Background: Recurrent Pregnancy Loss (RPL) is a syndrome recognizing several causes, and in some cases the treatment with Granulocyte Colony Stimulating Factor (G-CSF) may be successful, especially when karyotype of the previous miscarriage showed no embryo chromosomal abnormalities. In order to evaluate the effects of G-CSF treatment on the decidual and trophoblast expression of G-CSF and its receptor, VEGF and its receptor and Foxp3, specific marker of putative Tregs we conducted an immunohistochemical study. Methods: This study was conducted on three groups of patients for a total of 38 women: in 8 cases decidual and trophoblast tissue were obtained from 8 women with unexplained RPL treated with G-CSF that miscarried despite treatment; in 15 cases the tissue were obtained from 15 women with unexplained RPL no treated; 15 cases of women who underwent voluntary pregnancy termination were used as controls. Tissue collected from these patients were used for immunohistochemistry studies testing the expression of G-CSF, G-CSFR, VEGF, VEGFR-1 and Foxp3. Results: G-CSF treatment increased the concentration of cells expressing Foxp3, specific marker for Tregs, in the decidua, whereas in no treated RPL a reduction of these cells was found when compared to controls. Furthermore, G-CSF treatment increased the expression of G-CSF and VEGF in the trophoblast. Conclusions: Our study showed that G-CSF treatment increased the number of decidual Treg cells in RPL patients as well as the expression of G-CSF and VEGF in villus trophoblast. These finding may explain the effectiveness of this treatment in RPL, probably regulating the maternal immune response through Tregs recruitment in the decidua, as well as stimulating trophoblast growth. Recurrent Pregnancy Loss (RPL) is currently described as the loss of two or more pregnancies prior to the 24th week of gestation . It has G-CSF is a 177 aminoacid polypeptide with a molecular weight of about 25 Kd stimulating principally polymorphonuclear granulocytes . In earlVascular endothelial growth factor (VEGF) is a glycoprotein with angiogenic properties ,19 such Foxp3-Treg cells are a subset of suppressive CD4 T helper cells essential for immune tolerance in humans and mice . SeveralIn this study we evaluated the effects of G-CSF treatment on the maternal fetal interface using immunohistochemistry to assess the expression of G-CSF and its receptor, the VEGF and its receptor VEGFR-1, and Foxp3 in the trophoblast and decidua of first trimester miscarriages of RPL women treated with G-CSF that miscarried again despite the treatment, in no treated RPL and in normal first trimester pregnancies.In all three series of samples the syncytiotrophoblast and the cytotrophoblast did not show any specific staining for Foxp3, as well as villi stroma .p < 0.0001), , No staining was found when primary antibody was incubated with a 10-fold molar excess of the antigen used for immunization. No differences were found among samples showing chromosomal abnormalities and samples with normal karyotype in both groups of G-CSF treated and no treated RPL, where the intensity of staining was consistent in all samples.G-CSF was expressed in the epithelial cells of the decidua of normal first trimester pregnancy , as wellp < 0.001) was found among no treated RPL samples and the other two groups. In the cytotrophoblast G-CSF was expressed with a weaker intensity than in the syncytiototrophoblast in the three series of specimens with similar HSCORE values  for G-CSF , whereasE values . The strp < 0.001). In the syncytiotrophoblast, G-CSFR was expressed with weaker intensity than in the cytotrophoblast in all series of specimens with similar HSCORE values. The stroma of the villi in all three groups was negative for G-CSFR. No staining was observed when primary antibodies for G-CSF and G-CSFR were incubated with a 10-fold molar excess of the antigens used for immunization. No differences were found among samples showing chromosomal abnormalities and samples with normal karyotype in both groups of G-CSF treated and no treated RPL, where the intensity of staining was consistent in all samples.The cytotrophoblast of villi of normal first trimester pregnancies was positive for G-CSFR (116 ± 39) , as wellVEGF was expressed in the epithelial cells of the decidua of normal first trimester pregnancy A, as welp < 0.001) was found among no treated RPL women samples and the other two groups  , whereaso groups . In the The VEGFR-1 was expressed in the epithelial cells of decidua of normal first trimester pregnancies  as well p < 0.001). In the syncytiotrophoblast VEGFR-1 was expressed with weaker intensity than in the cytotrophoblast in all series of specimens with similar HSCORE values. The stroma of the villi in all groups was negative for VEGFR-1. No staining was observed when primary antibody was blocked by incubating the anti VEGF and anti VGFR-1 with a 10-fold molar excess of the antigens used in the immunization. No differences were found among samples showing chromosomal abnormalities and samples with normal karyotype in both groups of G-CSF treated and no treated RPL, where the intensity of staining was consistent in all samples.The cytotrophoblast of villi of normal first trimester pregnancies was positive for VEGFR-1 (99 ± 30) , as wellp < 0.001”). Similarly a statistical significant positive correlation was found (r = 0.87 “p < 0.001”), when a regression analysis was conducted among levels of G-CSF staining and the values of staining for VEGF in the trophoblast, as well as when the regression analysis was conducted between G-CSFR staining and VEGFR-1 staining values .Furthermore, when a linear regression analysis was performed among the levels of G-CSF staining and the number of Foxp3 positive cells in the stroma of decidua, a statistical significant positive correlation was found  from a total of 38 pregnant women. They were divided in three groups: eight