None of the samples had intrathecal IgG synthesis, as evaluated with quotient diagrams or oligoclonal band analysis. In cohort I, 98% of patient samples presented an intrathecal synthesis of FLC-k. In cohort II, all patients lacked intrathecal FLC-k synthesis. In the control group, 6.5% presented an intrathecal synthesis of FLC-k. The data support the concept that an intrathecal FLC-k synthesis is independent of the antibody class produced. In patients with an artificial intrathecal Ig synthesis due to blood contamination, FLC-k synthesis is lacking. Thus, additional determination of FLC-k in quotient diagrams helps to discriminate an inflammatory process from a blood contamination of CSF.In this retrospective, monocentric cohort study, we tested if an intrathecal free light chain kappa (FLC-k) synthesis reflects not only an IgG but also IgA and IgM synthesis. We also analysed if FLC-k can help to distinguish between an inflammatory process and a blood contamination of cerebrospinal fluid (CSF). A total of 296 patient samples were identified and acquired from patients of the department of Neurology, University Medicine Greifswald (Germany). FLC-k were analysed in paired CSF and serum samples using the Siemens FLC-k kit. To determine an intrathecal FLC-k and immunoglobulin (Ig) A/-M-synthesis we analysed CSF/serum quotients in quotient diagrams, according to Reiber et al. Patient samples were grouped into three cohorts: cohort I ( One method to distinguish blood derived from intrathecal, synthesized IgG, -A, or -M is the interpretation of IgG, -A, -M CSF and serum quotients  have recently gained new interest as a similar sensitive method to detect intrathecal inflammation compared to OCB detection . Some auFLC-k is synthesized in excess during the generation and assembly of Ig ,7,8. ConAn artifact mimicking intrathecal Ig synthesis in quotient diagrams can be present due to blood contamination of the CSF . IntrathThe primary aim of this study was to determine whether intrathecal FLC-k synthesis occurs in patients with an intrathecal IgA and/or IgM synthesis in the absence of OCB, compared to a control group of patients with no signs of inflammation. Secondly, we evaluated the use of FLC-k as a potential marker to distinguish true Ig synthesis in quotient diagrams from the artifact induced by blood contamination.The local institutional review board approved this study. This study was based solely on the use of coded surplus material. The use of coded surplus material without additional informed consent was based on the statement of the local ethics research committee . Between 2016 and 2018, we acquired samples for a prospective patient cohort as part of an already published study  compriseLaboratory analyses were performed in the interdisciplinary CSF laboratory of the University Medicine of Greifswald. Laboratory analyses were performed as described previously ,12.As a qualitative method to detect intrathecal IgG synthesis, the analysis of OCB was done by isoelectric focusing with a semiautomatic agarose electrophoresis system . As a quantitative method, the CSF serum quotients of FLC-k, IgG, IgA, and IgM were plotted against the albumin quotient in the already established quotient diagram ,3.IF) of Ig or FLC-k was calculated as described in Formula (1), with Qlim being the upper limit of the reference range:FLC-k in the sera and CSF were measured by nephelometry with the N Latex FLC kappa kit , using monoclonal antibodies on the BN Prospec analyser. CSF pre-dilution was set to 1:2, and serum pre-dilution was set to 1:100. The lower limit of quantification of the assay was 0.034 mg/L. Patients with highly elevated serum FLC-k values (FLC-k serum mean > 2 standard deviation) were excluded. The hyperbolic reference range, as well as the amount of intrathecally synthesized FLC-k, was calculated according to the formulas described by Reiber et al. [Cohort I: isolated IgM or A synthesis  or combined IgA and IgM synthesis. No quantitative or qualitative IgG synthesis, and erythrocyte count <500/µL;Cohort II: blood contamination by traumatic lumbar puncture with QIgM > QlimIgM, and if present, QIgA > Qlim-IgA and QIgG > QLim-IgG but QIgM > QIgA > QIgG. Erythrocyte count was >500/µL and/or elevated ferritin, which was done in cases of suspected subarachnoid hemorrhage and always if the erythrocyte count was >100/µL. Ferritin is a very sensitive, while not a specific marker to detect intrathecal blood. The absence of elevated ferritin is a strong indicator of artificial blood contamination . No patiControl group: no signs of inflammation in CSF profile; no pleocytosis or absence of intrathecal quantitative synthesis of IgM, IgA, IgG; and an absence of OCB in CSF.Patients were selected according to their CSF profile:Qlim–FLC-k). The mean amount of analysed CSF was estimated to be 2 mL. We used the individual serum albumin, IgG, IgA, IgM, and FLC-k values to simulate CSF contamination with 0.005 mL, 0.010 mL, 0.050 mL, and 0.100 mL of serum using the following formula:m represents the mass.To simulate artificial blood contamination, we used all patients of the control group that did not show an initial FLC-k synthesis  was used for statistical and graphical processing of the data. Statistical significance was assessed using Chi-square tests for nominal data. For a pairwise intergroup comparison, we performed the Kruskal–Wallis analysis of ranks test corrected for multiple tests; A total of 296 patients were identified retrospectively, the samples analysed and assigned to one of three cohorts based on their CSF findings. Cohort I was comprised of 41 patients with either a combined intrathecal IgA and IgM synthesis or isolated intrathecal IgA or IgM synthesis. No patient sample presented signs of blood contamination as defined in cohort II. Cohort II was comprised of 16 patients with artificial IgM and/or IgA synthesis through blood contamination. The control group was comprised of 239 patients with neither intrathecal Ig synthesis nor isolated OCB in the CSF. Details of the CSF data are described in Qlim–FLC-k. Furthermore, all patient samples with an isolated intrathecal IgA synthesis  had an FLC-k quotient > Qlim–FLC-k, meaning that FLC-k quotients are not elevated in cases of artificial Ig synthesis through blood contamination. One patient sample showed a QFLC-k below the lower limit of the reference range (Qlow–FLC-k).n = 220). The dot–dashed line represents the bisecting line (y = x), which is progressively approached by the patient samples with increasing simulated blood contaminations. Qlim–IgG/-IgA/-IgM/-FLC-k after simulating blood contamination of 2 mL CSF and with 0.005 mL, 0.010 mL, 0.050 mL, and 0.100 mL of serum. With 0.100 mL of serum contamination in the CSF, 99% of all patient samples would present artificial IgM or IgA synthesis, and 89% would present an artificial IgG synthesis, compared to no patient sample with artificial FLC-k synthesis.Qlim for IgG, IgA, IgM, and FLC-k, with increasing Qalb. Immunoglobulin G (IgG), IgA, and IgM indicate a similar progression, as already described by Reiber et al. [Qlim–FLC-k, though, is situated above the bisecting line, and shows a different progression than the immunoglobulins, resulting in a much higher slope.r et al. . The QliIn several studies, the determination of FLC-k is described as a similar sensitive method to the detection of OCB, with isoelectric focusing with lesser specificity ,14,15. AIt has already been shown that FLC-k is a potential biomarker for intrathecal inflammation in multiple sclerosis (MS) and clinical isolated syndrome (CIS), and that it can help to discriminate between inflammatory and non-inflammatory CNS diseases ,15,16. FQlim–FLC-k [Qlim–FLC-k, and a high level of IF FLC-k as a result.In recent works, FLC-k has been emphasised to have a higher sensitivity to the quantitative analysis of IgG in quotient diagrams, and a comparable sensitivity to detect intrathecal IgG synthesis by OCB analysis ,14,15,16im–FLC-k , the stiim–FLC-k ,14,15. Wim–FLC-k ,22. The Qlim–FLC-k. However, 6.5 % of the patients show intrathecal FLC-k synthesis. This FLC-k positivity of unknown origin could be due to the already-described low sensitivity of the quotient diagram for IgM or IgA. Moreover, the IgM quotient is prone to be influenced by measurement inaccuracy, due to the low CSF concentration [The majority of patients in the control cohort have FLC-k values below ntration  by whichntration  or IgD [ntration  synthesintration  and hencIn summary, the FLC-k quotient, accompanied by the established hyperbolic reference range, can not only be used to detect intrathecal inflammation, comprised of intrathecal IgG synthesis with a similar sensitivity to OCB analysis, but can also be an indicator for an inflammatory process with an intrathecal IgA or IgM synthesis.Qlim–FLC-k). This empirical data is supported by our simulated data  the linear function  while the quotient diagrams for IgM, IgA, and IgG result in apparent intrathecal Ig synthesis, are results of blood contamination. Adding FLC-k evaluation to CSF analysis can thus help to distinguish the effects of artificial blood contamination from intrathecal Ig synthesis in patient samples with QIgM > QIgA (>QIgG).FLC-k quotients above the upper reference limit in quotient diagrams (QFLC-k > Qlim-FLC-k) reflect intrathecal inflammation with high sensitivity . In addiOne major limitation of this study is the small sample size for the constellation of an isolated IgA or IgM synthesis, which is due to the rare appearance of this protein pattern.We could show that FLC-k synthesis can be found in patients with intrathecal IgA or IgM synthesis, with a sensitivity of 98%. However, in patients with artificial intrathecal Ig synthesis due to blood contamination, FLC-k synthesis is lacking. Thus, an additional determination of FLC-k in quotient diagrams helps to distinguish an inflammatory CNS process from an artificial Ig synthesis due to blood contamination of the CSF.


