + Calcd for C33H34N2ONa: 497.2564, Found : 497.2555.To a mixture of 2,7-dibromofluorenone , Pd(PPhIn this work, we designed a donor-acceptor-donor dye based on fluorenone, in order to achieve switching between TICT and excimer emission without ground state changes, by the simple expedient of changing the solvent. The dye was synthesized via a Suzuki-Miyaura coupling, and absorption and emission spectra in various solutions were obtained. The results indicate that excimer formation was enhanced in nonpolar solvents and that by increasing the solvent polarity, the excimer quantum yield decreased gradually and a TICT peak appeared. This unique phenomenon constitutes a very promising basis for the development of solvent polarity sensors.


TREM2 gene increase the risk for late-onset Alzheimer’s disease (AD). Soluble TREM2 (sTREM2) resulting from shedding of the TREM2 ectodomain can be detected in the cerebrospinal fluid (CSF) and is a surrogate measure of TREM2-mediated microglia function. CSF sTREM2 has been previously reported to increase at different clinical stages of AD, however, alterations in relation to Amyloid β-peptide (Aβ) deposition or additional pathological processes in the amyloid cascade (such as tau pathology or neurodegeneration) remain unclear. In the current cross-sectional study, we employed the biomarker-based classification framework recently proposed by the NIA-AA consensus guidelines, in combination with clinical staging, in order to examine the CSF sTREM2 alterations at early asymptomatic and symptomatic stages of AD.TREM2 is a transmembrane receptor that is predominantly expressed by microglia in the central nervous system. Rare variants in the TREM2 rare genetic variants, was conducted to measure CSF sTREM2 using a previously validated enzyme-linked immunosorbent assay (ELISA). ADNI participants were classified following the A/T/N framework, which we implemented based on the CSF levels of Aβ1-42 (A), phosphorylated tau (T) and total tau as a marker of neurodegeneration (N), at different clinical stages defined by the clinical dementia rating (CDR) score.A cross-sectional study of 1027 participants of the Alzheimer’s Disease Imaging Initiative (ADNI) cohort, including 43 subjects carrying TREM2 variants, whereas the p.R47H variant had higher CSF sTREM2, p.L211P had lower CSF sTREM2 than non-carriers. We found that CSF sTREM2 increased in early symptomatic stages of late-onset AD but, unexpectedly, we observed decreased CSF sTREM2 levels at the earliest asymptomatic phase when only abnormal Aβ pathology (A+) but no tau pathology or neurodegeneration (TN-), is present.CSF sTREM2 differed between Aβ pathology (A) and tau pathology/neurodegeneration (TN) have differing associations with CSF sTREM2. While tau-related neurodegeneration is associated with an increase in CSF sTREM2, Aβ pathology in the absence of downstream tau-related neurodegeneration is associated with a decrease in CSF sTREM2.The online version of this article (10.1186/s13024-018-0301-5) contains supplementary material, which is available to authorized users. TREM2 mutations strongly increase the risk of developing Alzheimer’s disease (AD) [TREM2 are sufficient to cause Nasu-Hakola disease (NHD) and FTD-like syndrome [The triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune receptor that is expressed on the plasma membrane of microglia in the central nervous system (CNS) . TREM2 iase (AD) , 8 and oase (AD) –12. Furtsyndrome , 14. TogTREM2 is a type-1 transmembrane protein that matures within the secretory pathway and its ectodomain is shed at the plasma membrane . Solubleknock-in mouse model bearing the Trem2 p.T66M mutation has decreased microglial activity [The fact that TREM2 is selectively expressed in microglia in the CNS and is associated with AD and neurodegeneration, let us hypothesize that sTREM2 in CSF may be a marker for microglia function and its response to Aβ and tau pathology and neurodegeneration. Specifically, sTREM2 may reflect the amount of signaling competent TREM2 on the surface of activated microglia. This idea is supported by the fact that the levels of sTrem2 in the brain of an Aβ mouse model correlate with TSPO small animal positron emission tomography (μPET) signal , a markeactivity .We and others have previously reported changes in the levels of CSF sTREM2 in AD compared to controls , 23–26. continuum and hence replicate ours and others findings in the ADNI cohort [An important unanswered question in this regard concerns the association between CSF sTREM2 and primary pathologies including Aβ and tau deposition, as well as neurodegeneration during the course of AD. Therefore, we used herein the biomarker-based A/T/N classification system , which iI cohort –26, 29.http://adni.loni.usc.edu) on 16/03/2018 and the data used in this study was downloaded on 21/03/2018. The ADNI project (http://www.loni.usc.edu/) is a multicenter longitudinal study led by Principal Investigator Michael W Weiner with the main goal to develop and validate biomarkers for subject selection and as surrogate outcome measures in late-onset AD [This is a cross-sectional study in which CSF sTREM2 was measured in 1031 participants of the ADNI project. Among them, 4 individuals did not have the AD core biomarkers measurements and were further excluded from the analysis, rendering a study sample of 1027 subjects. The CSF sTREM2 measurements were uploaded to the ADNI database  and neurodegeneration (N) groups in order to decrease the number of groups to be compared. TN negative (TN-) was defined as having both the aggregated tau (T) and neurodegeneration (N) biomarkers in the normal range . Participants were classified as TN positive (TN+) if either aggregated tau (T) or neurodegeneration (N) were abnormal . Only 5.4% of the individuals of the total differed between the T and N biomarkers groups.In line with the recently published 2018 NIA-AA “research framework” for the diagnosis of Alzheimer’s disease , we assicontinuum category, that is: ‘Preclinical AD A+/TN-’, ‘Preclinical AD A+/TN+’, ‘AD CDR = 0.5’ and ‘AD CDR = 1’. Since our aim was to study the Alzheimer's continuum, we excluded from this analysis those individuals that fall in the category of suspected non-Alzheimer’s pathology (SNAP) [1-42) and neurodegeneration/tau pathology (T-tau or P-tau181P).The combination of the biomarker profile (A/T/N scheme) and the clinical status (CDR) rendered 12 different groups that are displayed in Table y (SNAP) –38 and tIn the present study, we used the AD CSF core biomarkers measurements performed with the Elecsys® total-tau CSF, the Elecsys® phosphotau(181P) CSF and the Elecsys® β-amyloid(1–42) CSF immunoassays on a cobas e 601 instrument , 40. TheCSF sTREM2 measurements were done with a MSD platform-based assay, previously reported and validated , 26. A cTREM2 variants using Lipofectamine 2000 as the transfection reagent. TREM2 variant constructs were generated by site-directed mutagenesis (Stratagene) of a TREM2 wt construct bearing N- and C-terminal HA and FLAG tags, respectively, as previously described [HEK293T cells were cultured in DMEM with Glutamax I supplemented with 10 % (v/v) fetal calf serum (FCS) and 1 % penicillin/streptomycin. 24 hours after seeding, cells were transiently transfected with equal amounts of DNA coding for the different We measured the concentrations of HA-labeled TREM2 protein in the HEK293T conditioned media by two different ELISAs. First, sTREM2 concentrations were determined by the same ELISA used to measured sTREM2 in the human CSF samples, which includes a detection antibody against sTREM2  and were hence log10-transformed. After transformation, CSF sTREM2 followed a normal distribution as assessed by Kolmogorov-Smirnov test (P = 0.200) and visual inspection of the histogram. All the statistical analysis described in this study are performed with the log10-transformed values.CSF sTREM2 did not follow a normal distribution  was conducted to determine statistically significant differences on CSF sTREM2 between TREM2 rare variant carrier and 1 a non-carrier), 1 classified as ‘CDR = 0.5 A+TN-’ (TREM2 rare variant carrier), 1 classified as ‘AD CDR = 0.5’ (TREM2 rare variant carrier), 1 classified as ‘AD CDR = 1’ (non-carrier). Including or excluding these outliers do not change the findings of this study.The following analyses were conducted excluding outliers’ values of CSF sTREM2, defined as values differing 3 standard deviations from the mean. There were 5 outliers: 2 subjects classified as ‘Preclinical AD A+TN-’  with a multiple linear regression adjusted for age. The analysis was conducted separately in the healthy controls, Alzheimer’s continuum and SNAP groups. We performed the analysis both including or excluding outliers (defined as AD CSF core biomarkers 3 standard deviations below or above the group mean) in order to exclude that the associations were driven by extreme values. The analysis with and without outliers rendered similar results. For CSF Aβ1-42, the analyses were performed using both the truncated values at the upper technical limit and the exploratory measurements available based on the extrapolation of the calibration curve. In the main text, we report the results using the extrapolated measurements, but using the truncated ones yielded similar results.Finally, we studied the association between CSF sTREM2 and each of the CSF core biomarkers for AD . Figures were built using GraphPad Prism or free statistical software R. All tests were 2-tailed, with a significance level of α = 0.05.Statistical analysis was performed in SPSS IBM, version 20.0, and the free statistical software R  and Bonferroni’s post hoc comparisons test indicated that the p.R47H variant [P = 0.003) and the p.L211P variant [P = 0.002) than non-carriers. No differences in CSF sTREM2 were found between individuals with a p.R62H [TREM2 rare variant had relatively high CSF sTREM2  and mutated TREM2 and measured sTREM2 released in the media with the same ELISA used for the quantification of CSF sTREM2 and with an ELISA using an antibody against the epitope tag , we first