women with RPL treated with G-CSF that miscarried again despite the treatment, 15 women with RPL that miscarried during the clinical screening and did not receive any treatment and 15 healthy women underwent voluntary pregnancy termination used as controls. All women with RPL history (G-CSF treated and without treatment) underwent at the same clinical screening to exclude known causes of RPL . All patients had to be negative for all these tests for study inclusion. Furthermore, patients treated with G-CSF in their previous miscarriage had to show a normal embryo karyotype, mandatory condition to be treated with G-CSF. All the pregnancies included in this study were spontaneous pregnancies, since pregnancies obtained by ART were not included. The tissue obtained from D&C of all RPL patients miscarrying again were karyotyped by array-CGH to know the chromosomal status of embryos.A total of 8 cases with unexplained RPL (age 33.1 ± 2.0) were treated with recombinant G-CSF with a daily subcutaneous administration of Filgastrim , at a dosage of 1 mg /kg/day from the sixth day after ovulation until the occurrence of abortion . These mThe fifteen miscarriages of no treated women with unexplained RPL (age 31.6 + 2.3), showed in 8 cases an abnormal karyotype, whereas in 7 cases the karyotype was normal.Controls tissue specimens were obtained by 15 terminated voluntarily pregnancies from the 8th through the 12th week of gestation with the presence of fetal heart beat. These women had no history of previous abortions and they were 30.8 + 2.2 years old. Demographic data of the patients and controls are reported in All surgical procedures were carried out in the Center of Endocrinology and Reproductive Medicine (CERM) Rome, Italy. The project had the approval of the Institutional Ethical Committee . All patients signed an informed written consent for the use of their tissue samplesTissue samples were fixed in formalin at 4 °C overnight and subsequently paraffin embedded. Each sample of decidua and trophoblast were fixed without separation of embryonic and decidua tissues in order to have both trophoblastic villi and decidua on the same slide. Serial sections of the same samples 5 µm thick were used for immunohistochemistry. Commercially available monoclonal antibodies were used for the detection of G-CSF and G-CSFR , VEGF and VGFR-1 (Santa Cruz Biotechnology), and Foxp3 (Santa Cruz Biotechnology).Immunohistochemistry was performed on dewaxed and rehydrated sections, and endogenous peroxidase quenching was obtained with 0.3% hydrogen peroxide in methanol for 30 min incubation at room temperature. All slides were incubated for a non-immune block with normal horse serum for 30 min at room temperature before the incubation with the first antibody, carried out at room temperature for 1 h with a dilution of 1 to 100 for the all monoclonal mouse antibodies. Thereafter tissue sections were labeled with an avidin-biotin-peroxidase detection system ABC Vectastain . Each step was followed by three washing with phosphate buffer saline solution. Finally, 3,3’-diaminobenzidine was used as a chromogen. A light counterstaining was performed with Meyer’s hematoxylin with only 30 s bath in order to observe the eventual nuclear staining for the specific antigens. Controls were performed using the G-CSF antibody, G-CSFR antibody, VEGF antibody, VEGFR-1 antibody, Foxp3 antibody, preabsorbed overnight with a tenfold excess molar excess of the respective immunogen peptides purchased by the same Company . Furthermore, slides were incubated with nonimmune rabbit IgG at equal concentration as further control.For a semi-quantitative analysis of specific staining the HSCORE system was used according to the following equation: HSCORE = ∑Pi(i + 1), where i is the intensity of staining with a value of 1, 2, or 3  and Pi is the percentage of cells stained for each intensity, varying from 0% to 100%. Ten microscopic fields were counted by two of the authors independently for each slide (MS and FS). The intra-observer and inter-observer variation coefficient were 3.4% and 4.2% respectively. At least 3 slides of each sample were observed, for both antigens tested, under the microscope and each observer was blind to which sample it was (RPL or control slides). The slides, numbered progressively, were blindly analyzed by the two different observers and scored for HSCORE, and each value was recorded on the worksheet according to the corresponding patient for the given number. The HSCORE analysis was performed separately for the villous trophoblast, cytotrophoblast and syncytiotrophoblast, and the decidua, both component glandular and stromal cells (each observer performed 4 different HSCOREs for each slide). For Foxp3 quantification ten medium power fields were selected to count Foxp3 positive cells, the results was given in percent of total stromal decidua cells.p < 0.05. All statistical analyses were performed with SPSS 17.0.Statistical analysis. Differences between groups were determined using the Mann-Whitney U test. Significance was determined as