The concentration of MTX in blood is often measured quickly and easily by immunoassays. Thus, immunoassays may facilitate the easy determination of the concentration of MTX in the cerebrospinal fluid (CSF). In this study, we measured methotrexate (MTX) concentrations in the CSF using a high‐performance liquid chromatography (HPLC) method intended for analyzing CSF matrices and a chemiluminescence immunoassay (CLIA) method intended for assessing serum and plasma matrices and verified the differences in the results of the two methods.HPLC analysis for MTX in the CSF was performed using a Prominence UFLC system with a C18 column. The HPLC method was validated in accordance with the 2018 FDA guideline. The CLIA method was performed using an ARCHITECT i1000SR system intended for serum and plasma matrices. A total of 47 CSF samples  were analyzed using the two methods.HPLC = 1.205 × CLIA – 0.024.The HPLC method passed the validation criteria. The concentration of MTX in the same sample, determined using the HPLC and CLIA methods, differed proportionally; the percent difference in the concentrations averaged −23.0%  as revealed by the Bland‐Altman plot. The relationship between the measured values, evaluated using the Passing‐Bablok regression, was as follows: The equation deduced in this study can be used to correct the concentration of MTX measured using the CLIA method. MTX concentrations in the CSF were measured using a HPLC method intended for analyzing CSF matrices and a CLIA method intended for assessing serum and plasma matrices, and the differences in the results of the two methods were verified. The concentration of MTX in the same sample, determined using the HPLC and CLIA methods, differed proportionally. MTX, at high doses, is used clinically as a cytotoxic drug for the treatment of solid tumors and leukemia.The concentration of MTX in blood is often measured by immunoassays. The results can be obtained easily and quickly using this method, thereby greatly contributing to the management of delayed extracorporeal excretion. For the immunoassay of MTX concentrations in blood, fully automatic measuring devices are widely used, most of which use serum or plasma as the target matrix. Separation analyses methods, such as capillary electrophoresis The use of immunoassay as an alternative to the separation analyses methods can make the routine determination of the concentration of MTX in the CSF easy. Herein, we focused on the use of chemiluminescence immunoassay (CLIA) for this purpose. The CLIA method is now widely used to measure the concentration of MTX in blood.22.1Methotrexate and phosphate‐buffered saline (PBS) (pH 7.4) were obtained from Sigma‐Aldrich. 1,3,7‐Trimethyluric acid (137U) was obtained from Cayman Chemical. Acetonitrile (LC‐MS‐grade) was obtained from Merck. HPLC‐grade methanol and water were obtained from FUJIFILM Wako. Acetic acid, sodium acetate, sodium hydroxide (NaOH), and hydrochloric acid (HCl) were obtained from Nacalai Tesque.2.2HPLC analysis was performed on a Prominence UFLC system (Shimadzu). The Analyst software  was used to acquire and process the data. The CLIA method was performed on an ARCHITECT i1000SR system (Abbott).2.3Chromatographic separation was done at 40°C using an InertSustain AQ‐C18 column . The mobile phase consisted of sodium acetate buffer :acetonitrile . The flow rate was 0.9 mL/min. The detector wavelength was set at 307 nm.2.4MTX stock solution (100 mM) and the internal standard (IS)  were prepared in 0.01 M NaOH and HPLC‐grade water, respectively, and stored at −20°C. The concentration of the IS working solution was 100 µM.2.5For drug‐free CSF, the sample that remained after CSF testing was used. Matrix calibrators were prepared daily by the addition of properly diluted stock solution to drug‐free CSF. A series of calibration samples  was prepared. The lower limit of quantitation (LLOQ) (0.10 µM) and three quality control (QC) samples at low (0.20 µM), medium (0.60 µM), and high (1.20 µM) concentrations of MTX were prepared by the addition of appropriately diluted stock solution to drug‐free CSF.2.6The leftover CSF test samples collected from patients receiving MTX intrathecally were used as clinical specimens. Spiked specimens were prepared by the addition of properly diluted stock solution to drug‐free CSF.2O. Subsequently, the cartridges were washed with 1 ml of 5% methanol in 0.01 M HCl. Finally, MTX and IS were eluted with 1 ml of methanol. The eluent was collected in conical disposable glass tubes and evaporated under a gentle stream of nitrogen at 45°C. The residue was dissolved in 100 µl of PBS and centrifuged ; 50 µl of this solution was injected into the chromatograph.For the solid phase extraction procedure,2.7In accordance with the standard guideline 2.7.1Methotrexate‐ and IS‐free CSF samples were used to evaluate the selectivity. The lowest concentration on the calibration curve was regarded as the LLOQ. For MTX, interfering peak areas were <20% of the peak area of the LLOQ. For LLOQ samples, the mean accuracy was within ±20% of the nominal value and the coefficient of variation (CV) value did not exceed 20%.2.7.2The carry‐over effects of MTX and IS were evaluated by testing the response of a blank CSF sample immediately following the highest concentration of the calibration sample. The peak area of the blank CSF sample was <20% of the peak area of the LLOQ sample for MTX and 5% for the IS. The ordinary least squares method was used to fit the peak area ratio vs. the analyte concentration for linearity.2.7.3To evaluate the accuracy and precision, five replicates of LLOQ samples and QC samples at three levels were analyzed. The mean accuracy was within ±15% of the nominal values for the QC samples, except for the LLOQ, which was within ±20% of the nominal value. The CV value did not exceed 15% for the QC samples and 20% for the LLOQ samples.2.7.4To evaluate the dilution integrity for the clinical samples, a 10‐fold dilution of QC samples  using blank CSF was performed. Five replicates of diluted samples were analyzed. The accuracy and precision of the diluted samples were within ±15% and 15%, respectively. Stability tests were performed by evaluating the accuracy of QC samples at three levels under two conditions . Five replicates of QC samples at three levels were analyzed. The accuracy of QC samples for the stability test was within ±15%.2.8The CLIA was performed on the ARCHITECT i1000SR system (Abbott). The recommended matrices included serum and plasma. The sample volume was 60 µl. The calibration range was 0.040‐1.500 µM. Samples with MTX concentrations >1.500 µM were diluted 20‐fold with the ARCHITECT i1000SR system mechanically. Calibration  and QC samples  were routinely tested according to the manufacturer's instructions before the analysis of the samples.2.8.1Linearity of MTX detection in CSF was assessed using samples prepared by the addition of properly diluted stock solution to drug‐free CSF .2.8.2Four samples with MTX concentrations of 0.070, 0.450, 1.000, and 10.000 µM were prepared by the addition of properly diluted stock solution to drug‐free CSF. To evaluate the precision, five replicates of samples at each level were analyzed.2.9A total of 47 CSF samples  were analyzed using the two methods. For HPLC, samples with MTX concentrations >1.50 µM were diluted 10‐fold with the blank CSF. For CLIA, samples with MTX concentrations >1.500 µM were diluted 20‐fold with the ARCHITECT i1000SR system mechanically. A Bland‐Altman plot was used to evaluate the agreement between HPLC and CLIA for the analysis of MTX in human CSF.2.10This study was approved by the ethics review committee of the Faculty of Medicine at the University of Miyazaki, Japan .2.11www.r‐project.org) was used for statistical analyses. Results with a p‐value of <.05 were regarded as statistically significant.R v.3.5.1 .No carry‐over was observed. A typical calibration curve is shown in Figure 3.1.3The results for the accuracy and precision of the HPLC method are presented in Table 3.1.4For diluted samples, the mean relative error was 4.4% and the CV was 1.88%. The results of the stability tests are shown in Table 3.23.2.1r) was 0.997.The linearity of MTX detection in CSF by CLIA was excellent in the verified concentration range (0.1‐15.0 µM); the correlation coefficient   Figure . In addi) Figure .4In this study, we established an HPLC method for measuring the concentration of MTX in the CSF and successfully verified the concentration of MTX in the CSF measured using the CLIA method. The monitoring of the MTX concentration in the CSF is important for preventing its toxicity to the central nervous system and evaluating the therapeutic effects. The LLOQ in the HPLC method was determined to be 0.10 µM, which is considered sufficiently sensitive for the toxicity standard of MTX (1 µM).HPLC = 1.205 × CLIA – 0.024) obtained from the Passing‐Bablok regression, and this corrected value can be used in clinical practice. However, correction with this equation can only be performed in the concentration range (0.1‐15.0 µM) in which the linearity of the CLIA method was verified, and the correction accuracy should be verified in the future. Furthermore, this error in the determined values can be solved by constructing a CSF‐based calibration curve for the immunoassay. However, in a fully automatic analyzer, such as the ARCHITECT i1000SR system, the calibration curve is saved in the control system. For daily clinical practice, it is extremely useful to have an environment in which the concentration of MTX in serum, plasma, and CSF can be measured with one calibration curve . Nevertheless, it was shown that high and low MTX concentrations in CSF can be evaluated by the CLIA method. The adaptability of the correction value to the clinical criteria of MTX concentration in CSF requires further study, but irregular intraventricular drug clearance can be sufficiently evaluated. Moreover, the ARCHITECT i1000SR system can analyze more than 30 samples per hour, which is highly valuable for clinical applications requiring a rapid, simple, and convenient method.Because immunoassays are based on antigen‐antibody interactions, cross‐reactivity is an important issue. Drugs that are analyzed after in vivo administration undergo minute structural changes because of chemical reactions, such as metabolism, and can be converted into non‐analyte targets. If a drug is to be analyzed, these non‐analyte targets may be detected. Furthermore, when the sample to be analyzed is directly supplied to the reaction system, there is a possibility that the reaction is affected by the sample components. Although immunoassays are less versatile than separation analyses methods, they are very much applicable for clinical use, such as in commercial reagent kits. In this study, we measured the concentrations of MTX in different matrix samples using an immunoassay for which the intended matrix is serum or plasma. The concentration of MTX in the CSF sample measured using the CLIA method was slightly different from that measured using the HPLC method. The concentration determined using the HPLC method developed for the quantification of the MTX concentration in the CSF can be considered the actual value. We found that the concentration of MTX in the CSF could not be accurately measured by the CLIA method intended for serum or plasma matrices. Because of the cross‐reactivity, it is easily considered that the results obtained using the CLIA method would be higher than those obtained using the HPLC method. In addition, there is a concern that protein in the sample could influence drug quantification by the immune reaction. The protein binding rate of MTX in blood is about 50%.There are some limitations to our study. First, the number of clinical specimens used in the comparison of the two methods was few. Although a CSF sample spiked with MTX was used to ensure a wide range of comparative concentrations, data that are completely consistent with clinical samples might not have been obtained. Second, the accuracy of the regression equation used for correcting the values measured using the CLIA method could not be verified using a clinical sample different from the samples used to create the regression equation. This was because of the scarcity of specimens.In this study, we verified the accuracy of measuring MTX concentrations in the CSF using the CLIA method and deduced an equation that can correct the values with respect to the actual ones determined using the HPLC method. The evaluation of the existing immunoassays other than the CLIA method in the same manner may lead to further adoption of the measurement of the MTX concentration in the CSF in routine clinical practice. We believe such evaluations will contribute to providing safer and more personalized treatment.The authors declare that they have no conflict of interest.