assessed which demographic and genetic variables are associated with CSF sTREM2 , but not with gender  or APOE ε4 status . Consequently, all further analysis included age as a covariate, but not gender or APOE ε4 status.In the sample excluding the 181P were highly correlated, we merged the tau (T) and neurodegeneration (N) groups. ‘TN-’ profile was defined as both CSF P-tau181P and T-tau within the normal range, whereas ‘TN+’ was defined as abnormal levels of CSF P-tau181P or T-tau. Thus, we compared 4 different biomarker profiles within each clinical stage, namely: (1) A-/TN-, (2) A+/TN-, (3) A+/TN+ and (4) A-/TN+.In order to assess the impact of Aβ deposition or the downstream processes of the amyloid cascade (i.e. tau pathology and neurodegeneration), we applied the recently proposed A/T/N classification framework of AD, which proposes 3 binary biomarker groups : (1) agg3,286 = 21.3, P < 0.0001, ηp2 = 0.183). A Bonferroni post hoc test revealed that the A+/TN- profile had significant lower CSF sTREM2 compared to either other biomarker profile , a one-way ANCOVA showed a significant difference between the four biomarker profiles after adjusting for the effect of age  and the A+/TN- profile had also the significantly lowest CSF sTREM2 compared to either other biomarker profile , there was also a significant difference between the four biomarker profiles  and tau pathology status , that is A/T classification, or based only on their Aβ pathology  and neurodegeneration status , that is A/N classification. The results are shown in Figure S2 . A post hoc analysis using the Bonferroni criterion for significance indicated that the average CSF sTREM2 was significantly higher in the ‘AD CDR = 0.5’ group than in the ‘healthy controls’ and ‘Preclinical AD A+TN-’ groups  in the SNAP groups. Consistent with previous findings, CSF sTREM2 is associated with T-tau and P-tau181P in the three groups studied  is associated with decreased CSF sTREM2, tau pathology or neurodegeneration  are associated with an increase in CSF sTREM2. The higher CSF sTREM2 in the SNAP groups confirm that CSF sTREM2 rises with neurodegeneration without Aβ pathology.In the present study, we assessed the microglial-activity marker CSF sTREM2 within the early phases of AD. To this end, we applied the biomarker-based A/T/N classification in combination with clinical staging . The useTREM2 variants and they are increased in the p.R47H TREM2 variant compared to non-carriers, but decreased in the p.L211P variant and remained unchanged in the p.R62H variant, which is consistent with previous reports [Moreover, we show that the levels of CSF sTREM2 differ between  reports . Increas reports . A word 181P and CSF T-tau to be highly correlated , and consequently we merged the “T” (tau pathology) and “N” (neurodegeneration) groups. Importantly, the TN- group had normal levels of both T-tau and P-tau181P, which reasonably ensure that other comorbidities that may cause neural injury  are excluded. A plethora of other biofluid and neuroimaging markers  are in principle applicable for the implementation of the A/T/N classification, and future studies using these biomarkers are needed to confirm our results.Among the strengths of this study are the large and well-characterized sample size and the use of a reliable assay to measure sTREM2. Yet, this study has some limitations. First, this is a cross-sectional study and the results need to be confirmed in a longitudinal setting. Second, we used the CSF biomarkers to classify the ADNI participants in the A/T/N classification. Although the role of CSF biomarkers in AD is well-established and a complete A/T/N characterization is possible only with CSF biomarkers, CSF T-tau may not necessarily reflect neurodegeneration but could result from physiological production of tau . Here, w181P, but not with CSF Aβ1-42. A word of caution is needed in the SNAP category, given that this is an heterogenous group that most likely exhibits a non-AD related neurodegeneration. Well-powered future studies should address how CSF sTREM2 changes in neurodegenerative diseases different from AD.The A/T/N classification used herein is a descriptive biomarker-based classification that does not assume any temporal sequence of events in AD and is independent of the clinical stage of the disease. By applying this classification framework, we found an unexpected observation, namely a decrease of CSF sTREM2 in individuals with evidence of Aβ pathology but without signs of tau pathology or neurodegeneration. We did not observe this finding in previous studies, probably due to the low number of participants in the preclinical stage of late-onset AD and because we did not apply the A/T/N classification . NoteworThe flexibility of this new classification framework also enabled us to model the natural history of AD and confirm in the ADNI study our previous findings in participants of several European memory clinics  who wereet al. Nat. Neursci in press) and are therefore overrepresented in the Preclinical AD A+TN- group. Nevertheless, we are cognizant of the fact that this is an observational study and the findings reported herein do not elucidate precise mechanisms. Further work with longitudinal data is needed to address whether the stage-dependent changes in CSF sTREM2 predict a better or worse clinical outcome.The mechanism underlying the dynamic changes of CSF sTREM2 throughout the course of the disease still need to be investigated. Several studies have consistently demonstrated that microglia upregulate TREM2 expression in AD mouse models and in human AD brains , 47, 48.In conclusion, the present study represents the first attempt to study CSF sTREM2 based on the A/T/N classification framework. We demonstrate in the ADNI cohort that the increase in CSF sTREM2 which occurs in early stages parallels the increase in biomarkers of tau pathology and neurodegeneration. In contrast, Aβ deposition in the absence of tau deposition and neurodegeneration is associated with lower CSF sTREM2.Additional file 1:Figure S1. Effects of rare TREM2 variants on antibody affinities. Figure S2. CSF sTREM2 in the A/T and in the A/N classifications. Table S1. Demographic and clinical characteristics of the entire sample. Table S2. Associations of CSF sTREM2 with AD CSF core biomarkers including the biomarkers outliers. Table S3. Associations of CSF sTREM2 with AD CSF core biomarkers including subjects carrying a TREM2 rare variant. (DOCX 1992 kb)CSF sTREM2 measurement.


P=0.029) and shorter duration of drainage (P<0.001). Both groups displayed similar rates of vasospasm, hydrocephalus, intracerebral hemorrhage, the Glasgow Outcome Scale score one month after endovascular coiling and length of stay . In conclusion, both LD and serial LP are effective methods in the treatment of aneurysmal subarachnoid hemorrhage; besides, serial LP can reduce the incidence of CSF infection in draining hemorrhagic CSF in aneurysmal subarachnoid hemorrhage after endovascular coiling.The present study aimed to compare the complications and clinical outcomes of serial lumbar puncture (LP) and lumbar cerebrospinal fluid (CSF) drainage (LD) of patients with aneurysmal subarachnoid hemorrhage and provide more evidence to guide clinical management. In this retrospective study, 41 and 39 aneurysmal subarachnoid hemorrhage patients were enrolled in the LP and LD group, respectively. Clinical outcomes, including CSF infection, intracerebral hemorrhage, vasospasm, hydrocephalus, death, length of stay, duration of drainage and the Glasgow Outcome Scale score were compared between the two groups. By comparing with the LP group, the LD group showed a significantly higher rate of CSF infection ( The pathophysiology of aSAH is not completely known, while, undoubtedly, the presence of blood in the subarachnoid space is one important factor associated with these complications. Removal of bloody cerebrospinal fluid (CSF) plays an important role in the management of aSAH. Various studies have revealed that early removal of hemorrhagic CSF after the onset of aSAH can significantly cut down the incidence of vasospasm and hydrocephalus associated with aSAH[3‒.As a disease needing neurosurgery, aneurysmal subarachnoid hemorrhage (aSAH) is characterized with high morbidity and mortality. Although improved diagnostic techniques, emergency treatments and operations have lowered the mortality rate of aSAH, the overall outcome is still undermined by some complications, such as vasospasm and hydrocephalus, but it needs special surgical instruments and should be performed in the operating room, generally for patients with massive intraventricular hemorrhage or acute hydrocephalus. Even so, both serial LP and LD have been considered as conventional CSF drainage methods in the clinical treatment of aSAH and widely applied around the world. Unlike external ventricular drainage, serial LP and LD can be performed rapidly at bedside without the risk of sedation or general anesthesia.There exist three primary methods to drain hemorrhagic CSF: serial lumbar puncture (LP), lumbar CSF drainage (LD) and external ventricular drainage. External ventricular drainage, a standard approach in treating SAH-associated hydrocephalus, has been proven to be effective in CSF drainage[5‒. Recently, LD has gradually become another option to promote the clearance of blood from the subarachnoid space. Clinical studies have confirmed that LD is also an effective way to reduce aSAH associated incidence of vasospasm and hydrocephalus[3,.As a classical neurosurgery, serial LP has remained a basic method in clinical treatment of aSAH. Many studies have confirmed that draining hemorrhagic CSF by serial LP can significantly depress the complications of aSAH and improve the outcome of patients with aSAHHowever, each CSF drainage method has its advantages and disadvantages and comparisons between these methods are rare. As the two most common methods, it is necessary to compare the safety and efficacy of serial LP and LD in treating aSAH. Therefore, this study aimed to compare the complications and clinical