What is the value of nonculture cerebrospinal fluid infection testing in immune-competent children with normal cerebrospinal fluid cell counts?In this cross-sectional study of 4811 lumbar puncture specimens from immune-competent children aged 0.5 to 18.9 years, only 2 specimens with positive results from nonculture cerebrospinal fluid infection testing required clinical intervention, among all specimens with normal cell counts. Both children who required clinical intervention had other clinical signs of infection.Nonculture cerebrospinal fluid infection testing appeared to be common in immune-competent children with normal cerebrospinal fluid cell counts, but positive results seemed rare and unlikely to be independently associated with care. Nonculture infection tests of cerebrospinal fluid (CSF) samples using polymerase chain reaction and antigen or antibody assays are frequently ordered on lumbar puncture specimens concurrently with routine CSF cell counts, but the value of CSF infection testing in otherwise healthy children is unknown.To determine the value of nonculture CSF infection testing in immune-competent children with normal CSF cell counts.This cross-sectional study reviewed screening and diagnostic tests in the electronic medical record system of a large academic tertiary care children’s hospital. Records of children aged 0.5 to 18.9 years (n = 4083) who underwent lumbar puncture (n = 4811 procedures) in an inpatient or outpatient facility of Children's Hospital of Philadelphia between July 1, 2007, and December 31, 2016, were reviewed. Those with indwelling CSF shunts or catheters; those with active or past oncologic, immunologic, or rheumatologic conditions; or those taking immune-suppressing medications were excluded from analysis. This study was conducted from July 20, 2017, to March 13, 2019.Outcome variables included frequency of nonculture CSF infection testing and frequency of positive results in the entire cohort, and among those with normal cell counts. Normal cell counts were defined as CSF white blood cell counts lower than 5 cells/μL and CSF red blood cell counts lower than 500 cells/μL.In total, 4811 lumbar puncture procedures were performed on 4083 unique children, with a median (range) age of 7.4 (0.5-18.9) years, 2537 boys (52.7%), and 3331 (69.2%) with normal CSF cell counts. At least 1 nonculture CSF infection test was performed on 1270 lumbar puncture specimens with normal cell counts , and more tests were performed in the summer months. Only 18  of 1270 lumbar puncture specimens with normal cell counts had at least 1 nonculture infection test with a positive result; 2 of these 18 children required clinical intervention for their positive results, but each also had other clear clinical signs of infection.Nonculture CSF infection testing appeared to be common in immune-competent children with normal CSF cell counts, but positive results were uncommon and were not independently associated with clinical care; delaying the decision to send nonculture infection tests until CSF cell counts are available could reduce unnecessary diagnostic testing and medical costs, which may improve value-based care. This cross-sectional study examines the value of nonculture cerebrospinal fluid (CSF) testing in immune-competent children with normal CSF cell count. The presence of red blood cells (RBCs) in CSF samples represents either intrathecal blood  or introduction of peripheral blood into CSF during the performance of the lumbar puncture. Introduction of peripheral blood into otherwise sterile CSF may result in the potential contamination of a bloodstream pathogen, and it also makes the CSF WBC difficult to clinically interpret because an elevated count may reflect a true pleocytosis and/or peripheral blood WBCs.Acute central nervous system (CNS) infections, such as meningitis and encephalitis, require prompt recognition of treatable etiologies  to reduce potentially significant morbidity and mortality. Infections of the CNS are typically identified with lumbar puncture and evaluation of cerebrospinal fluid (CSF) cell counts, protein, glucose, Gram stain, bacterial culture, and nonculture infection tests. A CSF white blood cell (WBC) count lower than 5 cells/μL in immune-competent children older than 2 months is considered normal, whereas a count of 5 cells/μL or higher suggests infection and/or inflammation2 enterovirus PCR3) but are low for others . The positive and negative predictive values, as well as the clinical advantages of these tests in populations with a low pretest probability for CNS infection , are unknown.Polymerase chain reaction (PCR) of CSF and antigen or antibody assays are used to identify specific pathogens in suspected CNS infection; however, their overall diagnostic yield is variable. Nonculture CSF infection test sensitivity and specificity are high for some organisms  reporting guideline for cross-sectional studies. The study was conducted from July 20, 2017, to March 13, 2019.We reviewed the electronic medical record (EMR) of Children’s Hospital of Philadelphia, a large academic tertiary care children’s hospital. The Children’s Hospital of Philadelphia Institutional Review Board approved this study and waived the informed consent requirement and Health Insurance Portability and Accountability Act authorization because the research and use of personal health information poses no greater than minimal risk and the study could not be performed without the waiver, which did not adversely affect the rights or welfare of the patients. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  with indwelling CSF shunts or catheters; (2) with malignant oncologic, immunologic, or rheumatologic diagnosis that may have interfered with the ability to mount an appropriate pleocytosis in response to the infection at the time of the lumbar puncture; (3) who underwent lumbar puncture in an oncology department, when the location of the procedure was available; and (4) with EMR documentation of a medication, taken at the time of the lumbar puncture, known to suppress the immune system.We queried the EMR for any CSF cell count performed on a child aged 0.5 to 18.9 years at an inpatient or outpatient Children’s Hospital of Philadelphia facility between July 1, 2007, and December 31, 2016. Some children underwent more than 1 lumbar puncture during this study period and contributed multiple independent data points over time to the analysis. We defined exclusion criteria according to Patient data, including medical record number, demographic characteristics, epidemiologic information, and CSF test results were extracted from the EMR on children who met the inclusion and exclusion criteria. These data were manually reviewed in the EMR to verify suspected errors and missing data, confirm the