Gastric cancer (GC) is a malignancy with high morbidity/mortality, partly due to a lack of reliable biomarkers for early diagnosis. It is important to develop reliable biomarker(s) with specificity, sensitivity and convenience for early diagnosis. The role of tumour-associated macrophages (TAMs) and survival of GC patients are controversial. Macrophage colony stimulating factor (MCSF) regulates monocytes/macrophages. Elevated MCSF is correlated with invasion, metastasis and poor survival of tumour patients. IL-34, a ligand of the M-CSF receptor, acts as a “twin” to M-CSF, demonstrating overlapping and complimentary actions. IL-34 involvement in tumours is controversial, possibly due to the levels of M-CSF receptors. While the IL-34/M-CSF/M-CSFR axis is very important for regulating macrophage differentiation, the specific interplay between these cytokines, macrophages and tumour development is unclear.A multi-factorial evaluation could provide more objective utility, particularly for either prediction and/or prognosis of gastric cancer. Precision medicine requires molecular diagnosis to determine the specifically mutant function of tumours, and is becoming popular in the treatment of malignancy. Therefore, elucidating specific molecular signalling pathways in specific cancers facilitates the success of a precision medicine approach. Gastric cancer tissue arrays were generated from stomach samples with TNM stage, invasion depth and the demography of these patients (n = 185). Using immunohistochemistry/histopathology, M-CSF, IL-34 and macrophages were determined.+ TAMs were a good prognostic factor in some cases and could be used as an independent prognostic factor in male T3 stage GC.We found that IL-34 may serve as a predictive biomarker, but not as an independent, prognostic factor in GC; M-CSF inversely correlated with survival of GC in TNM III–IV subtypes. Increased CD68Our data support the potency of IL-34, M-CSF, TAMs and the combination of IL-34/TAMs as novel biological markers for GC, and may provide new insight for both diagnosis and cellular therapy of GC. Gastric cancer (GC) is an important disease with high morbidity and mortality. Due to a lack of relatively convenient and reliable biomarkers, large numbers of GC are diagnosed at an advanced stage, with poor prognosis . It is fThe inflammatory microenvironment surrounding a tumour is a complex ecology of immune cells interconnected with tumour cells. Among the leucocytes present at the tumour site, macrophages are abundantly present at all stages of tumour progression . Tumour-Macrophage colony stimulating factor (M-CSF) is a growth factor important in the regulation of differentiation, proliferation and survival of haematopoietic cell lineages . Circula+ monocytes in peripheral blood mononuclear cells. IL-34 stimulates the differentiation of monocytes into macrophages via the CSF-1 receptor [IL-34 was first identified by Lin et al. in 2008, as a protein that is able to bind to CD14receptor . Subsequreceptor . Using aIL-34 is also a ligand of the M-CSF receptor, and acts as a “twin” to the M-CSF cytokine, demonstrating overlapping and complimentary actions . IL-34 aStudies have long sought specific biological markers that could characterize GC . HoweverConventional chemotherapy kills cancers non-specifically, based on the high rate of cancer cell division. Precision medicine is becoming popular in the treatment of malignancy, which tailors intervention to the individual patient with customization of medical decisions and healthcare . However+-TAMs in GC. The relationship between M-CSF, IL-34 and CD68+-TAMs infiltration in GC was explored with a view to elucidate potential molecular targets.In our current study, we determined the production of M-CSF and IL-34, and the number of infiltrating CD68+-TAMs in GC were investigated. Following immunohistochemical staining , whereas CD68+-TAMs were increased 5.5 fold (p < 0.05)  Fig. .Fig. 1Re+-TAMs expressions were found to significantly differ between subgroups defined by a range of clinicopathological parameters (Table p < 0.05). Lower IL-34 (decreased 61%) was also observed in female compared to male GC patients (p < 0.05). In addition, there was a significant correlation between IL-34 and differentiation of GC (low differentiation group of GC had 74% decreased IL-34 compared to the high differentiation group) (p < 0.05), suggesting that IL-34 correlates with the state of differentiation of GC. There was no correlation between IL-34 and other parameters, such as tumour size, lymph node metastasis, tumour invasion depth and TNM stage of GC. Additionally, there was no correlation between clinical parameters and M-CSF or CD68+-TAMs , demonstrating a moderate to high accuracy, while IL-34 (AUC = 0.624) and M-CSF (AUC = 0.775) demonstrated only moderate accuracy [The area under the curve (AUC) derived from the ROC curves showed that CD68accuracy .+-TAMs , lymph node metastasis and tumour invasion depth T3. There was no significance in other subgroups between survival and M-CSF and CD68+-TAMs  was obtained from 180 GC patients undergoing gastrectomy without prior chemotherapy in Xuzhou Medical University, China (2008–2010). These GC patients were 140 males and 40 females (aged 23–85 years) (Table Sections (5 µm) from tissue microarray blocks were labelled with three antibodies, as described previously . The antBriefly, all the images were taken by an Olympus BX51 microscope with fixed exposure time and light sources to avoid any additional unwanted errors. The quantitative analysis was conducted using ImagePro Plus 7.1 software  as described by Liu et al. –45. The p < 0.05 was considered statistically significant.The SPSS 16.0 statistical software package was used for the statistical analysis. Comparison between two groups was performed via Mann–Whitney U test, as described , 45. ComAdditional file 1: Figure S1. Correlation of IL-34, MCSF and CD68+ TAMs expression with clinicopathological parameters of tumour size, lymph node metastasis, tumour invasion depth and TNM subtypes of GC. IL-34, MCSF and CD68+ TAMs all have no correlations with any clinicopathological parameters of tumour size, lymph node metastasis, tumour invasion depth and TNM subtypes of GC.Additional file 2: Figure S2. Correlation of MCSF and CD68+ TAMs with clinicopathological parameters of age, gender and differentiation subtypes of GC. MCSF and CD68+ TAMs both have no correlations with any clinicopathological parameters of age, gender and differentiation subtypes of GC.Additional file 3: Figure S3. Survival analysis of MCSF for prognosis of subtypes of GC patients. Kaplan-Meier survival analysis of GC patients: there were no correlations of MCSF with survival of gender, age, diameter and lymph node metastasis subtypes of GC patients.Additional file 4: Figure S4. Survival analysis of MCSF for prognosis of subtypes of GC patients. Kaplan-Meier survival analysis of MCSF for prognosis of GC in differentiation, tumour invasion depth and TNM stage subtypes.Additional file 5: Figure S5. Survival analysis of CD68+ TAMs for prognosis of subtypes of GC patients. Kaplan-Meier survival analysis of CD68+ TAMs for prognosis of GC in female, tumour diameter < 5 cm, no lymph node metastasis and T4 stage subtypes.Additional file 6: Figure S6. Survival analysis of CD68+ TAMs for prognosis of subtypes of GC patients. Kaplan-Meier survival analysis of CD68+ TAMs for prognosis of GC in age, tumour differentiation and TNM stage subtypes.Additional file 7: Table S1. Univariate analysis of MCSF and clinicopathological factors affecting survival of patients with GC in TNM III–IV.


Feline coronavirus (FCoV) infection initiates monocyte-associated viremia and viral persistence. Virus-infected, -activated monocytes also trigger feline infectious peritonitis (FIP), a fatal systemic disease of felids typified by granulomatous (peri)phlebitis. Currently, the exact mechanisms inducing monocyte activation and FIP are unknown. This study attempted to identify the potential immediate effect of virulent FCoV on colony-stimulating factor (CSF) (granulocyte (G)-CSF, monocyte (M)-CSF and granulocyte-monocyte (GM)-CSF levels through in vitro assessment, alongside prototypical pro- and anti-inflammatory mediators (interleukin (IL)-1, IL-6, IL-12p40, tumor necrosis factor (TNF)-α, and IL-10); this was assessed alongside the in vivo situation in the hemolymphatic tissues of cats euthanized with natural end-stage FIP. For the in vitro work, isolated monocytes from SPF cats were cultured short-term and infected with the FIP virus (FIPV) strain DF2. Mediator transcription was assessed by quantitative reverse transcriptase PCR (RT-qPCR) at 3, 6 and 9 h post infection (hpi), and in the post-mortem samples of bone marrow, spleen, and mesenteric lymph nodes (MLN) of cats with FIP. We observed limited and transient changes in cytokine transcription in monocytes after infection, i.e., a significant increase of IL-6 at 3 hpi and of GM-CSF over the 3 and 6 hpi period, whereas M-CSF was significantly decreased at 9 hpi, with a limited effect of age. The findings indicate that the infection induces expansion of the monocyte/macrophage population, which would ensure the sufficient supply of cells for consistent viral replication. In natural disease, the only upregulation was of G-CSF in the MLN, suggesting either immune exhaustion or an active downregulation by the host as part of its viral response.  Feline infectious peritonitis (FIP) is a widely distributed feline coronavirus (FCoV)-induced systemic disease in cats, characterized by a granulomatous to necrotizing phlebitis and periphlebitis, granulomatous inflammatory lesions in several organs, and fibrinous to granulomatous serositis with protein-rich body cavity effusions ,2,3. FolFeline monocytes constitutively transcribe cytokines, such as interleukin (IL)-1β, IL-6, IL-10, IL-12p40 and tumor necrosis factor (TNF)-α. Levels of transcription are variable but generally highest for IL-1β . In assoThe colony stimulating factors (CSFs) are members of the cytokine superfamily, first named numerically for their ability to act as growth factors for hematopoietic cells in vitro. They have since been renamed monocyte (M)-CSF, granulocyte monocyte (GM)-CSF and granulocyte (G)-CSF, respectively. The CSFs have overlapping but non-redundant roles. M-CSF is important for homeostasis, being detectable in plasma and constitutively expressed by many cell types, including macrophages, endothelial cells, and fibroblasts . GM-CSF Studies using isolated feline alveolar macrophages and/or monocytes have shown increases in G-CSF, GM-CSF, TNF-α and VEGF production at 2–3 days post infection with FIPV ,18,19. HPrevious studies have shown that constitutive cytokine transcription in isolated feline monocytes  is generally highly variable , or mockOverall, IL-1β was detected in all samples, TNF-α in 95.2% (118/124 samples), IL-6, M-CSF and GM-CSF in 90.3% (112/124 samples), IL-10 in 88.7% (110/124 samples), G-CSF in 87.9% (109/124 samples) and IL-12p40 in 74.2% (92/124 samples). 