outcomes of serial LP and LD of patients with aSAH and discover more evidence to guide management.A total of 80 patients with aSAH were included in this retrospective study. All the patients underwent endovascular coiling of intracranial aneurysm at the Department of Neurosurgery of the First Affiliated Hospital of Xi’an Jiaotong University between January 2009 and January 2014. The inclusion criteria were (1) spontaneous primary aSAH confirmed by CT scan and digital subtraction angiography (DSA); (2) Hunt & Hess grades Ⅱ to Ⅳ; (3) Fisher grades 2, 3, 4; (4) receiving the endovascular coiling within 36 hours after admission and receiving hemorrhagic CSF drainage less than 48 hours after onset. According to the different methods of hemorrhagic CSF drainage in the postoperative period, patients were divided into two groups. One group underwent serial LP, and the other LD. All other aspects of treatment were similar. This study was approved by the ethical committee of the First Affiliated Hospital of Xi’an Jiaotong University. Patient consent was not required because of the retrospective nature of the study.According to a consistent protocol with the exception of different CSF drainage management methods, all patients were given similar treatments. The diagnosis of aneurysm was confirmed by DSA and the initial CT scan was adopted to confirm and classify SAH using a Fisher grading system. Patients with ruptured aneurysm received early endovascular coiling within 36 hours after admission. Other treatments included blood pressure control, administration of nimodipine, maintenance of euvolemia and normal circulating blood volume, daily transcranial Doppler examination, rigorous monitoring and correction of electrolytes, blood gases and intracranial pressure (ICP), regular CSF examination, and CT scan.2O.Serial lumbar puncture was performed daily from postoperative day 1 with the following steps. The patient was placed in the lateral position. The puncture site was L4‒L5 or L3‒L4 intervertebral space. After regular skin disinfection, 1% lidocaine solution was injected from the skin into the interspinous ligament for local infiltrative anesthesia. A lumbar puncture needle was inserted into the subarachnoid space and hemorrhagic CSF was drained. Each time, a maximum of approximately 20 mL CSF was slowly drained; and the ICP was maintained with approximately 15 cmHLumbar CSF drainage was also performed since postoperative day 1. The procedure of lumbar CSF drainage was similar to that of lumbar puncture as described above. After the needle was inserted, a catheter was put through the needle into the subarachnoid space, and then the needle was removed. The catheter was then fixed to the skin and connected to an external drainage system. The collection rate was targeted at 5 to 10 mL per hour. CSF drainage in each group was stopped once the CSF was no longer visibly hemorrhagic.CSF was collected and analyzed every day. If leukocytosis in CSF was observed, CSF cultivation was performed; once positive, CSF infection was confirmed. Abnormal CSF findings (CSF/blood glucose ratio less than 0.5 and a neutrophilic CSF pleocytosis) and clinical manifestations (body temperature>38 °C) with negative CSF culture were defined as suspected infection. Both situations were defined as CSF infection.Regular CT scan was performed per 2 or 3 days. Additionally, patients received emergency CT scan anytime if neurological symptoms worsened or new symptoms appeared. Intracerebral hemorrhage was confirmed by CT scans.. Some patients with normal results by transcranial Doppler were also suspected of vasospasm if CT scans revealed a new region of cerebral infarction consistent with the distribution area of the intracranial artery. All patients underwent cerebral angiography when vasospasm was suspected on clinical evidence.Daily transcranial Doppler was performed in all patients; if the mean arterial velocity was higher than 120 cm/s, they were suspected of vasospasmThe diagnosis of hydrocephalus was based on neuro-imaging examinations. If CT scan showed that the dilated lateral ventricles and the third ventricle and the cella media index were above 0.25, and the third ventricle width was larger than 7 mm, hydrocephalus was confirmed.To evaluate the short term outcome, the GOS score was measured one month after endovascular coiling.The analysis of duration of drainage excluded those patients who suffered CSF infection, for they needed an extended period of lumbar CSF drainage. In addition, patients who died were excluded from analysis of length of stay and duration of drainage.t test was used to compare continuous data, which were presented as mean±standard deviation. P<0.05 was considered as statistically significant.Data were analyzed using SPSS 17.0. Chi-square test, Fisher’s exact test and Yates’ continuity correction test were applied to compare categorical variables between groups. Student Table 1. A total of 80 patients with aSAH were included in this study with 41 patients in the LP group and 39 in the serial LD group. Patient characteristics had no statistically significant differences between the two groups, including age, gender, Hunt & Hess grade, Fisher grade and aneurysm type .Patient characteristics, including age, gender, Hunt & Hess grade, Fisher grade, and aneurysm type in the two groups, are shown in Table 2, 6 patients had CSF infection during the removal of hemorrhagic CSF. All of them received lumbar CSF drainage (LD). Four of them were confirmed by CSF culture and two were suspected of infection. Two of these patients died of severe infection. No patient had CSF infection in the serial LP group. The CSF infection rate in the LD group was significant higher than that of the LP group (P=0.029).As shown in P=0.487).A patient in the LD group developed intracerebral hemorrhage and died of cerebral hernia. The rate of intracerebral hemorrhage in the LD group was 2.56%. No patient developed intracerebral hemorrhage in the serial LP group. Although there was a slight decrease in the risk of intracerebral hemorrhage with serial LP, no statistically significant difference was found (P=0.838).Thirteen patients had vasospasm, 7 in the serial LP group (17.07%) and 6 in the LD group (15.38%). Although the LD group had a lower rate of vasospasm, there was no statistically significant difference between the two groups (P=0.494).Two patients in the serial LP group developed hydrocephalus with a rate of 4.88%. No patient developed hydrocephalus in the LD group. There was no statistically significant difference between the two groups (P=0.222).As presented above, 3 patients died in the LD group. Two died of severe infection. One died of intracerebral hemorrhage, but there was no death in the serial LP group. The mortality showed no statistically significant difference between the two groups (P=0.532), but the duration of drainage in LD group was significantly shorter than that of the serial LP group .No significant difference was observed in the length of stay between the two groups (P=0.287).All patients were assessed with GOS scores 30 days after endovascular coiling. No significant differences in GOS scores between the two groups were found  suffered from CSF infection, 4 of whom (10.26%) were confirmed by CSF culture and the rate was similar to that reported previously. Some researchers considered LD as a significant risk factor for meningo-ventriculitis. As the risk of LD associated CSF infection was complicated, the patients with long term duration of catheter placement (more than 4 days) or with puncture site leakage are at greater risk. Other risk factors, including infection that originated from insertion procedure, the frequency of catheter manipulation and sampling, the presence of blood in the CSF, have also been identified. In contrast, our study demonstrated that serial lumbar puncture showed a lower infection rate during drainage of hemorrhagic CSF, which is consistent with previous report. The reasons may be related to the shorter duration of needle placement in each insertion procedure and the lower rate of puncture site leakage. LD related CSF infection is a preventable disease. Leverstein-van et al. have reported that they decreased the proportion of probable drain-related infection from 37% to 9% by strict adherence to accurately defined procedures, including improved awareness, focusing on the standard operating procedures, the diagnostic and therapeutic algorithm, timely administration of prophylaxis, and the improvement of the drainage system.Prevention and management of LD associated CSF infection pose a major challenge. The CSF infection rate of LD was 5%‒18% according to different studiesIn the present study, both LD and serial LP showed similar efficacy in the treatment of aSAH related vasospasm and hydrocephalus. Although there was no statistically significant difference, the LD group exhibited lower vasospasm and hydrocephalus rates. Meanwhile, even though GOS score and LOS in the two groups had no statistically significant difference, the LD group had significantly shorter drainage duration than the serial LP group. These results suggest that the main outcomes in the treatment of aSAH between the two groups are the same and lumbar CSF drainage seem to be more effective than the serial lumbar puncture.There are several limitations in this study. First of all, the selection bias is a potential problem in a retrospective non-randomized study. Secondly, the long term outcomes of these patients were not evaluated due to the loss of follow-up. Thirdly, patients with severe SAH were not enrolled in this study. Finally, this study is a single-center design with a long time span and only a small number of patients were included, which may affect the results. Therefore, the results of the present study still need randomized control trials to confirm.In conclusion, both lumbar CSF drainage and serial lumbar puncture are effective methods in the treatment of aSAH. Serial lumbar puncture reduces the CSF infection during the drainage of hemorrhagic CSF in aSAH after endovascular coiling.