accuracy of extracted data , and develop an abstract of clinical data from specimens with normal cell counts and a positive nonculture infection test result.Normal cell counts were defined as CSF WBC lower than 5 cells/μL and CSF RBC lower than 500 cells/μL; elevated cell counts were defined as CSF WBC 5 cells/μL or higher and/or CSF RBC 500 cells/μL or higher. When more than 1 set of cell counts were obtained for a given lumbar puncture specimen, we used the lowest value of each WBC and RBC count. We recorded CSF protein and glucose values less than a lower limit of detection  as the lower limit of detection.International Classification of Diseases, Tenth Revision, codes used to define exclusion criteria are summarized in the eMethods in the The 7Borrelia burgdorferi , cytomegalovirus ,8 Epstein-Barr virus ,9 enterovirus ,10 human herpesvirus 6 ,11 herpes simplex virus ,12 human parechovirus ,13 influenza A and B virus , human metapneumovirus ,14 mumps virus (sent to the Pennsylvania state laboratory), Mycoplasma pneumoniae ,15 parainfluenza virus 1-3 ,17 parvovirus B19 ,18 varicella zoster virus ,19 and West Nile virus (sent to ARUP Laboratories).Nonculture CSF infection tests we examined were PCR, antibody testing (IgG and/or IgM), and antigen testing. We included these tests in the analysis only if they were ordered on the same day that CSF cell counts were obtained; tests ordered on an existing specimen at a later date were excluded. Specific monoplex, real-time, qualitative CSF PCR tests we examined were those for the following: adenovirus ,20 Antigen or antibody CSF testing included arbovirus IgG or IgM panels , cryptococcal antigen , Epstein-Barr virus IgG panels (ran in house [Zeus Scientific]), lymphocytic choriomeningitis virus IgG or IgM (sent to ARUP Laboratories), Lyme total antibody level , Lyme Western blot IgG or IgM , measles IgG or IgM , rubella IgG or IgM , toxoplasmosis IgG or IgM , VZV IgG or IgM , and venereal diseases research laboratory assay (sent to ARUP Laboratories).The Children’s Hospital of Philadelphia does not use commercial multiplex PCR tests for CSF samples because of performance concerns.Nonculture CSF infection test results were recorded as positive, negative, or not performed. Medical records with either IgG- or IgM-positive antibody CSF test results were manually reviewed to determine the clinical interpretation as positive or negative for the child’s clinical presentation. We manually reviewed all indeterminate or equivocal antibody testing results in the EMR, and if repeated confirmatory testing was not performed, we conservatively considered the results positive. When any component of a multitest panel  had a positive result, we considered the overall panel results positive.2 test. Statistical significance was determined a priori as a 2-tailed P < .05.Data were analyzed using Stata, version 14.2 (StataCorp LLC). Nonparametric methods were used because not all continuous variables (such as age) were normally distributed and to minimize the effect of outliers with statistical associations. Continuous variables were described using medians and interquartile ranges (IQRs), and intergroup differences were compared using Wilcoxon rank sum tests. Categorical variables were described using counts and frequencies, and intergroup differences compared using the χAfter the overall cohort was analyzed, the subset of infants aged 6 to 12 months was also examined separately, given our a priori concern that their relatively immature immune system could potentially be a factor in the test results being different from those of older children.P = .005) and African American  compared with children with normal CSF cell counts  are summarized in l counts ; median Among the total 4811 lumbar puncture specimens, 2038  had at least 1 nonculture CSF infection test sent: 270 specimens had both PCR and antibody or antigen testing, 1613 had only PCR testing, and 155 had just antibody or antigen testing. Among those specimens that had at least 1 nonculture CSF infection test, 1368  had more than 1 test . Testing was more frequent in the summer months compared with the remainder of the year A but wasOf the 4811 lumbar puncture procedures performed, 3331 specimens  performed on 3033 unique children had normal CSF cell counts, of which 1270  had at least 1 sent for nonculture CSF infection test. Among those 1270 specimens with normal cell counts sent for a nonculture CSF infection test, 827  were sent for more than 1 test . Only 18  of 1270 specimens with normal cell counts tested, or 19  of the 3021 total nonculture CSF infection tests performed on these specimens, had a positive result . PositivTo explore the role of CSF RBC values in these data, we performed a sensitivity analysis to include children with elevated CSF RBC in the original normal cell count subgroup. This inclusion added 191 lumbar puncture specimens to the sensitivity analysis, for a total of 3522 specimens. Among these 3522 specimens with normal CSF WBC alone, 1348  were sent for at least 1 nonculture CSF infection test, of which 22  had a positive result. However, none of the additional 4 procedures with positive results  had active treatment implications.Of the 4811 lumbar puncture procedures that met inclusion and exclusion criteria, 537 (11.2%) were performed on 460 unique infants between 6 and 12 months of age. Among the 537 procedures performed on these infants, 169  had specimens sent for at least 1 nonculture CSF infection test, for a total of 352 nonculture infection tests. Only 13  of the 169 lumbar puncture procedures performed on infants who had nonculture CSF infection testing, or 13  of the 352 total nonculture infection tests performed on these procedures, had a positive result. These proportions are each slightly lower than seen in the overall cohort. Three hundred fifty-one  of 537 lumbar puncture specimens in infants had normal cell counts, a similar