2 = 0.175, F = 6.27, p < 0.01). When transcription was examined pairwise for each time point using a t-test, for G-CSF, transcription levels were significantly higher at 6 hpi (mean ΔCt = 2.010) than at 3 hpi (mean ΔCt = 5.460) . For M-CSF, the opposite was observed; transcription levels were significantly lower at 6 hpi compared to 3 hpi  and 9 hpi . In uninfected monocytes, IL-1β was transcribed at the highest relative levels, followed by TNF-α, G-CSF, M-CSF, IL-10, IL-6, GM-CSF and IL-12p40 . The tim2 = 1.09, F = 3.61, p < 0.05), G-CSF , M-CSF  and GM-CSF . When transcription was examined for each step, IL-6 monocytes exhibited significantly higher levels at 3 hpi (mean ΔCt = 4.322) than at 9 hpi . For G-CSF, the transcription levels were significantly higher at 6 hpi than at 3 hpi . For M-CSF, the opposite was observed; transcription levels were significantly higher at 3 hpi (mean ΔCt = 2.859) than at 6 hpi  and at 9 hpi . GM-CSF transcription was significantly higher at 6 hpi (mean ΔCt = 3.223) compared to both 3 hpi  and 9 hpi . In FIPV-infected monocytes, IL-1β (mean ΔCt: −0.954) was again transcribed at the highest relative levels, followed by TNF-α (mean ΔCt: 3.836), G-CSF (mean ΔCt: 3.89), M-CSF (mean ΔCt: 5.349), IL-6 (mean ΔCt: 5.513), GM-CSF (mean ΔCt: 5.609), IL-10 (mean ΔCt: 6.101) and IL-12p40 (mean ΔCt: 11.646) . We founPrevious in vitro studies have shown that FIPV infection of isolated alveolar macrophages, peripheral blood mononuclear cells (PBMC) and/or monocytes can induce the production of cytokines, such as TNF-α, IL-1β, G-CSF, GM-CSF and VEGF, after 1–3 days, an effect enhanced by incubation with an anti-S (spike) protein antibody ,19,23,24p < 0.01) and 6 h  time points , but had returned to levels comparable to those of uninfected monocytes at the later time points (p < 0.05); they had progressively dropped over time, as shown by the fact that levels were significantly lower at 6 hpi and at 9 hpi compared to 3 hpi   compared to 9 hpi  . No modep < 0.001, 6 hpi: t = 4.054, p < 0.001, 9 hpi: t = 2.255, p = 0.185). This is illustrated in The age of animals whose monocytes had higher IL-10 transcription post infection than mock infection was significantly lower at 3 hpi and 6 hpi compared to 9 hpi, when examined by univariate analysis  and 6 hpi . For G-CSF, they were found to be significantly higher at 6 hpi compared to 9 hpi , and for TNF-α they were significantly higher at 3 hpi than at 6 hpi .The relative transcription (ΔΔCt) of M-CSF showed significantly lower levels at 9 hpi than at both 3 hpi .Mean viral loads were lowest in the BM, and increased in spleen and MLN in cats with FIP . A compap < 0.05, p < 0.001). When the different groups of MLN were examined in pairs, both groups without FIP (FCoV-positive and -negative) had significantly lower G-CSF transcription levels than cats with FIP , whilst there was no significant difference between the non-FIP groups themselves. This showed that there was no significant influence of FCoV infection in the absence of FIP. The results are illustrated in The G-CSF transcription levels were significantly lower in the BM of cats with FIP (Wilcoxon rank sum (Mann–Whitney) (Wrs): z: 2.211, p < 0.05) and spleen , with no significant differences between any subgroups for the MLN (p < 0.05), as for G-CSF.M-CSF was significantly lower in cats with FIP in both the BM   C. No sig < 0.05) .p < 0.0001), which is compatible with both being lower in FIP; no correlations between FCoV and CSF levels were observed. The spleen showed a negative correlation between FCoV and M-CSF , as well as positive inter-CSF correlations between GM-CSF and the other CSFs . In the MLN, the elevated G-CSF transcription in FIP corresponded to a positive correlation with viral load (p = 0.0001), whilst M-CSF correlated positively with both other CSFs . The latter correlations were in spite of it being only G-CSF transcription that was significantly upregulated in the MLN in FIP, suggesting that whilst M-CSF and GM-CSF mRNA levels were not altered significantly by the presence of FCoV or by FIP, the stimuli to produce CSFs in the MLN may be linked. The results are presented in Levels of CSFs and FCoV were correlated with each other across all cats. Within the BM, G- and M-CSF correlated with each other  and late, i.e., end-stage FIP (in vivo)—thus avoiding the use of experimental in vivo infection. Immediate post-infection responses were assessed in isolated feline monocytes within the first hours after infection, after the first replication cycles, with FIPV DF2, whilst the late phase response was studied in the hemolymphatic organs of clinical cases euthanized with FIP.FIPV DF2 is a serotype II coronavirus isolate able to induce FIP experimentally ,29. FeliWe observed limited and transient changes in cytokine transcription after infection, i.e., upregulation of IL-6 at 3 hpi, and of GM-CSF at 3 and 6 hpi, and downregulation of M-CSF at the latest time point (9 hpi). The age of the animals at the time of blood collection had a limited effect, showing the more intense IL-1β response of monocytes from younger and older cats, and higher IL-10 mRNA levels in the monocytes of younger cats during the first six hours after infection. These findings reflect the observations made regarding the constitutive transcription of these cytokines . The finFocusing on the changes occurring in vivo during late-stage disease, there was variation between organs. Both M- and G-CSF were lower in the BM of cats with FIP, but the difference was significant only in FIP cats with FCoV-negative BM samples. This may indicate a systemic attempt at the downregulation of inflammatory mediators that is slightly counteracted locally when the virus is present within the monocyte population of the BM; the BM exhibits an increase in monocytes with systemic FCoV infection and, even more so, with FIP . M-CSF aWithin these hemolymphatic organs, in FIP there is an increasing viral load from BM to spleen to MLN. BM alterations reflect most closely the systemic background response, as this organ has not been reported to develop FIP lesions (possibly as the immature monocytes are not susceptible to infection—which instead occurs in circulating and fully differentiated cells) . The splThis part of the study was performed on 14 barrier-maintained cats, ranging in age from 10 months to 7 years 9 months at the time of sampling. Four cats were male neutered and had been purchased at the age of 1.5 years , another four were female and aged 8 months when purchased . The remaining six cats were male neutered and had been purchased at Ciba Geigy, Switzerland, at the age of 4 months.All cats tested negative for feline leukemia virus, feline immunodeficiency virus, feline herpesvirus, feline calicivirus and parvovirus. Throughout the experiment, cats were housed under barrier-maintained conditions at the Small Animal Clinic, University of Giessen, Germany and at the Animal Facility of the Vetsuisse Faculty, University of Zurich, Switzerland. They were monitored daily for any signs of illness. The animal work was undertaken with permission of the Regierungspräsidium Giessen (II 25.3/19c20/15c) and the Veterinäramt Zürich (118/99).®  within 2 h of death and stored at −80 °C until use. The Bristol cases were part of a biobank, built up as a resource for multiple studies, many of which have been previously utilized [All cats in this part of the study had been seen initially as patients at the university small animal clinics or local veterinary practices of Bristol, UK, or Zurich, Switzerland, and were euthanized with or without FIP for clinical reasons unrelated to this study. A post-mortem examination was performed on each cat with owner consent and samples of BM, spleen and MLN were collected into RNAlaterutilized ,40,41. Autilized . Not allBlood samples were taken from donors at 4-week intervals. From most animals, two samples were taken at different time points, with two animals being sampled three times. The mean age of the cats whose samples were examined at 3 hpi (n = 25) was 2.64 years (95% CI: 2.00–2.91), at 6 hpi (n = 19) was 2.37 years (95% CI: 1.61–3.14) and at 9 hpi (n = 18) 2.29 years (95% CI: 1.46–3.12).50; [2, followed by washing with RPMI medium without FCS and further incubation in RPMI medium with 10% FCS for another 2 h, 5 h and 8 h, respectively. The other culture received the same treatment with the exception of a sham infection step (uninfected controls). After further washing with medium without FCS, cells were collected in a total of 700 μL RLT buffer  with 1% mercaptoethanol and frozen at −80 °C.Each time, 14 mL of peripheral blood was collected and divided into two. From each half, peripheral blood mononuclear cells (PBMCs) were isolated and cultured short-term for 15 to 18 h in Roswell Park Memorial Institute (RPMI) (PAA Laboratories GmbH) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories GmbH) and 1% Pen/Strep (PAA Laboratories GmbH), followed by thorough washing of plates to eliminate non-adherent cells and obtain pure monocytes, as previously described ,27. One 50;  for 1 h After thawing and addition of 700 μL ethanol, RNA was extracted from monocytes using a commercially available kit . TheFor the demonstration of FCoV infection, from both monocytes and tissue samples, a TaqMan RT-qPCR detecting the FCoV 7b gene was used as previously described ,28.Contaminating genomic (g)DNA in RNA extracts was digested and complement (c)DNA synthesized according to published protocols . Real-tiThe relative quantification of cytokine signals was done by the comparative Ct method . The Ct −ΔΔCt [Differences in cytokine transcription between uninfected monocytes and infected monocytes for the different time points were also evaluated as follows: For each cytokine, the Ct value obtained from uninfected monocytes was subtracted from the ΔCt values obtained from infected monocytes . Cytokine transcription was calculated as the difference in transcription of the respective cytokine from control monocytes and experimental monocytes: 2−ΔΔCt ,27,43.−ΔΔCt.Tissue sample results were calculated slightly differently in that there was no paired control sample. Here, the mean bone marrow ΔCt from the non-FIP group was used as the calibrator to obtain ΔΔCt, following the same initial step of comparing the target and GAPDH Ct values. Analysis and graphical representation were performed on 2t-test and ANOVA) to examine preliminary associations with time of infection and differences between infected and uninfected cells. Further analysis of the associations between age and infection time with the cytokine transcription used log10 transformation of the ΔΔCt values of the difference between control and infected cell cultures, which led to approximate normal distributions. Log-transformed values above zero indicated increased cytokine expression in the infected cells with negative values indicating a decrease. Cytokine expression was examined against time from the point of infection and the age of the animals using univariate analysis (t-test). To examine and visualize the association between age and upregulation of cytokine transcription, paired animal samples were organized in six age groups of approximately equal number (group 0 (n = 10): 10 to 13 months old; group 1 (n = 7): 14 to 15 months old; group 2 (n = 14): 16 to 21 months old; group 3 (n = 10): 22 to 28 months old; group 4 (n = 10): 29 to 36 months old and group 5 (n = 11): 59 to 93 months old). Both linear and quadratic multivariate regression models were examined using cluster  robust standard errors. Model fitness was assessed by examining the distribution of residual values. The threshold for statistical significance was established at p < 0.05.All statistical analyses were completed using Stata 13.1 (StataCorp LP). For the in vivo study, non-parametric methods (Mann–Whitney/Wilcoxon rank test and Spearman’s correlation) were used to examine differences in viral load and cytokine expression between FIP and non-FIP specimens. For the in vitro study, initial analysis of the ΔCt values used univariate analysis (In conclusion, our data suggest that FIPV infection of feline monocytes has limited immediate effects on cytokine expression by these cells. A potential increase in the monocyte/macrophage pool and limited, short-term neutrophil activation appear to be the most likely main consequences. This may however be associated with increased phagocytosis, and hence contribute to an increased cell uptake and increased viral load even in the absence of notably increased cell numbers . These r