Samples from healthy controls (n = 23) and patient controls (n = 27) served as baseline information. Sandwich enzyme-linked immunosorbent assay (ELISA) for proteasomes and their complex formation with complement factor 3 or 4, and Western blot for C3 activation were performed on CSF samples. Increased compleasome formation, both presenting as an initial formation and showing exposure of subunit AF1 in the compleasomes, was found in CSF samples drawn from patients with HSE compared with samples from the control groups (p < 0.0005). The total protein CSF concentration was equal in all groups. The levels were higher in the acute phase compared with late in the disease course (p < 0.0005). Complement 3 breakdown product iC3b was detected in CSF samples of the HSE patients. The early increased formation of compleasomes in CSF suggests that this complex may be involved in host defence against HSE.Herpes simplex encephalitis (HSE) is a common cause of viral encephalitis (HSV-1) characterised by pronounced inflammation and elevated intracranial pressure. We have shown in a rat model that HSV-1 infection causes an interaction between complement factors and proteasomes, leading to formation of proteasome/complement complexes (compleasomes). Exposure of the proteasome regulatory subunit antisecretory factor 1 (AF1) leads to a decrease in intracranial pressure. The aim of this study was to evaluate the acute and prolonged formation of compleasomes in cerebrospinal fluid (CSF) from patients with HSE. Cerebrospinal fluid samples ( Herpes simplex encephalitis (HSE), most often caused by herpes simplex virus type 1 (HSV-1), induces an acute focal, necrotizing inflammation with a predilection for the frontotemporal regions of the brain. Although acyclovir treatment has been reported to reduce mortality . Polyclonal antibody (pAb) against proteasome 19S subunit was produced as previously described M monoclonal antibody (mAb) against AF1/RPN10 was produced as previously described  and sufficient lumbar CSF (55 samples). We defined acute infection (“early samples”) as days 0–10 after onset of neurological symptoms. Samples after day 10 were defined as continued inflammation/late infection (“late samples”). For included HSE patients, number of days after onset of neurological symptoms was noted and the medical records were examined. Treatment data were retrieved, and outcome, measured after 6 months, was reported according to the Glasgow Outcome Scale (GOS)  or computed tomography (CT) findings compatible with herpes encephalitis. The diagnosis was confirmed on admission to hospital by detection of HSV-1 DNA using quantitative in-house TaqMan PCR methods  and patient controls . The healthy controls were recruited as next of kin to patients suffering from amyotrophic lateral sclerosis (n = 23), and samples were collected at the Department of Neurology, Sahlgrenska University Hospital, Gothenburg, Sweden. The healthy controls had no history or symptoms of signs of psychiatric, neurological, malignant, or systemic disorders. The patient controls were subjects who were seeking medical care and were lumbar punctured because of headache and/or psychoneurotic symptoms and in whom CNS infection initially was suspected, but later excluded. Exclusion of CNS infection was based on a non-pathological neurological status and lack of pleocytosis in the CSF. Analysis of patient control samples revealed normal CSF cell count and, for most, normal CSF albumin concentrations. Characteristics of the clinical sample collection are presented in Table Decoded samples of CSF were obtained from healthy controls .n = 23–24), PCs (n = 25 or 27), and HCs (n = 23) were titrated in PBS with 0.2% BSA and 0.05% Tween 20, and shaken for 2 h. A pAb (anti-C3 or anti-C4) at 1:2000 dilution was used as the detecting antibody. An anti-rabbit-AP secondary antibody was applied after 1 h of incubation, and AP substrate was added after a further hour. Absorbance was read at 405 nm in a photometer, and the difference between antibody-coated samples and controls (PBS) was estimated.A sandwich enzyme-linked immunosorbent assay (ELISA) for detection of compleasomes (proteasome/complement complexes) in CSF was performed as previously described  and from patients with HSE (n = 3) by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting onto nitrocellulose membrane. The membrane was blocked with 1% BSA in PBS at 4°C for 16 h. The primary polyclonal anti-C3  was then added to the membranes and incubated for 1.5 h. The blot was developed with AP-conjugated goat anti-rabbit IgG followed by 5-bromo-4-chloro-3-indolyl phosphate and 4-nitro blue tetrazolium .The content of C3 products was determined in CSF samples collected from healthy individuals (C3c was detected and quantified in CSF from HSE patients (ten early samples and 18 late samples) and patient controls (13 samples) using a C3c assay kit , following the manufacturer’s instructions. The C3c concentration of selected CFS samples, which were run parallel with standards, was determined from the standard curve.n = 23) was performed as previously described  and healthy controls (n = 9), PCs (n = 10) and HCs (n = 10) was determined with the Pierce BCA protein assay kit  reading absorbance at 562 nm using a NanoDrop photometer .The protein content in CSF from patients with HSE  for two means was assessed using an unpaired Student’s t test; for three means, analysis of variance (ANOVA) was used and p < 0.05 was considered significant.GraphPad Prism version 7  was used for statistical analysis and graph construction. Data are presented as mean ± standard deviation (SD). Statistical significance of difference (p < 0.0005). The compleasome levels found after using catching antibody against proteasome subunit 20Sα6 represent the intact compleasome in CSF. Use of mAb AF1 as catching antibody in the sandwich ELISA indicated increased exposure of the specific subunit AF1 in these samples obtained from HSE patients. The levels detected in samples collected from PCs were also somewhat increased compared with CSF obtained from HCs. However, the induction of the compleasomes exposing AF1 was lower compared with formation of initial compleasome . The late samples were then divided into two subgroups, collected at days 11–30 (14 samples) and between 31 and 523 days (28 samples) after onset of symptoms. Levels were compared with the samples from early HSE , compared with the highly significant decrease in the compleasome complex detected in the samples collected very late (p < 0.0005).The formation of compleasomes after HSV-1 infection in early and late CSF samples was examined by sandwich ELISA. As shown in Fig. n = 7). A tendency (p = 0.0541) of higher levels of compleasome was seen in samples taken from patients (n = 4) with good recovery (GOS 5) compared with patients (n = 3) with worse outcome (GOS 1–4).We compared patients’ outcome according to GOS and the level of compleasome in CSF during acute HSV-1 infection where data were available  by constructing a graph. The compleasome levels in the follow-up samples showed a distinct pattern whereby early formation of compleasomes was followed by a decline over time  while degradation products were absent in HC samples (n = 2). The reaction was strongest in samples collected during early infection (day 7), compared with a weaker signal in samples taken on day 16 and day 173 after onset of symptoms.Since C3 degradation is involved in the formation of compleasome, the level of C3 breakdown products in CSF from HSE patients was investigated by Western blot using C3 antibody. As Fig. n = 10) and late (n = 18) in HSE, together with samples taken from PCs (n = 13) using the C3c assay kit. Samples collected early in the infection at acute phase had significantly higher levels of specific C3c compared with samples collected more than 10 days after onset of symptoms (p = 0.0036) and samples from PCs (p = 0.0033) (Table We quantified the level of C3c in CSF in samples collected early (3) Table .Table 2Lp = 0.339 and p = 0.280). This finding suggests no leakage of proteins from blood to CSF in the HSE patients. Therefore, low levels of proteasomes were detected in CSF collected from HCs (1 ± 1 ng/ml), while the HSE samples contained a high level of proteasomes (50 ± 13 ng/ml).There was no significant difference in protein concentration in CSF in the PCs and HCs compared with the HSE patients, the respective levels being 0.72 ± 0.18 mg/ml, 0.71 ± 0.17 mg/ml and 0.86 ± 0.36 mg/ml  compared with patients with worse clinical outcome (GOS 1–4). This suggests that a rapid and strong formation of the innate compleasome complex may be important in the control of brain HSV-1 infection. The retrospective design of this study and analysis of frozen CSF samples imply limitations, i.e. lack of standardised timing of lumbar punctures and selected bias when using stored samples. However, the acute and late CSF samples in this study were from patients with both good and bad outcome, since the patients had previously participated in studies with long-term follow-up. The clinical data with outcome measurements (GOS) after 6 months were accurately documented in the patient’s records. Intrathecal complement activity has been examined in other CNS infections such as cryptococcal meningitis, although no test of correlation with the clinical outcome was performed  domain. It has been described elsewhere that proteins incorporating von Willebrand factor (vWF) motifs participate in several biological events via interaction with other proteins (Edwards and Perkins The present results demonstrate that AF compleasomes are formed in CSF during HSV-1 infection. The levels of compleasome in CSF were correlated to the number of days after onset of symptoms; at acute phase of infection, the HSE patients demonstrated higher levels of CSF compleasomes compared with the later stage of infection. The rapid formation of compleasomes in response to the HSV-1 infection suggests an important role in the host defence immune system of this specific complex.


Primary central nervous system lymphoma (PCNSL) is the most prevalent brain, spinal cord, eyes, and leptomeningeal lymphoma. It is often misdiagnosed due to an unspecific presentation or unavailable biopsy and results in a poor prognosis. Although the craniocerebral imaging examination of PCNSL has some characteristics, it is limited, and atypical cases are especially difficult to identify with intracranial tumours and other diseases. The biopsy, as the gold standard for PCNSL diagnosis, is not eligible for all patients suspected of having PCNSL.This report documents a woman who presented with a three-month history of numbness and weakness in the right leg. She was treated with drugs at a local hospital for one month. She developed demyelination lesions and her symptoms were aggravated. The patient was admitted to the Department of Nerve Infection and Immunology at Tiantan Hospital. Head magnetic resonance imaging (MRI) enhanced scanning indicated significant inflammatory demyelinating disease, and lymphoma was not excluded. CSF revealed a high protein level and CSF cytology detected abnormal cells, PCNSL was eventually presumed according to positive CSF cytology and cytological detection of the cerebrospinal fluid flow.PCNSL is a highly invasive tumour. With the development of technologies such as cerebrospinal fluid cytology and flow cytology, CSF analysis has become one of the definite diagnosis methods, and the tumour cell finding in CSF is the only reliable basis for diagnosis. Flow cytometric analysis and gene rearrangement testing also provide objective evidence. Lymphoma is a malignant tumour of the lymphohaematopoietic system that can affect all of the tissues and organs throughout the entire body. It is divided into two categories: non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Primary central nervous system lymphoma (PCNSL) is the most prevalent brain, spinal cord, eyes, and leptomeningeal lymphoma. Most PCNSL is non-Hodgkin lymphoma, which originates from B lymphocyte monoclonal proliferation . MisdiagA 54-year-old woman with symptoms of numbness and weakness in the right leg with no obvious cause presented to the field hospital. The diagnosis was the possibility of metastatic tumour after head CT and MRI examination. Pathological findings at another hospital suggested demyelinating disease. The patient was treated with hormone drugs in the field hospital and her symptoms were relieved. But the numbness in her right leg was aggravated, and symptoms of dizziness and nausea appeared two months later. The patient was eventually admitted to the Department of Nerve Infection and Immunology at Tiantan Hospital for concentric sclerosis. The patient had a history of hypertension, hyperthyroidism, and mammary gland hyperplasia but no history of diabetes, heart disease, hepatitis, tuberculosis, or drug allergy.Physical examination after admission found a decrease in her calculation capacity. She had instability of pointing at her nose with her right finger and the tibial experiment with her knee in the nervous system examination. She had no other obvious abnormalities.9/L. Her glucose (2.93 mmol/L), sodium (124 mmol/L), and chlorine (87 mmol/L) levels were decreased. Her fibroproteinogen (1.62 g/L) level was decreased. A routine CSF examination revealed her CSF had a yellow and clear appearance, and the total number of CSF cells was 514/μl. The number of leukocytes was 14/μl. Her CSF protein was high (82.54 mg/dl), and her 24-h IgG intrathecal synthesis rate was increased (13.54). The IgG oligoclonal band of her CSF was negative. Her cytokine interleukinin-10 level was 641.00 pg/ml .Laboratory examination after admission found that her leukocyte level in a routine blood examination was 13.52 × 10The patient’s cytomegalovirus IgG of the CSF was positive. Neuronal antigen spectrum antibody IgG (CSF and blood) were negative. Tumour markers  were negative. Autoimmune antibody tests and protein electrophoresis were normal. The result of a CSF culture indicated Bacillus (suspected contamination). CSF gram stain, acid fast stain, and ink stain were negative Tables , 3, 4, 5Colour Doppler ultrasound of the patient’s abdomen, urinary system, thyroid, lower extremity veins, and superficial lymph nodes were performed, with no abnormal findings. PET-CT indicated no metastatic tumour lesions.Head MRI enhanced scanning demonstrated multiple abnormal signals in the double frontal cortex, subcortical, basal ganglia, temporal lobe, and callosum. Inflammatory demyelinating disease was more likely, and lymphoma was not excluded. Abnormal signals of the left parietal lobe were demonstrated after biopsy, and there was an ischaemic infarct in the patient’s right cerebellar hemisphere Fig. .Fig. 1HeCSF cytology conducted in the laboratory at Tiantan Hospital showed that a group of cells were suspected lymphoma cells. They had larger bodies and irregular nuclei, visible nucleolus, abundant cytoplasm, and deep staining, and this type of cell accounted for 11% Fig. . We promAccording to the CSF cytology results, the patient’s clinicians immediately prescribed flow cytometry and CSF DNA sequencing. CSF flow cytometry showed that 14.86% of her cells (129 karyocytes) expressed CD38, CD22, CD19, CD20, kappa, CD79b, CD180, CD54, and CD44. Some cells were considered malignant monoclonal mature B cells. They expressed K167 (40%), CD9, and CD200 and did not express CD56, CD2, CD7, CD3, CD4, CD8, CD138, CD30, lambda, FMC7, CD103, CD25, CD11C, and CD34  and transferred from the Department of Nerve Infection and Immunology to the Department of Haematology for further treatment.In the Department of Haematology, the initial treatment was rituximab combined with a chemotherapy regimen (R-MAD). On the second day, the patient was administered a lumbar puncture and intrathecal injection of 50 mg cytarabine and 5 mg of dexamethasone, and then the routine, biochemical, and cytological examinations were repeated. CSF cytology showed that the lymphoma cells had decreased to 1%. On the third day, the patient had a clear state of mind but her spirit was weak, and there was no nausea and vomiting and no obvious adverse reactions to chemotherapy.Primary central nervous system lymphoma (PCNSL) is the most prevalent brain, spinal cord, eyes, and leptomeningeal lymphoma. Most PCNSL is non-Hodgkin lymphoma, which originates from B lymphocyte monoclonal proliferation. Its morphological and pathological features are similar to those of diffuse large B cell lymphoma (DLBCL). The WHO’s (2008) classification of haemopoietic and lymphatic tissue tumours classified DLBCL as an independent category originating in the central nervous system (CNS) .PCNSL accounts for 3% of brain tumours, and more than 95% of cases are DLBCL, occurring in 50~70-year-old patients. The time from onset to presentation is within 2~3 months. The main symptoms of PCNSL are changes in mental state and intracranial hypertension such as headache, nausea and vomiting, papilledema, and local compression symptoms, including epilepsy, memory loss, unstable gait, visual impairment, blurred speech, and mild haemiplegia. In addition to brain damage, 10%~ 20% of patients have eye damage characterised by blurred vision or floating objects . AlthougNeurological Clinical Cerebrospinal Fluid Cytology by Su Xiuchu and Kong Fanyuan. This report documented a large number of atypical lymphocytes in the CSF of patients with central nervous system lymphoma. Atypical cells have small strong basophilic and blue cytoplasm around the nucleus and no particles. The nuclei are round or oval and often twisted, with chromatin clusters, visible nucleolus, and often mitotic phases. The tumour cells are sometimes large and irregular in shape. The amount of cytoplasm is more or medium with vacuoles, and the nucleus has a pea-like shape. Lymphoma cells in the CSF of PCNSL patients should be distinguished from activated lymphocytes. In general, lymphoma cells have irregular nuclei that are large and obvious, and vacuoles are common in the cytoplasm, but the activated lymphocytes demonstrate no signs of malignant cells.PCNSL is a highly invasive tumour. It tends to spread along the CSF with the changing of the CSF composition or the shedding of tumour-causing cells into the CSF. Therefore, the CSF analysis has become one of the most definitive diagnostic methods. Tumour cell findings in CSF are the only reliable basis for the diagnosis, and 80% of patients with PCNSL have leptomeningeal involvement. PCNSL is described in Immunological methods contribute to the identification of lymphoma cells and activated lymphocytes. B cell lymphoma is the main type of lymphoma, and activated lymphocytes caused by infection account for a considerable proportion of T cells . TherefoCytological detection of the cerebrospinal fluid may have high specificity in the diagnosis of PCNSL. However, due to the spread range of PCNSL, CSF volume, and other factors, lymphoma cells might be undetectable in some of PCNSL cases. Thus, this method has certain limitations. In routine practise, our laboratory staff find it easier to obtain first-hand information than clinicians. Clinicians should actively communicate when diagnostic indications are present to promote further examinations for an accurate diagnosis and prompt treatment.


Somatostatin (SOM) and Neuropeptide Y (NPY) are two neuropeptides which are expressed in GABAergic interneurons with different fates in AD the former only being markedly affected. The aim of this study was to analyze CSF SOM, NPY and CSF Aβ1–42; T-Tau, P-Tau relationships in 43 elderly mild cognitively impairment (MCI) participants from the Biomarker of AmyLoïd pepTide and AlZheimer’s disease Risk (BALTAZAR) cohort. In these samples, CSF SOM and CSF Aβ1–42 on the one hand, and CSF NPY and CSF T-Tau and P-Tau on the other hand are positively correlated. CSF SOM and NPY concentrations should be further investigated to determine if they can stand for early AD biomarkers.A combination of low cerebrospinal fluid (CSF) Amyloid βwww.ClinicalTrials.gov, identifier #NCT01315639.Clinical Trial Registration:  The combination of low Aβ1–42 and high T-Tau or p-Tau, which occurs at a prodromal stage of AD  neuropathology is characterized by intraneuronal protein clusters of hyperphosphorylated Tau protein (neurofibrillary tangles) and extracellular amyloid beta (Aβ) protein aggregation that start decades before the occurrence of clinical symptoms . The BALTAZAR study aimed to study the relationships between plasmatic biomarkers  and determine the relevance of these biomarkers on the risk of conversion of patient aged 70 years and older with mild cognitive impairment (MCI) according to international criteria  study was a longitudinal, multicenter study including 23 French memory clinics. It was approved by the local ethics committee  and Magnetic Resonance Imaging (MRI) scans at baseline. Subjects with geriatric depression scale (GDS) score over 20/30  and treatments were recorded by a physician. The educational level was scored as elementary school, secondary school and high school diploma and above. Each participant had a physical examination performed by a physician including the calculation of the body mass index (BMI).Fasting blood samples were collected for all subjects . APOE was genotyped in a single centralized laboratory  using a classical polymerase chain reaction (PCR) digestion method.1–42 concentrations were measured in the same laboratory  using the commercial kits: INNOTEST®hTAU Ag, INNOTEST®β-Amyloid(1–42)  . The quality of the results was ensured by using validated standard operating procedures  an axial FLAIR T2W, an axial EG T2W and an axial T2W FSE. MRI analysis was centralized and analyzed by the Centre d’acquisition et de traitement d’images . R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, AustriaGeneral, medical and cognitive characteristics were first presented in the whole sample with percentages and numbers for categorical variables and means and standard deviations (SD) for continuous variables. Means and SD of variables of interest for the different values of each categorical variable were compared by analysis of variance. Pearson’s correlation coefficients and Demographic, medical and cognitive characteristics of the 43 participants included are summarized in Table p = 0.004), diabetes (p < 0.0001), low plasma total cholesterol (p = 0.004) and LDL cholesterol (p = 0.002), high CSF NPY concentrations (p = 0.03) and high CSF Aβ1–42 concentrations  and imaging characteristics are summarized in Tables  p = 0.00, high CSp = 0.04), diabetes (p = 0.01), low plasma total cholesterol (p = 0.005), HDL cholesterol (p = 0.003) and LDL cholesterol (p = 0.02), high serum creatinine (p = 0.01); high CSF T-Tau (p = 0.02) and P-Tau concentrations (p = 0.01) and high CSF SOM concentrations  and blood total cholesterol concentrations (p = 0.03) and CSF NPY remained significantly associated with CSF T-Tau (p = 0.03). CSF SOM and CSF NPY were no more significantly associated. The Figure 1–42 and T-Tau in included subjects.The results of the multivariate analysis are presented in Tables 1–42 concentrations on the one hand and high CSF NPY and high CSF Tau and P-Tau concentrations on the other hand, are correlated in a small population of elderly subjects suffering from MCI. Moreover, high CSF SOM concentrations are also related to diabetes and hypertension and low plasma cholesterol level. High CSF NPY concentrations are related to male gender, diabetes, and low plasma cholesterol.The present results indicate that high CSF SOM and high CSF AβTau and P-Tau are released in the CSF very early in the AD process when neuronal damage occurs  in an exhaustively phenotyped population.Nevertheless, this is the first report of a relationship between CSF SOM and NPY, two peptides produced by subsets of GABergic interneurons, and the CSF AD markers (CSF Aβ1–42 deposition and hyper phosphorylated Tau aggregation. The present study needs to be replicated in a larger sample size. If those results are confirmed additional interest of CSF SOM and NPY to already known AD biomarkers could be tested.We report for the first time an independent relationship between CSF SOM and NPY, two peptides known to be affected in AD pathophysiology, and CSF markers of the two pathophysiological processes in AD: AβED, DG, AG, CB, BA, and SL performed measurements and collected data. J-SV performed statistical analysis. FP, LB, SS-M, A-SR, OH, and JE supervised the study. SB collected data revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study was to establish the validity and reliability of the newly developed surface electromyography (sEMG) device (PSL-EMG-Tr1) compared with a conventional sEMG device (BTS-FREEEMG1000).  