proportion to the overall cohort. One hundred fifteen  of 351 lumbar puncture specimens in infants with normal cell counts had at least 1 nonculture CSF infection test sent, 3  of which had a positive result, a slightly higher proportion than the overall cohort .From a value perspective, if nonculture CSF infection testing on lumbar puncture specimens with normal cell counts was not performed in this cohort, we would have eliminated testing on 1270 lumbar puncture specimens, a total of 3021 of the 5054 total tests sent  of all nonculture CSF infection tests sent for this cohort. However, we would have missed the 19 positive test results in 18 children outlined in P < .001), but this is only a 27% difference in magnitude, suggesting that cell counts alone are not a factor in the decision to send specimens for a nonculture CSF infection test in most children. Furthermore, once 1 nonculture infection test was sent for testing, both groups had a similar proportion of multiple tests sent (65.1% vs 67.1%), suggesting that once the decision is made to send for nonculture infection testing, CSF cell counts are not a factor in the number of tests ordered. Annual testing frequency has not substantially changed in the past 10 years at Children’s Hospital of Philadelphia. Seasonal testing frequency increases annually in July to September, which may be associated with seasonal enteroviral transmission (and subsequent increased testing practice) or with the relative inexperience of new house officers, as proposed in previous CSF PCR testing epidemiologic studies.22 Testing practices were similar in infants 6 to 12 months of age compared with testing in older children.Lumbar puncture specimens with normal CSF cell counts were common in this cohort of immune-competent children but were rarely associated with nonculture CSF infection tests with a positive result and implications for clinical care. The proportion of these specimens sent for nonculture infection testing was lower compared with the proportion observed in those with elevated cell counts  in the cohort were discharged before the results were available. One child underwent a lumbar puncture as an outpatient, and the positive nonculture infection test result (CSF measles IgG) was believed to be associated with prior intravenous immunoglobulin and not clinically significant. One child had a positive CSF enterovirus PCR result that prompted an acyclovir sodium discontinuation. The 2 children with positive test results that required clinical intervention  each had other clear clinical and neuroradiographic signs of infection. The remaining 6 children had positive results that did not affect their medical treatment.Cell count results from CSF are typically available within hours, but other nonculture CSF infection tests can take days or weeks to complete. Implementing a testing strategy to delay the decision to send specimens for additional nonculture CSF infection testing until CSF cell counts were available could have reduced up to 60% of all nonculture CSF infection testing for this cohort. This type of change could reduce unnecessary diagnostic testing and medical costs, without substantially delaying the results in children with elevated cell counts or those with normal cell counts and a specific clinical concern based on other clinical data, thus improving value-based care.24 Identifying sources of low-value care and minimizing it are critical: small changes applied across populations can be associated with meaningful reductions in domestic health care expenditures.23Low-value care  is believed to constitute up to one-third of health care costs in the United States.22 demonstrated that restricting HHV PCR testing to specimens with CSF WBC count higher than 5 cells/μL and protein level greater than 45 mg/dL would have saved almost one-third of costs without reducing sensitivity. Hanson et al25 demonstrated that limiting HSV PCR testing to requests on specimens with CSF WBC count higher than 5 cells/μL or protein level greater than 50 mg/dL, specimens obtained from an immunocompromised individual (HIV positive or recipient of organ transplant) or from a child younger than 2 years, reduced testing by 22% (363 of 1659 specimens) and missed only 2 positive CSF HSV PCR results, which in retrospect did meet the criteria for testing. Hauser et al26 demonstrated that, among 6357 CSF HSV PCRs ordered on adults at a Veterans Affairs hospital over a 13-year period, no clinically significant positive results would have been missed by applying the Hanson et al25 criteria. In a split retrospective observational study with a screening and validation cohort, Wilen et al27 demonstrated that limiting the 8791 HSV, VZV, cytomegalovirus, and enterovirus PCRs ordered during the screening phase to immune-competent adults with CSF WBC count higher than 10 cells/μL would have reduced testing by about 55%, missing only 1 clinically significant result. In their validation cohort, 17 of 2126 tests were positive for HSV, VZV, cytomegalovirus, or enterovirus; applying the same criteria would not have missed any clinically significant test results.Several groups have previously examined various interventions to reduce unnecessary nonculture CSF infection testing in different populations. In a retrospective study of 732 adult CSF specimens submitted for testing at the Mayo Clinic, Tang et alThis current study adds to the growing body of literature on CSF testing and low-value care. It provides pediatric data on diagnostic yield of nonculture CSF infection testing across a broad battery of common tests. Unlike the adult studies, protein levels were largely normal among the children in this cohort with positive nonculture CSF infection testing with the exception of 1 child  and for neurosurgical concerns  remained in the cohort, likely because the pertinent diagnosis had not yet been added to their EMR. These findings may imply that our conclusions