Pythium insidiosum, an aquatic oomycete. Therapeutic protocols based on antifungal drugs are often ineffective because the cytoplasmic membrane of P. insidiosum does not contain ergosterol. Therefore, the treatment of pythiosis is still challenging, particularly making use of natural products and secondary metabolites from bacteria. In this study, xanthyletin and substances obtained from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 exhibited anti-P. insidiosum activity and, moreover, xanthyletin was non-toxic against human cell lines. The hyphae of P. insidiosum treated with these three substances exhibited lysis holes on a rough surface and release of anamorphic material. Therefore, xanthyletin could be considered a promising alternative agent for treating cutaneous pythiosis in the near future.Pythiosis is a harmful disease caused by  Pythium insidiosum is an aquatic oomycete, or fungus-like microorganism. Its morphology is similar to that of filamentous fungi but a phylogenetic analysis of this pathogen has shown a closely related to diatoms and algae than to true fungi1. P. insidiosum can infect both humans and animals through the eyes or skin wounds, causing pythiosis, an endemic disease with high morbidity and mortality rates3. Human pythiosis manifests clinically as cutaneous and subcutaneous, vascular, ocular or disseminated infection. The treatment includes antifungal agents, radical surgery and immunotherapy. However, these options have not always been successful and generally accepted therapeutic protocols have not yet appeared4. Most of the common antifungal drugs are ineffective because the cytoplasmic membrane of P. insidiosum does not contain ergosterol or this pathogen possesses an incomplete ergosterol biosynthetic pathway5. Therefore, the treatment of pythiosis is still challenging. Previous studies demonstrated that natural compounds from plants and secondary metabolites from microorganisms had relatively good in vitro antimicrobial activity against P. insidiosum7. Xanthyletin, a natural compound of green plants, is classified in a group of pyranocoumarins, coumarin derivatives. Antifungal activity of xanthyletin against Candida albicans, Aspergillus fumigatus and Fusarium solani as well as its anti-P. insidiosum effect have been reported8. Besides, its anticancer and antibacterial activity were also described9. Bacterial species belonging to the genus Pseudomonas, especially those isolated from the environment, are often used as biocontrol agents in economic crops since the pathogen excretes substances with antibacterial and antifungal activity10. Moreover, microorganisms of the genus Klebsiella produce secondary metabolites with antibacterial and antifungal activity as well11. An in vitro study of secondary metabolites excreted from bacterial environmental strains included Pseudomonas stutzeri and Klebsiella pneumoniae from 16 strains that expressed anti-P. insidiosum activity7. However, the mechanisms of action of both xanthyletin and secondary metabolites from bacteria with anti-P. insidiosum activity remain unclear. Fibroblasts are one of the most common cell types, widely present in many structures, particularly in connective tissue and predominantly in the human dermis. Fibroblasts are of mesenchymal origin, exhibiting a spindle or stellate shape morphology. These cells play an important role in the cutaneous wound healing process13. As one of the clinical manifestations of human pythiosis is cutaneous and subcutaneous infections, toxicity testing of substances with anti-P. insidiosum effects on these cell lines are important in terms of their safety in the treatment of pythiosis. Therefore, this study evaluated the anti-P. insidiosum effect of xanthyletin and secondary metabolites from P. stutzeri and K. pneumoniae on P. insidiosum by broth dilution susceptibility testing and scanning electron microscopy (SEM). The toxicity of these substances to the cell lines prepared from normal human dermal fibroblast (NHDF) cells was also assessed.P. stutzeri ST1302 (brown liquid) and K. pneumoniae ST2501 (dark brown liquid) showed anti-P. insidiosum activity as screened by the disc diffusion method. After that, the crude extracts were fractionated by activity-guided separation liquid column chromatography. The crude extracts from K. pneumoniae ST2501 were divided into 5 fractions; fraction number 1 was brown semisolid, fraction number 2 was brown oil, fraction number 3 was brown solid, fraction number 4 was black oil and fraction number 5 was black semisolid. The crude extracts from P. stutzeri ST1302 were divided into 14 fractions; fraction no. 1 was yellow oil, fraction no. 2 was orange oil, fractions no. 3, 4, 5, 7 and 9 were deep yellow solid, fraction no. 6 was yellow solid, fractions no. 8, 12, 13 and 14 were brown oil, and fractions no. 10 and 11 were white solid. Fractions no. 1 to 4 of the crude extracts from K. pneumoniae ST2501 and fractions no. 3, 4, 6, 9 and 12 of the crude extracts from P. stutzeri ST1302 exhibited anti-P. insidiosum activity (Table P. insidiosum activity (the largest inhibition zone) was exhibited by fraction no. 6 of the crude extract from P. stutzeri  and hyphae changes when P. insidiosum was treated with thimerosal  and treated with three anti-P. insidiosum substances. The negative control showed cylindrical hyphae with a smooth surface 6. SEM analysis of P. insidiosum treated with anti-P. insidiosum substances revealed lysis holes on a rough surface and anamorphic material released from hyphae, suggesting effects on protein composition in the cell wall or cell membrane of this oomycete. Our previous study using Fourier transform infrared spectroscopy found anti-P. insidiosum activity of xanthyletin and secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501, characterized by clearly changed proteins in P. insidiosum21. Thimerosal is a well-known compound used as a preservative in various cosmetics, vaccines and drug products because of its bactericidal and antifungal properties including anti-P. insidiosum activity. However, it is toxic to humans, causing mortality in the fetus, birth defects and neurodevelopmental disorders as evidenced by a case of a pregnant woman who received a vaccine preserved with thimerosal. Besides, cellular apoptosis and abnormal functions of T- and B-cells that affected cytokine production, cell growth and proliferation following chronic exposure to low levels of methyl mercury have also been reported23. Recently, we used 0.02% (w/v) thimerosal as a positive control because it can kill P. insidiosum although this amount is not suitable for use in humans and animals. The mechanism of its action is not fully understood yet and we suspected it to be related to interactions with some proteins in P. insidiosum leading to the death of the microorganism21. Xanthyletin is a pyranocoumarin that has been shown to be cytotoxic in vitro against tumor cells such as Caco-2, HCT-8 and HEp-2 cell lines using an MTT assay24. Moreover, xanthyletin showed cytotoxic activity against human cancer HeLa cells without toxicity to normal cells9. The NHDF cell culture was selected for cytotoxicity assay as these cells are a common part of the human dermis and toxic effects of drugs on them are undesirable. Moreover, one of the important clinical signs of human pythiosis is skin damage. In the present study, xanthyletin at 0.003 mg/mL showed the best anti-P. insidiosum activity and exhibited low toxicity for NHDF cells. However, this study evaluated anti-P. insidiosum activity against only 11 strains of P. insidiosum isolated from pythiosis patients in Thailand. Therefore, further studies on geographically and genetically diverse P. insidiosum strains and anti-P. insidiosum activity against pythiosis in animal models should be conducted to confirm the properties of these anti-P. insidiosum substances.It is well known that the therapy of pythiosis has posed a challenge in recent decades as the current treatment options are highly variable and often lead to failureP. insidiosum effects and toxicity of xanthyletin and secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501, xanthyletin showed excellent anti-P. insidiosum activity as well as the lowest toxic effects in vitro on NHDF cells. Therefore, it could be considered a promising alternative agent for treating cutaneous pythiosis in the near future.As seen from the evaluation of anti-P. insidiosum isolates (n = 11) from human pythiosis were identified by molecular analysis in a study by Chaiprasert et al.25. The cultures were maintained on Sabouraud Dextrose Agar  at room temperature and subcultured once a month. According to a study by Thongsri et al., they were rechecked by their morphology on the SDA at 25 °C, microscopically by detection of perpendicular sparsely septate hyphae and induced zoospore formation and by PCR identification7.Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 which produced anti-P. insidiosum substances were isolated from a water sampling area around Khon Kaen University and identified biochemically using the VITEK 2 system (bioMérieux)7. They were stored in 10% Oxoid Skim Milk Powder (Thermo Fisher Scientific) solution with 15% glycerol at −20 °C.It was obtained as a powder  from ChemFaces (Wuhan).P. stutzeri ST1302 and K. pneumoniae ST2501 were prepared according to McFarland Standard No.1 (3×108 CFU/mL) in saline; 700 µL of this concentration were inoculated into 700 mL of Brain Heart Infusion broth  and incubated in a rotary shaker  for 3 days. Each culture broth was then centrifuged and the supernatant was filtered through a membrane filter (Millipore). The cell-free filtrate was concentrated ten-fold in a rotary evaporator  and mixed three times with a two-fold volume of dichloromethane (Thermo Fisher Scientific) in case of P. stutzeri or with ethyl acetate (Thermo Fisher Scientific) in case of K. pneumoniae. Both dichloromethane and ethyl acetate layers were dried with anhydrous Na2SO4 (Merck) and concentrated in a rotary evaporator. Then the crude extracts were fractionated by activity-guided separation liquid column chromatography using a protocol described by Wittayapipath et al.21.The inoculations from P. stutzeri and K. pneumoniae were tested for anti-P. insidiosum activity using the disc diffusion method. Stock solutions of each testing solution were prepared as 500 mg/mL. Paper discs  were placed onto SDA plates with P. insidiosum aged 2 days grown on the plates. Twenty microliters of testing solutions were applied to the discs (dichloromethane and ethyl acetate were used as controls) and then the testing plates were stored at room temperature for 2 h in a laminar flow biosafety cabinet to test the solution diffusion. Subsequently, they were incubated at 37 °C for 3, 6 and 9 days and inhibition zones were measured when the growth of P. insidiosum reached the control discs21.Xanthyletin and the crude extracts from et al. was used with some modifications20. Briefly, blocks measuring 5 mm in diameter were cut out from SDA plates with P. insidiosum cultures with a cork borer. These mycelia-containing blocks were transferred into 1.95 mL of Sabouraud Dextrose Broth  and incubated at 37 °C for 3 days. Dilutions of xanthyletin were prepared from 0.100 to 0.001 mg/mL, dilutions of P. stutzeri ST1302 and K. pneumoniae ST2501 crude extracts from 12.5 to 0.1 mg/mL by double dilution. Each 50 μL of diluted testing solutions were added to the cultures and incubated at 37 °C for 24 h. A P. insidiosum block in 2 mL of SDB was incubated at 37 °C for 4 days as a growth control. After expiration of the incubation time, the P. insidiosum agar blocks with mycelium were placed on SDA plates and incubated at 37 °C for 3, 6 and 9 days (depending on the growth speed) to evaluate the hyphal growth and determine the minimum fungicidal concentrations (MFCs). The MFCs were assessed as the lowest concentration of the solution without any apparent