In total, 20 healthy participants  were recruited. EMG signals were recorded simultaneously on two devices during three different isometric contractions ). Two trials were performed, and the same session was repeated after 1 week. EMG amplitude recorded from the dominant biceps brachii (BB) and rectus femoris (RF) muscles was analyzed for reliability using intrasession intraclass correlation coefficient (ICC). Concurrent validity of the two devices was determined using Pearson's correlation coefficient. r = 0.943 (BB), r = 0.940 (RF)) and 80% MVIC (r = 0.983 (BB); r = 0.763 (RF)).  Nonnormalized sEMG data showed moderate to very high reliability for all three contraction levels (ICC = 0.832–0.937 (BB); ICC = 0.814–0.957 (RF)). Normalized sEMG values showed no to high reliability (ICC = 0.030–0.831 (BB); ICC = 0.547–0.828 (RF)). sEMG signals recorded by the PSL-EMG-Tr1 showed good to excellent validity compared with the BTS-FREEEMG1000, at 40% MVIC ( The PSL-EMG-Tr1 was performed with acceptable validity. Furthermore, the high accessibility and portability of the device are useful in adjusting the type and intensity of exercise. Sarcopenia is defined as decreased skeletal muscle mass and muscle strength with age. Muscle mass and strength gradually decrease after reaching a peak in early adulthood, and the degree of decrease varies among individuals . Elderly2; females, age 22.3 ± 2.7 years, height 162.1 ± 5.0 cm, weight 56.4 ± 5.0 kg, BMI 21.5 ± 1.9 kg/m2; mean ± SD) were recruited; all participants who provided informed consent prior to the study were recruited. Ethical approval was granted, and the informed consent form was approved by the Ethics Committee of Pusan National University Hospital, Busan, Korea (IRB number: 1703-018-052). Exclusion criteria included musculoskeletal disease, cardiopulmonary disease, and other diseases that could prevent exercise.In total, 20 healthy participants  between the ages of 21 and 34 years  25.4 ± 3.33 kg/mAt each session, participants were first required to perform three maximal voluntary isometric contractions (MVIC) for 5 seconds each, with a 5-minute rest between contractions. Each session consisted of two trials. After three MVIC measurements, 15-second isometric contractions were performed at different intensity levels. In the pretest, it took at least 10–15 seconds to maintain the same intensity isometric contraction through visual feedback. First, 40% MVIC was performed, followed by 80% MVIC after 5 minutes of rest. In the second trial, the placement of the electrodes for the two devices (BTS-FREEEMG1000 and newly developed device) was interchanged, and contractions were again measured by the same method . During The participants sat on a Biodex System 3 PRO dynamometer  with a visual torque feedback monitor. Each participant sat in an upright posture and was strapped firmly to the chair with adjustable belts across the arm, trunk, and thigh. To evaluate BB muscle contractions, the participant sat with the dominant arm flexed at 90° and the forearm flexed at 120° relative to the upper arm. To evaluate quadriceps (RF) contractions, the hips were flexed at 90° and the tested knee was flexed at 45°. The axis of the dynamometer was positioned at the center of the tested elbow or knee joint. The lever arm was fixed by the precalibrated force sensor .The EMG signal was recorded simultaneously using two different sEMG devices. The BTS-FREEEMG  was set to a sampling rate of 1,000 Hz per channel, and the signals were band-pass filtered from 20 to 500 Hz. The newly developed sEMG device  was set to a sampling rate of 30,000 Hz, and signals were amplified with a 3–2,000 Hz bandwidth .Adhesive hydrogel surface electrodes  were used, and the interelectrode distance, electrode placement procedure, and skin preparation followed standard Surface Electromyography for the Non-Invasive Assessment of Muscles (SENIAM) guidelines . Two paiThe root mean square (RMS) value was used to analyze and process the recorded electrical signals in the muscles. Based on the square root calculation, the RMS reflects the mean power of the signal and is the preferred recommendation for smoothing. The RMS value can be used as a parameter to reflect the physiological activities of the motor unit during muscle contraction .∗Power software . In this study, the number of subjects required for a null-correlation (R0) = 0, alternative correlation (R1) = 0.6, alpha = 5%, power = 80%, and two-tailed test value was 19. Statistical analysis was performed using SPSS software . To determine concurrent validity between the two sEMG machines, Pearson's correlation coefficient (r) was used for the average of two trials in each device. Interpretation of the correlation coefficients was based on guidelines for Pearson's coefficients suggested by Portney and Watkins [r > 0.75, good to excellent correlation; r = 0.50–0.75, moderate to good correlation; r = 0.25–0.50, fair correlation; and r = 0.00–0.25, little to no relationship [Sample size was calculated using Gt-test was also conducted comparing the RMS (μV) and torque (N·m) between two trials of each week. Biodex is a device that has proved its reliability and validity. Therefore, Biodex was used only to evaluate the exact intensity during muscle contraction, and validity was compared between two sEMG devices [The intraclass correlation coefficient (ICC) of two trials performed on each week was used to indicate the relative reliability of the measurements. For the test-retest reliability, ICC using a two-way mixed-effects model and absolute agreement definition is used . Munro's devices .Twenty participants completed a total of four trials over 2 weeks. μV) of the nonnormalized RMS values recorded by the PSL-EMG-Tr1 devices at the three contractions levels for the first and second weeks are shown in Recorded sEMG data were processed for RMS analysis. The amplitudes  is shown in r values were used to evaluate validity because all of the % MVIC values measured in the BB and RF muscles were normally distributed. The two sEMG devices were compared using averages of all four trials for two weeks of 40% MVIC and 80% MVIC. The 40% MVIC displayed excellent validity for BB (r = 0.907) and RF (r = 0.965), and the 80% MVIC showed good to excellent validity for BB (r = 0.781) and RF (r = 0.757). r = 0.781–0.907 for BB; r = 0.757–0.965 for RF) and showed nonsignificant results on the paired t-test (p > 0.05). Especially in the BB muscle, there was little reliability because of the low ICC of normalized RMS values.In this study, we confirmed the reliability and validity of the newly developed sEMG device for monitoring muscle activity during exercise and in daily life. The nonnormalized RMS values measured on both devices showed high reliability . The normalized RMS values showed good to excellent validity  using sEMG. In this study, a relatively low ICC (range: 0.57–0.80) was seen in the BB in intersession single measurement. However, the average of repeated sEMG measurement was considered to obtain high reliability rather than single measurement . KollmitIn elderly populations, physical activity is reduced, with less than one-fifth of elderly individuals engaging in the recommended level of physical activity . CommercTo evaluate the accuracy of the new device, we recruited young people free of disease and disability. However, elderly individuals may have chronic diseases that could cause peripheral neuropathy or myopathy due to disuse atrophy, so the quality of surface EMG data may vary. However, such difficulties would not indicate a problem with the accuracy of the device itself. In other words, it is possible to compare muscle activity within one individual, but caution is needed when comparing individuals with one another. Despite these limitations, it is very encouraging that there is a tool that allows easy and objective evaluation of muscle quality in elderly people. For the clinical application of the device, further studies will be necessary for healthy elderly or sacropenic patients. This study had the other limitation that should be addressed: the electrodes were attached according to the SENIAM guidelines, but it is considered that the 2.0 cm interelectrode distance was the main cause of the lower ICC in this study. However, this problem will not affect the clinical application of muscle monitoring over time for people undergoing monitoring or engaging in exercise, as only one device will be used. This is because ICC was also lower in clinically widely used sEMG device (BTS-FREEEMG1000), and good to excellent validity was found in normalized RMS values of the two sEMG devices.The newly developed sEMG device is wired for a single channel, although a wireless model for two or more channels is under development. A high sampling rate was used for the sEMG device in this study. Since most signals from the human muscles have frequency characteristics that are valid at less than 400 Hz, we recommend that the signal analysis sample more than twice the major quard of interest frequency. While we do not need this sampling frequency for existing RMS and MDF analysis, we are interested in signal characteristics that we have not known before by increasing the maximum sampling frequency. This will be used for further study of the characteristics of EMG signals of damaged muscles. Therefore, the newly developed instrument is measuring at a sampling frequency much higher than the frequency of interest. However, since many burdens are expected in the signal processing during commercialization, the sampling rate will be reduced to 2,000 Hz; it will enable fast signal processing and long recording time. In addition, if the EMG signals from the patient's muscles are automatically stored and the system is programmed so that this information can be delivered to medical staff located elsewhere, the therapeutic value of the device will increase still further.Signals from the BB and RF muscles, recorded by the newly developed PSL-EMG-Tr1 device, showed good to excellent validity and moderate to high ICC values with nonnormalized RMS values. However, low ICC values were seen with the normalized RMS values. Although the sEMG itself may have limitations, it can be overcome somewhat through repeated measurements and appropriate muscle selection. Since it has a high correlation compared to conventional sEMG devices, it can be used as an alternative to conventional sEMG devices. The newly developed device may be effective to evaluate and monitor the condition of individuals' muscles during repetitive daily activities or exercise, and it has higher accessibility and portability than do conventional sEMG devices.