are more generalizable to broader populations, but further focused study is warranted.This study has several limitations. First, although we suspected that CSF cell counts were not available at the time the decision was made to perform nonculture CSF infection testing, we did not know the definite timing of clinical decision making for each lumbar puncture in this large cohort. For this reason, we excluded nonculture infection tests ordered on days after the lumbar puncture, to exclude tests that were ordered on the basis of preliminary CSF data. Second, given the large size of the cohort, we based the exclusion criteria on 29 Thus, the interpretation of CSF data in younger infants compared with older infants is a different and unique clinical challenge. Fifth, given the variation in CNS pathogen prevalence in different source populations, these data may not be completely generalizable to all tertiary care pediatric hospitals. However, the potential to reduce nonculture CSF infection testing in children with normal CSF cell counts should be applicable in any setting.Third, the data we used were produced by EMR query, making it possible that some CSF tests ordered may have been missed. To minimize this risk, we manually reviewed all 3331 records of children with normal cell counts to ensure accurate documentation of nonculture CSF infection tests performed and their results. Fourth, we did not include infants younger than 6 months, limiting the study’s application to this population. This decision was a priori, given that the CSF WBC profiles of infants in the face of infection have substantial variability.This study’s findings suggest that lumbar puncture specimens with normal cell counts are common in immune-competent children older than 6 months but are rarely associated with nonculture CSF infection tests with positive results that are independently associated with clinical care. Implementing a hospitalwide intervention to delay the decision to order nonculture CSF infection testing, or to hold specimens in the laboratory until CSF cell counts are available, could dramatically reduce unnecessary testing and associated costs, improving value-based care. A nuanced approach is needed in the decision to send specimens for nonculture CSF infection testing. This approach should include community prevalence of infection, known pathogen risk factors, and patient-specific clinical judgment.


Cerebrospinal fluid (CSF) analysis is a crucial method in the diagnostic process for suspected subarachnoid hemorrhage (SAH), especially when cerebral imaging is negative or inconclusive. CSF cytology (detection of erythrophages or siderophages) is used to determine whether a bloodstained CSF resembles a genuine SAH. Whether erythrophages may develop in vitro after a traumatic puncture in case of delayed CSF analysis is unclear. An in vitro development of erythrophages after traumatic puncture would diminish the diagnostic properties of CSF analysis. We assessed whether erythrophagocytosis is detectable in CSF after an imitated traumatic lumbar puncture.We mimicked a traumatic lumbar puncture by mixing surplus CSF with whole blood from the same patient. From this mixture, cytological specimens were obtained immediately and repeatedly at time intervals of 1 h, until 7 h after mixing, or until the mixture was exhausted. Each cytological specimen was microscopically examined independently by four experienced CSF cytologists for the presence of erythrophages.We studied 401 CSF cytological specimens of 96 punctures in 90 patients. We could not identify any erythrophages in all cytological specimens. Fleiss’ Kappa for interrater-reliability was 1.0.We did not find evidence for an in vitro erythrophagocytosis after a mimicked traumatic lumbar puncture. Therefore, the occurrence of erythrophages in CSF cytology can be regarded as a reliable sign of an autochthonous bleeding in the subarachnoid space. Our results support the crucial role of CSF analysis in clinical practice in case of a suspected SAH but negative cerebral imaging.The online version of this article (10.1186/s12987-018-0116-3) contains supplementary material, which is available to authorized users. Cerebrospinal fluid analysis is a crucial method in the diagnostic process if a subarachnoid hemorrhage (SAH) is clinically suspected but findings from cerebral imaging are negative or inconclusive. This is especially important in cases with an increased time interval between the suspicious clinical event and the cerebral imaging as the sensitivity of cerebral imaging declines significantly over time , 2. WhenFour hours after a subarachnoid hemorrhage, monocytes begin phagocytosis with ingestion of other cell types, mainly erythrocytes, and develop into erythrophages. Erythrophages are detectable approximately 4 h after a subarachnoid hemorrhage in CSF cytology. Erythrophages develop into siderophages by degrading ingested erythrocytes containing hemoglobin within the next 3–4 days , 8.Whether erythrophages may develop in vitro after a traumatic lumbar puncture (LP) in case of a delayed CSF analysis is a matter of controversy in clinical practice. CSF should be analyzed quickly after LP. The quality of a cytological specimen decreases over time, as cells disintegrate in vitro over a period of several hours after the puncture. It is unsure whether surviving monocytes in CSF tubes could start to ingest erythrocytes and develop into erythrophages. This process would then lead to a false positive diagnosis of a subarachnoid hemorrhage after cytological CSF analysis. Evidence of in vitro development of erythrophages after traumatic puncture would therefore significantly diminish the diagnostic properties of CSF analysis in the setting of a suspected subarachnoid hemorrhage. We assessed whether an in vitro erythrophagocytosis after a mimicked traumatic LP is detectable in a CSF cytological specimen.We studied CSF specimens