hyphal growth. All tests were performed in triplicate.The method by Trolezi et al.20 and Mendoza et al.3 were followed with some modifications. For SEM analysis, hyphae in blocks from each testing solution evaluated as MFCs were selected. P. insidiosum hyphal fragments from the growth control in broth dilution susceptibility testing served as a negative control, that is the agent survived. By contrast, P. insidiosum hyphal fragments treated with 0.02% (w/v) thimerosal (Sigma-Aldrich) were used as a positive control. Thimerosal is well known as a preservative in various cosmetics, vaccines and drug products to prevent harmful contamination with microorganism. Therefore, thimerosal completely killing P. insidiosum was used as a positive control in this study. Hyphal fragments were collected and washed three times in 0.1 M phosphate buffer solution (PBS). Then, they were fixed in 2.5% glutaraldehyde (Merck) in 0.1 M PBS at 4 °C for 1 h and washed in the same buffer. Subsequently, the samples were dehydrated with a graded ethanol series. The Emitech K500X sputter coater (Quorum Technologies) was used for gold sputtering of SEM samples. Finally, the hyphal morphology was evaluated by the S-3000N scanning electron microscope (Hitachi).The protocols described by Trolezi P. insidiosum substances from P. stutzeri ST1302 and K. pneumoniae ST2501 were evaluated for cytotoxicity using NHDF cell lines (Promocell). A colorimetric assay with 3-−2,5-diphenyltetrazolium bromide (MTT) was applied to investigate the cytotoxicity27. Briefly, NHDF cell lines were counted with the Neubauer hemocytometer using trypan blue solution . Then, the cells were cultured in the wells of 96-well flat-bottom microplates (Thermo Fisher Scientific) containing 100 μL of Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% solution of antibiotics  at 37 °C with 5% CO2 and 95% humidity for 24 h. Subsequently, 100 μL of each anti-P. insidiosum substance double diluted with fresh medium was added to each well with the final concentrations in the wells ranging from 0.125 to 0.004 mg/mL in case of xanthyletin and from 12.5 to 0.391 mg/mL in case of both secondary metabolites from P. stutzeri and K. pneumoniae. The plates were then incubated under the same conditions. Further, the solutions in the wells were carefully discarded and cells were washed with PBS. Then, the MTT solution  was added at an amount of 100 μL each into the wells and the mixture was kept at 37 °C with 5% CO2 and 95% humidity for 4 h. After removal of the supernatant, the formazan crystals were dissolved by adding 100 μL of dimethyl sulfoxide per well and the absorbance was determined at a wavelength of 570 nm. The experiment was performed in triplicate and repeated three times under the same conditions. The rate of cytotoxicity was determined using the following formula:Xanthyletin and two anti-Additionally, the half-maximal inhibitory concentration (IC50) was measured.


Headaches attributed to low cerebrospinal fluid (CSF) pressure are described as orthostatic headaches caused by spontaneous or secondary low CSF pressure or CSF leakages. Regardless of the cause, CFS leaks may lead to intracranial hypotension (IH) and influence cerebral blood flow (CBF). When CSF volume decreases, a compensative increase in intracranial blood volume and cerebral vasodilatation occurs. Sinking of the brain and traction on pain-sensitive structures are thought to be the causes of orthostatic headaches. Although there are many studies concerning CBF during intracranial hypertension, little is known about CBF characteristics during low intracranial pressure. The aim of this review is to examine the relationship between CBF, CSF, and intracranial pressure in headaches assigned to low CSF pressure. According to the International Headache Society, headaches attributed to low cerebrospinal fluid (CSF) pressure are described as orthostatic headaches caused by spontaneous or secondary low CSF pressure or CSF leakages, usually accompanied by neck pain, tinnitus, changes in hearing, photophobia, and/or nausea. These symptoms remit after normalization of CSF pressure or successful sealing of the CSF leak . Due to CBF is commonly defined as the volume of blood delivered to the brain tissue per minute, while ICP is defined as the pressure inside the skull and is the result of the interaction between the brain, CSF, and cerebral blood volume. Cerebral autoregulation (CA) is the mechanism responsible for maintaining constant CBF over a wide range of arterial blood pressures. CBF regulation is possible through neurogenic, myogenic, metabolic, or endothelial control to support CBF at an appropriate level for the brain’s metabolic oxygen requirements . HoweverThe Monroe–Kellie doctrine describes the following principle: “with intact skull, sum of volume of brain plus volume of CSF plus volume of intracranial blood is constant, and therefore decrease or increase in one will result in increase or decrease in one or both of the remaining two” ,17. In IMultiple methods have been developed to accurately measure CBF metabolism. Each method has its unique advantages, as well as limitations . CurrentSee In a study of 66 patients who required diagnostic lumbar punctures, CBF was assessed 24 h before and within 24 h after the procedures . In patiPomeranz et al. measured regional CBF utilizing the hydrogen clearance method in the cerebral cortex and subcortical nuclei in 11 cats with stable blood pressures and intracranial hypotension of at least 15 torr. They noticed that regional CBF was unchanged relative to the baseline, and concluded that cerebral vascular autoregulation is maintained during significantly increased perfusion pressure due to negative intracranial pressure. Additionally, the symptomatology of clinical intracranial hypotension is not due to decreased cerebral perfusions . ContrarChaves et al. reported a patient who developed multiple cerebral infarcts from reversible cerebral vasoconstriction (RCVS) after intracranial hypotension caused by an iatrogenic lumbar CSF leak. These authors hypothesized that the cerebral vasoconstriction was caused by the sinking of the brain and associated structures, which was caused by CSF loss . MechaniDespite a small number of studies, there is some evidence indicating CBF changes in low intracranial pressure headaches, but the results are incoherent. Most studies revealed the dilatation of intracranial vessels, especially veins with CBF increases and flow velocity decreases, whereas some data showed IH-related vasoconstrictions and flow velocity increases. These ambiguous results may have arisen from different methods used for CBF assessments or heterogeneous groups of patients. Although there is a link between low intracranial pressure and cerebral blood flow, a larger study should be performed to assess possible connections between intracranial pressure, CBF, CSF, vein and artery flow, CVR, and cerebral autoregulation in low intracranial pressure headaches.


Leptomeningeal metastasis (LM) is a terminal stage cancer manifestation to whole neuraxis via cerebrospinal fluid (CSF). Up to now, LM has no solid biomarkers for disease progression or treatment response. Extracellular vesicles (EVs) in biofluids have been recently studied to evaluate cancer diagnostics and prognostics. Here, we measured nanoparticles in human CSF from 472 patients with both Dynamic Light Scattering and Nanoparticle Tracking Analysis. We found that the size distribution and concentration of nanoparticles in LM-disseminating CSF were significantly different from those in non-LM CSF samples. Changes in EVs concentration showed a potential biomarker for the therapy response in patients undergoing intra-CSF chemotherapy. Our suggestion of combined biomarker of EVs concentration and onco-miR for LM chemotherapy could help physicians to perform this possible neurotoxic treatment with appropriate monitoring tools for the effectiveness.8 /mL vs. 1.49 × 108 /mL, p < 0.01). Changes in NTA-measured nanoparticles concentration after intra-CSF chemotherapy were further examined in 33 non-small cell lung cancer patients with LM. Overall survival was longer for patients with increased EV than the others . Markers of extracellular vesicles (CD9/CD63/CD81) significantly decreased in the EV-decreased group. MicroRNA-21 expression decreased in this favorable prognostic group, whereas it increased in the EV-decreased group. In conclusion, the elevated concentration of extracellular vesicles in cerebrospinal fluid in patients with LM may be a predictive marker for survival duration. Moreover, EV changes combined with microRNA-21 might be a biomarker for monitoring the efficacy of intracranial chemotherapy of LM in non-small cell lung cancer patients.Leptomeningeal metastasis (LM) has a poor prognosis and is difficult to diagnose and predict the response of treatment. In this study, we suggested that the monitoring of changes in the concentration of extracellular vesicles in cerebrospinal fluid could help diagnose or predict outcomes for LM. We measured nanoparticles in 472 human cerebrospinal fluid (CSF) from patients including LM with both Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) after two-step centrifugations. NTA revealed that the concentration of CSF nanoparticles was significantly increased in LM compared to other groups (2.80 × 10 Cerebrospinal fluid (CSF) circulates in the central nervous system (CNS) and transports neurotransmitters, hormones, and other actively or passively secreted metabolites from brain cells . AnalyziLeptomeningeal metastasis (LM) is a terminal-stage cancer that is devastating to patients, with cancer cells spreading through the CSF and adhering to the entire CNS. The overall survival of LM patients is approximately 6–8 weeks, and there is no definitive treatment for LM except intra-CSF chemotherapy, which has a questionable cost/benefit profile due to both low response rate and neurotoxicity ,14. AlthThere have been several studies to establish these nanoparticles as a biomarker for CNS tumors, but this has yet to be established mainly due to lack of general existence, reference values from many samples, and difficulty in setting standard method to measure quantitatively. Here, we measured nanoparticles in human CSF from 472 controls and patients with various CNS diseases to provide a reference value and to testify nanoparticles as a biomarker for LM diagnosis and treatment response monitoring. We also tracked changes in EVs concentration in CSF from LM patients after intraventricular chemotherapy and analyzed its relationship with overall survival. Furthermore, we measured the expression of a well-known onco-miRNA, miR-21, in CSF from non-small cell lung cancer patients with LM after intraventricular chemotherapy.Demographic characteristics of the 472 patients in the proportion analysis are summarized in Relative proportions of nanoparticles in CSF samples by Dynamic Light Scattering (DLS) based on intensity particle size distribution (PSD) were obtained using Zetasizer Nano and built-in software. Although the Zetasizer Nano can detect PSD at the millimeter range, we focused on intensity peaks in the 1–1000 nm range for the relative proportion calculation. The majority of the samples  exhibited two peaks of various PSD proportion A,B. A mip < 0.0001). Upon further analysis, we found that the large peak was associated with a significantly larger particle size in LM patients than in the other patient groups  nearly abolished the small peak and increased the proportion of the large peak in DLS measurement .8 particles/mL  and different among patients groups . Cancer control (CC) and brain metastasis (BM) patients exhibited mean EV concentrations of 2.68 × 108/mL  and 2.49 × 108/mL , respectively. LM patients exhibited the highest mean EV concentrations of 7.15 × 108/mL , which