Cerebrospinal fluid (CSF) metabolomic investigations are a powerful tool for studying neurometabolic diseases. We aimed to assess the effect of CSF contamination with blood on the concentrations of selected biomarkers.CSF samples were spiked in duplicate with increasing volumes of whole blood under two conditions: (A) pooled CSF spiked with fresh whole blood and frozen to cause red blood cell (RBC) lysis; (B) pooled CSF spiked with fresh blood and centrifuged . CSF concentrations of amino acids, biogenic amines, pterins, and vitamins were analysed by HPLC coupled with tandem mass spectrometry, electrochemical and fluorescence detection.Aspartate, glutamate, taurine, ornithine, glycine, citrulline, pyridoxal 5´-phosphate, 5-methyltetrahydrofolate, and thiamine showed higher values when RBCs were lysed when compared with those of CSF with no RBC, while arginine, 5-hydroxyindoleacetic and homovanillic acids showed lower values. When RBCs were removed from CSF, only some amino acids, thiamine and pyridoxal 5´-phosphate showed moderately higher values when compared with the non-spiked CSF sample.CSF-targeted metabolomic analysis is feasible even when substantial RBC contamination of CSF has occurred since CSF centrifugation to remove RBC prior to freezing eliminated most of the interferences observed. Cerebrospinal fluid (CSF) is a biological fluid that is mainly produced by the choroid plexus, which constitutes the interface between blood vessels and CSF , 2. The CSF metabolomic investigations have been demonstrated to be a powerful tool for studying specific neurometabolic pathways and related diseases and for exploring metabolic transport from the blood into the brain . SeveralWith this background, we aimed to assess the effect of CSF contamination with blood on the concentrations of selected molecules which are biomarkers for the study of different neurometabolic conditions.g 10 min at 4 °C, with the clear supernatant frozen at − 80 °C. Details of the protocol are stated in Additional file CSF samples were collected from patients where lumbar puncture was done to rule out meningoencephalitis, and stored at − 80 °C, following a previously reported protocol . The remInitially, to identify a cut-off value at which blood contamination can cause substantial interference in the measurement of the above-mentioned metabolites, CSF was spiked with whole blood volume range from 1 to 0.02%. No relevant effects were detected in most metabolite concentrations studied under these conditions (data not shown).The concentration of albumin in CSF, used as a surrogate biomarker of CSF RBC contamination or impaired blood–brain and blood-CSF barriers, was analysed using an Abbot automated analyser (Architect c8000) by spectrophotometric procedures. Other biomarkers of blood contamination such as haemoglobin concentration were analysed by an automated procedure . CSF amino acids were analysed by UHPLC coupled to tandem mass spectrometry detection in a Xevo QT Waters system, as previously reported . BiogeniThe precision of the different techniques was initially calculated using the coefficient of variation (CV = standard deviation/average × 100%) from 20 replicates and was below 10% for all of the metabolites studied, as previously reported –24. ThusCSF anonymized samples from remnants were collected in our Hospital following our diagnostic protocols, and the study was conducted only once such investigations were concluded. In every case, informed consent was obtained from each patient before performing the lumbar puncture and CSF collection. The Ethical committee of Sant Joan de Déu Hospital approved the study. All samples from the patients were obtained following the 2013 revised Helsinki Declaration of 1964.The absolute values of the different metabolites studied are depicted in Fig. In Table Results of the surrogate biomarkers of CSF blood contamination  are stated in Additional file CSF metabolomic analysis is a good analytical tool for the study of the neurometabolic conditions stated here . FurtherCSF RBC contamination is frequent and has been recognized as a substantial confounding factor for proper interpretation of CSF analysis data describing concentrations of amino acids and other molecules , 27. HowAlbumin, a protein synthesized in the liver, is a good surrogate biomarker for compromised permeability of the blood–brain barrier and also for blood contamination. However, since its concentration in the blood largely exceeds that of the CSF (by approximately 100-fold), its concentration may remain elevated even in the case of low RBC/plasma contamination . Haemoglobin measures are an alternative surrogate marker when RBC lysis has occurred, and in our hands, values around 0.35 g/dL of haemoglobin may be a signal for cautious interpretation of the data presented here Table , since iWith regards to amino acids, several reports have indicated differences between blood and CSF compartments , but to Biogenic amines and pterins are synthesized peripherally in some tissues but also in the brain, and no substantial transport has been documented between blood and brain (only OAT3 transporters efflux both 5-HIAA and HVA from CSF to blood ). Since Vitamins displayed unpredictable results, except for folate. Folate forms  are highly concentrated in RBC when compared with plasma and this would explain the positive interference observed when RBC lysis occurred, but not after RBC removal. Only thiamine and PLP had increased values when comparing spiked CSF samples at 2.5% with non-spiked samples under both experimental conditions, although it was less remarkable when RBC removal was performed. These effects were minimized when the CSF blood contamination was 1% (data not shown). Regarding thiamine, active conversion of free thiamine, TMP and TDP occur inside cells  . This woCSF-targeted metabolomic analysis is feasible even when remarkable RBC CSF contamination occurs since CSF centrifugation to remove RBC prior to freezing prevents most of the biases observed. However, data should be cautiously interpreted, especially for some metabolites. CSF albumin, haemoglobin, and TDP can be used as surrogate biomarkers of the potential confounding effect of CSF plasma/RBC contamination.Additional file 1: Table S1. Percentage of blood contamination, albumin and haemoglobin levels from CSF spiked with increasing amounts of blood.Additional file 2: Figure S1. CSF blood spiking protocol. A total of 20 CSF aliquots were analysed. In the picture, the colours of the 5 CSF sample are presented. Even in the 2.5% spiking condition, the red colour was intense. The median CSF blood contamination observed in our laboratory typically ranged from 0.01 to 0.035 g/dL of a haemoglobine, which is lower than the 0.35 g/dL observed in the 2.5% blood contamination condition.Additional file 3: Figure S2. Typical chromatograms of the different metabolites analysed in non-spiked CSF samples: (1) Amino acids. (2) Biogenic amines. (3) Pterins. (4) 5-methyltetrahydrofolate. (5) Pyridoxal 5´-phosphate. (6) Thiamine.


Dryophytes chrysoscelis) is one of few freeze-tolerant frogs that mobilize glycerol as a cryoprotectant, yet cold and freezing-induced accumulation of this and other osmolytes has received little attention in this species. This study investigated the development of freeze tolerance in D. chrysoscelis, analyzing the response of the cryoprotectant system to cold acclimation, freezing, and thawing. Glycerol production was low and unresponsive to acclimation temperature, or duration of acclimation to 5 °C, except for one cold-acclimated frog that presented elevated glycerol in plasma, liver, and skeletal muscle. Curiously, glycerol concentration in skeletal muscle was higher than that of plasma and liver, in both warm- and cold-acclimated frogs, suggesting glycerol synthesis in muscle. Urea concentration in plasma doubled in response to cold acclimation but did not change during freezing. Freezing induced hepatic glycogen catabolism and an increase in glycerol and glucose in several tissues, although the mobilization dynamics differed between these cryoprotectants, possibly as a result of different transport mechanisms. Although hepatic glucose mobilization was of considerable magnitude, glucose accumulation in peripheral tissues was low and was surpassed by that of glycerol and urea. The muscle production of glycerol and the cold-induced accumulation of urea imply a role for skeletal muscle metabolism in the mobilization of cryoprotective solutes in D. chrysoscelis. The cryoprotectant system of D. chrysoscelis is complex, highly variable, and unique, with glycerol, glucose, and likely urea serving as cryoprotectants.Cope’s gray treefrog ( Among temperate vertebrates, a small number of anurans survive the low temperatures of winter by tolerating freezing of up to 65% of their body fluids, despite the several physical, biochemical, and molecular challenges associated with internal ice formation .Dryophytes chrysoscelis were acclimated to different temperatures as previously described  was sampled after a minimum of 12 weeks under warm acclimation . In late October, a random subset of animals was transferred to a refrigerated room and was cold-acclimated by being progressively cooled to 5 °C: during a 2-month period the environmental temperature changed from 22, to 20, to 15, to 8 °C, decreasing every 2 weeks, and finally remained at 5 °C for 2 weeks. Cold acclimation was accompanied by a shift to an 8:16 h light–dark regime. Frogs held at 5 °C were not kept in constant darkness  remained at 5 °C for an additional 3 weeks before being sampled in early January (hereafter “cold frogs”).N = 5) was cold-acclimated and kept at 5 °C for an additional 12 weeks (referred to as “long-term cold frogs”), and plasma glycerol levels were monitored in this group through repeated blood sampling. Animals were sampled for blood when their acclimation temperatures were 20, 8, and 5 °C, which resulted in frogs being sampled approximately at 4 week intervals, except for two sample points at 5 °C, which were 11 weeks apart. Blood was sampled by puncturing an axillary blood vessel, followed by collection (of approximately 30 µl) into heparinized microcapillary tubes. Blood was also obtained following terminal sampling of this group at the end of the 12 weeks at 5 °C. Following collection, blood was centrifuged  to isolate the plasma, and frozen in liquid N2.A subset of frogs (Acclimation experiment served as the unfrozen controls (N = 5–6). Animals to be frozen were kept in individual plastic containers and were placed in an incubator set at 2 °C. The temperature of the incubator decreased 1 °C day−1 until it reached − 2 °C. Once the incubator reached − 2 °C, the unfrozen frogs were each quickly placed on top of moist gauze and ice was added to the individual containers to ensure crystallization. Immediately after ice was added to the containers, the