from patients who underwent an LP as a part of a routine clinical workup for several neurological diseases or had repeated therapeutic LPs for diseases like benign intracranial hypertension. All patients gave their informed consent to LP and for the use of surplus CSF for this project. The study protocol was approved by the ethics committee of the Medical Center, University of Freiburg (EK-Fr 271/13). All data generated or analysed during this study are included in this published article. Clinical and demographic data  was obtained from the medical records for each patient, as well as CSF routine parameters (white blood cell count (WBC), total protein, and, if available, albumin quotient and intrathecal immunoglobulin synthesis). Patients were excluded if they had a history of a subarachnoid or intracerebral hemorrhage. If the routine CSF cytology showed erythrophages in the initial routine analysis, the respective patient was excluded from further analysis.Surplus CSF not needed for routine clinical analysis was harvested for immediate further handling at room temperature. To imitate a traumatic LP, surplus CSF was mixed with whole blood from the same patient collected at the same time point. Whole blood was mixed to CSF in a proportion of 2 μL whole blood/1000 μL CSF. If the LP happened to be traumatic by chance no additional whole blood was added to the CSF.From this mixture, a cytological specimen was obtained immediately after mixing whole blood and CSF; additional single samples of cytological specimens were obtained at time intervals of 1 h until 7 h after mixing had passed or until the mixture was exhausted. During the incubation period the mixture was stored at room temperature. Each cytological specimen was performed using 1.2 mL of the mixture. Cytological specimens were stained using May–Grunwald staining.Each cytological specimen derived from the mixture was microscopically examined by four experienced CSF cytologists  independently, for the presence of erythrophages. If one cytologist detected erythrophages in a cytological specimen, the corresponding specimen was reviewed by all cytologists and the assessment was discussed accordingly. Concordance of the assessment was calculated using Fleiss’ Kappa.Initially, CSF specimens from 96 patients were screened. Six patients had to be excluded due to various reasons. One patient withdrew consent after the LP was performed. One patient showed erythrophages in the initial routine CSF cytology and was therefore excluded from further analysis. Due to artificial changes, cytological specimens from four other patients could not be analyzed, as no leukocytes were detectable in the CSF sample. These patients were excluded from further analysis. In total, 401 CSF cytological specimens of 96 LPs in 90 patients were studied. The mean number of specimens per patient was 3.8 (SD 1.9). Demographic characteristics and results of routine CSF analysis of the included patients regarding their medical conditions are shown in Additional file We obtained 50 cytological specimens at baseline directly after the puncture, 69 one hour after puncture, 88 two hours after puncture, 93 three hours after puncture, 65 four hours after puncture, 26 five hours after puncture, 9 six hours after puncture, and 1 seven hours after puncture. Of the included patients, 28 were suffering from neuroinflammatory diseases and 17 CSF samples showed inflammatory changes in terms of a pleocytosis in the initial routine analysis. In the cytological assessment, we did not identify erythrophages in any specimen. Fleiss’ Kappa for interrater-reliability was 1.0.Whether an in vitro erythrophagocytosis exists in bloodstained CSF is a matter of controversy. An in vitro development of erythrophages after traumatic puncture would drastically reduce the diagnostic value of CSF analysis in clinical practice if a subarachnoid hemorrhage is suspected. Our study did not find any signs of erythrophagocytosis in CSF cytology after a mimicked traumatic LP in 401 CSF cytological specimens of 96 LPs from 90 patients. Fleiss’ Kappa for interrater-reliability was 1.0.Whether in vitro erythrophagocytosis exists after traumatic LP has been scarcely investigated. The only available study used a comparable paradigm to our study to investigate in vitro erythrophagocytosis was in 1973 [Methodological differences, like the use of the now-outdated sedimentation technique by Oehmichen and Schütze, may account for these differences. By using the sedimentation technique, the quantity of large cells like monocytes in CSF cytology is higher than in specimens obtained with a modern centrifuge. This may lead to more doubtfully altered cells which might be misinterpreted as erythrophages that are absent in cytological specimens obtained with current techniques.Why erythrophages develop in SAH and not in CSF after an artificial traumatic lumbar puncture remains speculative. In SAH monocytes could be specifically activated by damaged choroid plexus epithelium or ependymal cells. Another explanation could be differences in temperature in vivo as compared to storing CSF samples at room temperature after lumbar puncture. Another issue could be longer incubation time of monocytes in CSF with contact to erythrocytes in vivo compared to the limited time after lumbar puncture until monocytes start to disintegrate in vitro.We did not find any erythrophages in CSF cytology after a mimicked traumatic LP. Therefore, the occurrence of this cell type in CSF cytology should be regarded as a reliable sign of an autochthonous bleeding in the subarachnoid space. Thus, our results support the crucial role of CSF analysis in clinical practice in case of a suspected subarachnoid hemorrhage and negative cerebral imaging.Additional file 1: Table S1. Pathological conditions, demographic data and results from routine CSF analysis from included patients. Data is shown as mean with standard deviation.