was significantly higher than all the other groups (p < 0.0001) except the other CNS disease group. Patients with other CNS diseases (OD) showed the widest distribution of EV concentrations. The OD group consisted of patients with autoimmune disease (n = 25 out of 27 patients with multiple sclerosis), intracranial hemorrhage (n = 13), or CNS infection (n = 9). Patients with autoimmune disease exhibited a mean EV concentration of 1.31 × 108/mL , whereas patients with intracranial hemorrhage or CNS infection exhibited higher mean EV concentrations of 15.32 × 108/mL  and 14.86 × 108/mL , respectively  in CSF from different patient groups by Nanoparticle Tracking Analysis (NTA) using NanoSight NS300 at the same camera condition as described in the Methods. The measured EVs concentration of 472 samples was a mean of 3.46 × 10s groups A,B. Healectively .p < 0.001). The mean EV size of the other CNS disease group was also significantly smaller than other groups (p < 0.001). In detail, the subgroup of intracranial hemorrhage and autoimmune disease  drove the EV size of OD groups into the smallest . Meanwhile, the proportion of 50–150 nm sized EV was higher in BT and control groups than that of LM (p < 0.05). To compare the relative EV size distribution between groups, empirical cumulative distribution function (ECDF) plots were generated. LM groups showed the largest distribution as depicted by ECDF plot D, followWe compared these values by NTA to those measured by DLS. In DLS measurement, we assumed the large peak to be EV-sized and calculated a mean of the large peak size by intensity PSD to be a mean EV size. The discrepancy of the mean values between DLS and NTA was various among patients groups .Although the protein level is relatively low in CSF compared to serum, it has been known that various lipoproteins could be observed at the EV size range in human biofluids . To idenhttp://cris.nih.go.kr, Identifier: KCT0000082) of ventriculolumbar perfusion (VLP) chemotherapy with methotrexate [p < 0.05, p < 0.001, Among LM patients, 41 were enrolled in a prospective clinical trial  patients among those with increased and decreased EV concentration of the above survival analysis .n = 10) and decreased (n = 19) EV concentrations (p < 0.001), whereas the increased groups showed no significant exosome concentration change (ratio 1.13).Beads bearing each CD9/CD63/CD81 antibody-capturing exosome in CSF were measured regarding their mean fluorescence intensity (MFI) in patients with increased .As changes in EV concentration after the treatment were related to patients’ OS, we next measured the expression of a well-known onco-miRNA, miR-21, in NSCLC (adenocarcinoma). Among those above 33 patients with NSCLC, fourteen samples were available for this further analysis. The miR-21 expression measured by droplet digital polymerase chain reaction (ddPCR)  was normSince CSF is filtered from blood through the “blood-CSF barrier”, it contains clinically important biomolecules and extracellular vesicles of CNS disease ,26. Our 6–109 particles/mL) without specific EV enrichment process, and also, it provided a reference EV concentration for unaltered CSF from hundreds of patients with various CNS diseases. Counts of EVs in CSF by NTA can be varied by camera level and detection threshold [The merit of our measurement of EV concentration by NTA is that we minimized the sample manipulation and transfer before measurement . Instead, we directly counted the number of EVs in clinically available CSF samples, requiring only two-step centrifugation to discard waste cells and debris. Our study proved that the EV concentrations of CSF is suitable for the NTA measurement range 10hreshold . However9 particles/mL followed by CNS infection. Spaull at al., who measured an EV concentration of unaltered CSF from preterm intracranial hemorrhage patients by NTA, reported 2.89 × 1010 to 1.31 × 1011 particles/mL in three samples [Our observation of significantly higher EV concentrations in LM patients compared with other patients could be explained by the fact that the CSF of LM patients harbors many cells, including cancer cells and white blood cells, which is known to be a major source of secreted EVs . Althoug samples . Aside fOur observation of changes in EV concentration after chemotherapy demonstrates the possibility of conveniently analyzing patient CSF samples by commercially available NTA to provide a useful biomarker of LM treatment response. In general, increased EV secretion could reflect the removal of toxic materials such as chemotherapeutic drugs from cells, increased intercellular communication, or a modulation of the microenvironment for migration, immune regulation, or cancer metastasis ,29,31. PUnfortunately, we could not separate exosomes, microvesicles, and apoptotic bodies in this study. However, the decreased EV concentration in this study was proved to reflect a decreased concentration of exosome surface marker bearing EVs by both MACSPlex APC-conjugate beads and ExoView Tetraspanin Chips. We evaluated the implications of changes in EV concentration in a discrete cohort of LM patients with the same primary cancer of NSCLC, adenocarcinoma, who underwent the same protocol of phase II ventriculolumbar perfusion methotrexate chemotherapy. The prognostic value of miR-21 in NSCLC patients has been examined in many studies ,33,34,35It remains a technical problem of evaluating CSF microRNA expression level due to both the absence of endogenous control for normalization and the origin of microRNA  ,12. ReceIn this study, we also tracked changes in EV concentration and miR-21 expression in the CSF of LM patients after the intraventricular chemotherapy. Decreased post-treatment EV concentration and increased miR-21 expression was associated with poor prognosis in NSCLC patients with LM, who underwent intra-CSF methotrexate chemotherapy. We believe that our observation could be used as reference values for future CSF nanoparticle study and developed as a method in clinical practice to monitor the treatment response in these patients with LM.This observational study was approved by the Institutional Review Board of the National Cancer Center , followed all rules and regulations regarding the protection of human subjects in clinical research, and was in accordance with the ethical guidelines outlined in the Declaration of Helsinki. All patients were signed informed consent. For patients below 18 years, the informed consent was also obtained from a parent and/or legal guardian. We analyzed CSF samples from six groups of patients. Control groups without CNS tumors were composed of patients with systemic cancer  and patients without any cancer . The second category of patients was LM from systemic cancer. The third group of patients was having parenchymal brain metastasis but no LM . The 4th group of patients was with primary brain tumors other than brain metastasis . The last group of patients was from various CNS diseases but no cancer . To include CC, recent imaging  or bone marrow reports were evaluated to detect cancerous lesions. All LM patients were cytologically diagnosed and had positive neuroimaging studies  .CSF samples were obtained after Institutional Review Board approval of the National Cancer Center (Identifier: NCC-2014-0135) for the purpose of identifying CSF biomarkers. CSF samples were obtained via lumbar puncture during spinal anesthesia (CC), CSF cytology examination , or CSF chemistry (OD). Other CSF samples were obtained from the cisternal/subarachnoid space during craniotomy . A total of 669 CSF samples was consecutively obtained from six groups of patients between 2014 and 2019 . As prelg for 20 min for cells down at room temperature as storage in refrigerator (4 °C) increased nanoparticle concentration  and kept frozen at −80 °C for genomic profiling and nanoparticle evaluation. We also confirmed that one time freezing–thawing did not alter EV concentration significantly, but EV concentration increased significantly after three times of freezing–thawing cycles  was placed in a disposable cuvette for Zetasizer Nano measurement following the manufacturer’s protocol . The Zetasizer Nano system determines particle sizes (0.3 nm to 10 µm) by measuring their Brownian motion in the sample using dynamic light scattering. Of the measurement display modes, intensity particle size distribution (PSD) was chosen to represent the relative proportion of nano-sized particles.6 to 109 particles/mL). Pure CSF samples were loaded into the laser module sample chamber manually with a confirmation of no air bubbles, and an automated camera module tracked the Brownian motion of particles in the liquid sample. The in-built software  calculated the EV size and concentration of triplicate of each sample with 30 s video capture. For the consistency of the observed values, we used the sCMOS  camera type and fixed camera level at 8 of 10, and set the temperature at 25 °C. When the concentration was more than 109 particles/mL, the sample was diluted, and the initial concentration was calculated according to the dilution factor.Pure CSF samples without dilution or concentration were used for the quantitative measurement of EV-sized particles using the NanoSight instrument , which is a laser-based light scattering system that provides general nanoparticle characterization in terms of size and concentration , and 0.2 mL of concentrate was recovered. The concentrate was measured for protein concentration using a BCA protein assay kit . The concentrate was diluted with sample dilution buffer . U-87 MG (human glioblastoma cell line) were purchased from ATCC , and they were culture in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. After washing with PBS, cells were lysed with lysis buffer  and centrifuged at 12,000× g for 10 min. Cell lysate and supernatant were diluted with sample dilution buffer. The samples were used for protein expression reference. First, 15 mL of pre-centrifuged CSF was concentrated with AmiconSamples were resolved by 10% or 15% SDS-PAGE, transferred onto nitrocellulose membrane, blocked with 5% nonfat dry milk in TBST  followed by incubation with primary antibodies against exosome membrane CD63 or CD81  or cytosolic protein cytochrome c or GM130 . Blots were washed and incubated with HRP-conjugated secondary antibody. Antibody complexes were visualized using an enhanced chemoluminescence Western blotting detection system . Relative luminescence intensities were calculated using Image J .