incubator temperature was lowered to − 2.5 °C. Frogs were visually inspected 24 h post-inoculation for signs of internal ice formation and a group (N = 5) was sampled shortly thereafter (“frozen frogs”). A subset of frozen frogs (N = 6) was sampled after thawing for 24 h at 5 °C (“thawed frogs”).Cold frogs were used in freezing and thawing experiments in early January. The cold frogs from the Acclimation and Freezing experiments were euthanized following approved LACUC protocols. Warm, cold, long-term cold, frozen, and thawed frogs were dissected in a 4 °C room after being weighed and their bladder fluid removed (bladder fluid was not removed in frozen frogs). Blood was collected from severed vessels, or from the heart (in frozen frogs), as described above. The liver and skeletal muscle from the thigh were excised, and samples of these organs were immediately frozen in liquid N2 and stored at − 80 °C before metabolite analyses were carried out. Other portions of liver, thigh skeletal muscle, and the frog carcasses, were blotted to remove excess moisture, weighed, placed in a 65 °C oven, and reweighed after drying for 5 days. Initial water concentration in the tissues and carcass, the latter serving as a measure of body water content, was determined by dividing the mass lost after drying by the mass of the remaining dried tissue. Water content was expressed in mass (g) of water per mass (g) of dry tissue so that the magnitude of change in this variable could be readily interpreted. This was preferred over reporting water content as percent of body mass, a form of quantification that may lead to erroneous interpretation of results, as the relationship between this variable and the expression of water on a mass basis is not linear, but asymptotic.Frogs in the −1 sodium acetate buffer, pH 4.8, with 1 mg ml−1 amyloglucosidase (Sigma Aldrich). The reaction was stopped by adding cold 7% (w/v) perchloric acid and the free glucose was determined as described above. Glycogen concentration was expressed as glucosyl units (µmol g−1 dry tissue) after subtraction of the initial free glucose. Liver and skeletal muscle metabolite levels were expressed per g of dry tissue.Plasma osmolality was measured by freezing point depression osmometry . Liver and skeletal muscle extracts were prepared by homogenizing samples in cold 7% (w/v) perchloric acid, followed by neutralization of the aqueous portion of the homogenate with KOH. Neutralized extracts and plasma were assayed for glycerol using a colorimetric glycerol assay reagent  following manufacturer’s instructions. In addition, levels of glucose, urea, and lactate in plasma and in neutralized tissue homogenates were determined using spectrophotometric assay kits . Extracts of liver and skeletal muscle were also assayed for glycogen using an enzymatic procedure as previously described  were converted to µmol ml−1 of tissue water. This expression of metabolite concentration serves as an estimate of intracellular cryoprotectant levels and allows for direct comparison of hepatic and muscle levels with plasma values , where tissue was the repeated factor (as multiple tissues from the same frog were compared). If there was a significant interaction between factors (treatment and tissue), or a significant effect of one factor, data were analyzed for simple effects with a Bonferroni correction for multiple comparisons. Comparisons among three treatments were performed using a one-way ANOVA, followed by a Tukey’s HSD test. As necessary, data were transformed to fulfill the parametric tests’ assumptions. Analyses were performed using JMP ; significance was accepted at P < 0.05.Means ± standard error of the mean (SEM) are the descriptive summaries used for the variables measured. Comparisons between two treatments were performed using two-sample The data that support the findings of this study are available from the corresponding author on request.Frogs were acclimated to distinct thermal regimes to determine the effect of temperature on the cryoprotectant system. Warm frogs fed readily throughout their period in captivity. In contrast, cold frogs ceased feeding when acclimation temperatures reached 8 °C. All frogs survived the acclimation treatments; all were males, with the exception of one cold frog, but the data from this frog did not differ, qualitatively, from that of other frogs in the group and the animal was kept in the analyses.Cold 3), had a plasma glycerol concentration of 44.5 µmol ml−1 and was a statistical outlier regarding all glycerol measurements , as well as plasma osmolality. This frog was excluded from analyses pertaining to glycerol and plasma osmolality. Exclusion of Cold 3 did not change the outcome of the statistical analyses reported, with one exception noted below.Warm and cold frogs did not differ regarding body mass, body and tissue water content, hepatic and skeletal muscle glycogen levels Table . Plasma −1 and plasma glycerol levels were below 2 µmol ml−1 in warm and cold frogs, averaging 0.4 ± 0.3 µmol ml−1 in the latter  in plasma glycerol levels, which were below 2 µmol ml−1 in all frogs (data not shown).Long-term cold and cold frogs were indistinguishable regarding all variables except water content in skeletal muscle, which was higher in long-term cold frogs Table . RepeateF1,9 = 1.2, P = 0.3) with cold acclimation, and glycerol content of cold frogs averaged 2.0 ± 0.3 and 7.7 ± 1.1 µmol g−1 of dry tissue in liver and muscle, respectively  in both warm and cold frogs.Tissue glycerol levels did not change  hepatic and muscle glucose content, although glucose levels did not differ between tissues  with cold acclimation, in liver  but not skeletal muscle ; nonetheless, urea levels were not distinct  between these tissues.Cold acclimation decreased  following 24 h of thawing . Freezing induced an increase in glycerol content, which reached 155 ± 27 and 207 ± 51 µmol g−1 of dry tissue in liver and muscle, respectively, and remained elevated during thawing.Plasma glycerol concentration increased by almost 370-fold, from 0.4 ± 0.3 to 149 ± 40 µmol ml−1 in cold frogs, to 59 ± 18 µmol ml−1 in frozen frogs (Table P = 0.0028) that of unfrozen frogs during thawing. Hepatic and skeletal muscle glucose levels changed during freezing with distinct dynamics . Hepatic glucose content increased by approximately 180-fold, from 0.8 ± 0.2 to 143 ± 26 µmol g−1 of dry tissue after 24 h of freezing, and decreased (P = 0.0034) with thawing, remaining above cold frog levels (P < 0.0001). Glucose levels in skeletal muscle increased during freezing, reaching an average of 27.9 ± 7.6 µmol g−1 of dry tissue, and remained elevated during thawing (P < 0.0001). Plasma, liver, and skeletal muscle content of urea did not change with freezing or thawing.Plasma glucose concentration increased from an average of 1.8 ± 1.1 µmol mlgs Table , and rem−1 to determine the direction of the concentration gradients during warm and cold acclimation, freezing, and thawing.We calculated cryoprotectant concentrations in liver and skeletal muscle in µmol mlF2,4 = 158, P = 0.0002) and cold frogs , being highest in muscle in both treatments , and muscle (P = 0.2), although plasma and muscle values differed (P = 0.0006). In thawed frogs glycerol concentration did not differ  among the three tissues.Glycerol levels varied among tissues in warm  and cold frogs , plasma levels were generally higher than those in muscle , being lowest in muscle and approximately tenfold higher in liver. During thawing, glucose levels varied among tissues with a pattern identical to that in warm and cold animals, with hepatic and plasma levels being similar (P = 0.09).Although intracellular glucose concentration varied among tissues in warm , cold , and frozen , but not thawed  frogs.The tissue concentration gradients differed between glycerol and glucose in warm  but not cold  frogs  and thawed  animals.Intracellular levels of urea varied among tissues of warm , plasma concentrations of these two cryoprotectants did not differ in cold , frozen , or thawed frogs . Moreover, hepatic intracellular concentrations of glycerol and glucose were similar in all treatments except in thawed frogs, in which hepatic glycerol concentration surpassed  that of glucose. Intracellular muscle glycerol concentrations were higher than those of glucose in all treatments .Levels of freezing-mobilized glycerol and glucose were overall similar in all the tissues sampled , following Costanzo et al. D. chrysoscelis, our ability to predict the effect of bound water on the dynamics of cryoprotectant mobilization is somewhat limited.The dynamics of cryoprotectant mobilization differed between glucose, glycerol, and urea. Somatic freezing resulted in the increase of plasma osmolality, partly as a result of cryoprotectant production, accumulation of lactate, and of freeze concentration. Urea levels did not respond to freezing, although intracellular concentrations of this metabolite differed between liver and muscle in frozen and thawed frogs, possibly due to different degrees of tissue dehydration. In contrast, concentrations of glycerol and glucose increased in all tissues during freezing, although the glucose concentration gradient between liver and plasma was more pronounced than that of glycerol, despite similar hepatic concentrations, possibly as a result of the different export system for each metabolite. Glucose export from the liver occurs via facilitated diffusion through glucose transporters  was greater than that of glucose (27.9 ± 7.3 µmol g−1 of dry tissue), a pattern also observed in R. sylvatica (Costanzo et al. Glycerol likely serves as a systemic cryoprotectant in y Yancey . The accR. sylvatica (Costanzo et al. Cryprotectant levels remained elevated in plasma of thawed frogs, rendering the animals susceptible to urinary loss of these metabolites (Layne et al. in Dryophytes chrysoscelis by promoting, under certain conditions, glycerol accumulation and, as seen in this study, the accumulation of urea, a putative cryoprotectant in this species. Variation in responses among individuals, populations, and studies suggests that in addition to cold exposure, light reduction, and aphagia, other variables may contribute to the physiological responses to cold and freezing. The differential patterns of accumulation of cryoprotectants during cold acclimation, freezing, and thawing likely represent the combined results of specific responsiveness to hormonal regulation, unique roles in cellular metabolism, and solute- and tissue-specific transport processes engaged during low-temperature exposure. The presence of such a complex cryoprotectant system probably allows Dryophytes chrysoscelis to augment its freeze-tolerance capacity by not only mobilizing cryoprotective glucose, but also by relying on both glycerol and urea for systemic cryoprotection.Cold acclimation contributes to the development of freeze tolerance