®, Merck, Darmstadt, Germany) to remove debris and protein aggregates. Cell-free CSF were further concentrated with 100 kDa MWCO centrifugal ultrafiltration device . Intact forms of exosomes in 8 mL concentrated CSF were purified by a method that comprises exosome precipitation with metal-affinity and further purification with a spin-based size-exclusion chromatography. The purified CSF exosomes were subsequently subjected on transmission electron microscopy to determine their shapes and ultrastructures. Then, 5 µL of exosome preparation at 1 × 109 particles /µL were adsorbed onto glow-discharged carbon-coated copper grids  for 5 min. After excess liquid removal, the grid was washed 10 times with PBS and subsequently stained with 2% uranyl acetate . The grid was finally examined in JEM 1011 microscope , and images were recorded with an ES1000W Erlangshen CCD Camera .Exosomes in cell-free CSF were isolated using the ExoLutE exosome isolation kit  according to manufacturer instructions. Briefly, thawed CSF samples were pre-filtered by a 0.45 µm syringe filter  and analyzed with a BD LSRfortessa™ . For setting up the instruments, the Exosome setup beads were used, and the flow cytometry data were analyzed using FlowJo software . The median fluorescence intensity (MFI) values of APC for CD9, CD63 and CD81 bead populations were used to determine the expression of exosome markers.CSF samples after two-step centrifugation were subjected to bead-based multiplex EV analysis by flow cytometry with an MACSPlex Exosome Kit  as a provided protocol . BrieflyQuantitative measurement of EVs was tested with ExoView Tetraspanin kits  as previously decribed . CSF wasg for 5 min to separate the aqueous phase. After phase separation, a 1.25 volume of 100% ethanol was added to the aqueous phase, and the solution was loaded into the column and centrifuged at 12,000× g for 30 s. Extracted RNA was washed and eluted with 25 µL (95 °C) RNase-free water. The purity (A260/280 and A260/230 ratio) and concentration of dissolved RNA samples were measured using a Nanodrop spectrophotometer .We used mirVana PARIS  to extract extracellular RNA from cleared CSF according to the manufacturer’s instructions. Briefly, 0.5 mL CSF was gently mixed with 2× denaturing solution, acid-phenol:chloroform was added, and the mixture was centrifuged at 10,000× To evaluate differences in miRNA expression between LM and normal CSF samples, we measured the expression of hsa-miR-21-5p by ddPCR. First, 2 µL purified total RNA was reverse-transcribed into cDNA using a Taqman advanced miRNA cDNA Synthesis Kit  according to the manufacturer’s instructions. A total of 5.5 µL undiluted cDNA was mixed with 2× ddPCR Supermix  and Taqman advanced miRNA Assay probes . Each PCR sample was partitioned into up to 20,000 nl-sized droplets by a Bio-Rad QX299 droplet generator. Then, 40 µL of each PCR mixture underwent the following cycling protocol: 95 °C for 10 min (DNA polymerase activation), followed by 40 cycles of 94 °C for 30 s (denaturation) and 60 °C for 1 min , followed by post-cycling steps of 98 °C for 10 min (enzyme inactivation) and an infinite 4 °C hold. The amplified PCR product of the nucleic acid target in the droplets was quantified in the FAM channel using a QC200 Droplet Reader  and analyzed using QuantaSoft v.1.7.4.0917 software .p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 18.0 , GraphPadPrism 6 , and R software 3.6.0 .Categorical data were analyzed using Chi-square, Fisher’s exact, or Mann-Whitney U tests as appropriate. Continuous data were analyzed using Student’s t-tests or analysis of variance (ANOVA) with parametric scheffe’s test and nonparametric Dunn’s tests for post hoc comparisons. The Kolmogorov-Smirnov test was used for the comparison of Ecdf analysis. A two-tailed We measured nanoparticles in human CSF from 472 controls and patients including leptomeningeal metastasis (LM), which has neither effective treatment nor biomarkers for disease progression or treatment response, by both Dynamic Light Scattering and Nanopaticle Tracking Analysis. We analyzed the difference of size and concentration according to different CNS disease status. In addition, we found the change of EVs concentration after intra-CSF chemotherapy in patients with LM to correlate with patients overall survival, and the expression level of onco-microRNA (miR-21) was in inverse proportion to EV concentration change in non-small cell lung cancer. Our measurement of combined EVs concentration and onco-miR for LM chemotherapy could be performed in moderately equipped institution and help physicians monitor this possible neurotoxic treatment.


Progressive multifocal leukoencephalopathy (PML), caused by JC polyomavirus, is a demyelinating disease of the central nervous system that primarily affects oligodendrocytes. It can cause significant morbidity and mortality. An early diagnosis is of high relevance as timely immune reconstitution is essential. However, diagnosis can be challenging if virus detection via cerebrospinal fluid (CSF) PCR remains negative. Hence, identifying CSF biomarkers for this disease is of crucial importance. We applied a targeted metabolomic screen to CSF from 23 PML patients and eight normal pressure hydrocephalus (NPH) patients as controls. Out of 188 potentially detectable metabolites, 48  passed the quality screen and were included in the analyses. Even though there was a tendency towards lower concentrations in PML (mostly of phosphatidylcholines and sphingomyelins), none of the differences between PML and controls in individual metabolite concentrations reached statistical significance (lowest p = 0.104) and there were no potential diagnostic biomarkers (highest area under the ROC curve 0.68). Thus, CSF metabolite changes in PML are likely subtle and possibly larger group sizes and broader metabolite screens are needed to identify potential CSF metabolite biomarkers for PML. Progressive multifocal leukoencephalopathy (PML) is a rare, opportunistic, demyelinating disease of the central nervous system (CNS) caused by the human JC polyomavirus HPyV-2). HPyV-2 is a small ubiquitous DNA polyomavirus with a 5.13 Kb circular enclosed double-stranded DNA. Antibodies against this virus are detectable in about half of all people worldwide . HPyV-2 . As PML As demonstrated in studies on neurodegenerative disorders  and also in the context of neuroinflammation, metabolomic profiling of CSF has a considerable potential for identifying biomarker candidates and elucidating disease-associated metabolic networks –15. FurtIn the present study, we applied the AbsoluteIDQ®-p180 kit  for CSF analysis as a targeted metabolomics approach based on liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate differences in CSF metabolic profiles between patients with PML and controls with normal pressure hydrocephalus (NPH). The aim was to identify and validate potential biomarkers for the diagnosis of PML, especially in HPYV-2-PCR negative patients.The study subjects comprised 23 patients with PML and 8 patients with normal pressure hydrocephalus (NPH) as controls. All patients were treated at the Department of Neurology at Hannover Medical School during the years 2007 and 2019. CSF samples were obtained via lumbar puncture. The following routine CSF parameters were determined within 2 hours: cell count, lactate concentration, protein concentrations (via Bradford dye-binding assay). HPYV-2 DNA concentrations were measured with a real-time PCR assay. Determination of HPYV-2 DNA concentrations by PCR and HPYV-2 antibody specificity index in CSF were performed as routine tests at the Institute of Virology, Hannover Medical School, Germany, and at the Institute of Virology, University of Düsseldorf, Germany, as described before [The study was approved by the institutional review board of Hannover Medical School (file no. 2413–2014). All patients gave written informed consent to the use of their data.All CSF samples were shipped on dry ice and analyzed in duplicate using a targeted metabolomics kit . This approach allows for the simultaneous quantification of 188 metabolites from different compound classes  using a combination of liquid chromatography  and mass spectrometry . The lipids, acylcarnitines, and the hexoses were determined via flow injection analysis-tandem mass spectrometry (FIA-MS/MS), while the amino acids and biogenic amines were measured by LC-MS/MS. Sample preparation and measurements were performed according to the manufacturers’ instructions. It has to be noted that some of the CSF samples had been stored for more than ten years. Knowledge about possible effects on long-term storage of CSF samples on metabolite profiling is rare. Regarding serum and plasma, metabolomic profiles are affected by repeated freeze/thaw cycles –24.After normalization and pre‐processing of the data, MetIDQ™ software (Biocrates) was used for peak integration and calculation of metabolite concentrations. All analytes that were above the limit of detection (LOD) in ≥50% of patients were selected for further investigation. Values below the LOD were replaced by the reference value provided by the manufacturer (LOD/2), and all values between LOD and LLOQ were replaced by the value LLOQ/2. Nine analytes  passed the initial screen based on LOD but were subsequently excluded because most of the values >LOD were <LLOQ. The concentration data were entered into the Metaboanalyst 3.0 software for multivariate analyses, including partial least squares discriminant analysis (PLS-DA), classical univariate receiver operating characteristic (ROC) curve analysis and multivariate ROC curve based exploratory analysis.The clinical characteristics of all patients and the relative concentrations of the CSF metabolites are presented as mean ± standard deviation (SD). For demographic and clinical characteristics, outcome comparison in PML and control groups was achieved using the independent sample t test for continuous variables and chi-square test for categorical variables. The Wilcoxon Mann-Whitney U test, as an alternative for not normally distributed variables, was applied to determine whether there was a significant difference between the two groups regarding the concentration of each metabolite. P values are 2-tailed and P < 0.05 was considered statistically significant. All analyses were performed using SPSS version 24.0 .The demographics and clinical characteristics of the PML subjects are shown in Of the 188 potentially detectable metabolites, 48 passed the 2-step quality assessment mentioned above. These metabolites comprised 13 amino acids, 4 biogenic amines, 1 acylcarnitine, 21 phosphatidylcholines, 8 sphingolipids, and the sum of hexoses.All 48 detected metabolites were analyzed by multivariate statistical analysis, using PLS-DA to identify differentially abundant metabolites. The PLS-DA score plot suggested that there were some differences between PML and NPH metabolite abundance patterns . MetabolA similar analysis was performed with the PML group being divided into HPYV-2 PCR positive (PML-P) and HPYV-2 PCR negative (PML-N) patients. Using PLS-DA, compared to the control group, we found 18 metabolites with VIP scores ≥1.0 in PML-P patients and 20 in PML-N patients. In the subsequent non-parametric test and ROC curve analysis all of them failed to meet the criteria for significance or biomarker potential .In this study, we employed a targeted LC-MS/MS approach to investigate the metabolic profile of CSF samples from PML patients in comparison to a control group. CSF is an important source of biomarkers for neurodegenerative and neuroinflammatory diseases because it has close contact to the damaged tissue. Therefore, metabolic changes within the brain are likely to be reflected in the CSF composition , 26.Although the CSF routine examination revealed that the CSF cell count and the CSF protein concentrations in PML patients were significantly higher than those in control patients, this was mostly driven by patients with HIV as the underlying cause for immunodeficiency. This only reflects that an elevated cell count or protein concentration in the CSF does not exclude PML and that the routine CSF parameters are normal in the majority of PML cases. Thus, there is a need for new biomarker for PML, in particular in patients with negative HPYV-2 PCR.As the CNS contains a high concentration of lipids, it is assumed that metabolites belonging to lipid/fatty acids, glutathione, and energy metabolism have a strong association with autoimmune or degenerative neurological diseases, such as multiple sclerosis or Parkinson’s disease –29. To oThis study has some limitations. Firstly, it captured only a fraction of the more than 430 metabolites that have been reported for CSF , and we Mass spectrometry is a key technique in CSF biomarker research, involved in discovery, verification, validation and the establishment of reference methods, and it should not be abandoned as a tool for biomarker discovery for PML. Our results suggest that any changes in CSF metabolites in PML may be subtle and that larger sample sizes and broader metabolite screens are needed to identify clinically useful CSF metabolite biomarkers for this rare and insidious disorder.