Around one million patients present to hospital with chest pain every year, accounting for approximately 5% of all emergency department attendances in the UK. However, only one in five patients are found to have had a myocardial infarction.Cardiac troponin is the biomarker of choice for the detection of myocardial injury, and international guidelines recommend concentrations are measured using a high-sensitivity assay.et al report results from a prospective cohort study evaluating the diagnostic accuracy of the Troponin-only Manchester Acute Coronary Syndrome (T-MACS) decision aid when applied using a contemporary point of care cardiac troponin I assay. The Abbott iSTAT point of care assay has a reported 99th centile of 80 ng/L and limit of detection of 20 ng/L, with a coefficient of variation of 16.5% at the 99th centile diagnostic threshold.One potential strategy to improve efficiency is through the use of point of care devices. Similar to blood glucose testing, the delivery of rapid and accurate measurements of cardiac troponin at the bedside could allow earlier diagnosis or rule out of myocardial infarction in practice see et al are encouraging, there are important limitations to consider. First, the majority of patients were male (74.3%), and only those with symptoms for less than 12 hours were included. Although this may reduce the generalisability of the findings, focusing on patients with a shorter duration of symptoms would be expected to reduce diagnostic accuracy, as troponin release is a time-dependent phenomenon. Blood samples were obtained at the bedside but were processed by research nurses not involved in patient care, so the impact on diagnostic efficiency is unmeasurable. Finally, in keeping with the majority of studies in this area, the results are based on observational data. Patients were not managed on the basis of test results and may have undergone additional investigations and management which could have influenced their outcomes. Importantly, local validation of this approach is essential to adjust for differences in population level characteristics and the pretest probability of disease which may influence diagnostic performance.While the findings of Body It should be acknowledged that it is difficult to conduct observational cohort studies in patients with suspected acute coronary syndrome that replicate real world clinical practice. Indeed, in the majority of studies evaluating cardiac troponin use, the symptom onset to sample time is around 6 hours. With the availability of bedside testing, this time may significantly fall and diagnostic performance may be less robust. Furthermore, the availability of easily accessible bedside troponin testing may lead to less selective, non-judicious testing which could lead to an increase in the diagnosis of type 2 myocardial infarction or myocardial injury.et al is on the in-hospital use of point of care testing, there are a number of novel approaches which could be transformative for clinical practice. Prehospital use may facilitate the evaluation of suspected myocardial infarction in the community, allowing redirection of patients with a higher probability of myocardial infarction to cardiac centres, and low risk patients to district general hospitals. The Pre-hospital Evaluation of Sensitive Troponin study will evaluate the performance of pre-hospital cardiac troponin testing (NCT:03561051). The study aims to recruit 700 patients who have phoned the emergency services with symptoms suspicious for acute coronary syndrome before they arrive at hospital. Blood samples will be obtained in the ambulance and on arrival to hospital. While tests will not be run live, this study will obtain samples as near to symptom onset as practicable, giving important insight into the diagnostic accuracy of point of care testing and allowing for validation of the T-MACS clinical risk score in this population.Although the focus of the study of Body et al in a pilot study of 354 patients. While not formally designated as a high-sensitivity assay, the Abbott TnI-Nx is capable of reporting concentrations from 1 to 1500 ng/L.STAT high-sensitivity cardiac troponin I laboratory platform. Whether the addition of this novel assay to the T-MACS strategy could allow more patients to be identified as low risk in the emergency department remains unclear, but would be an important evaluation in future.In future, the diagnostic performance of point of care cardiac troponin assays is likely to improve. Recently, a novel point of care cardiac troponin I assay was described by Pickering et al should lead to prospective validation studies to demonstrate the safety and efficacy of this approach.Contemporary point of care cardiac troponin testing in combination with a clinical risk score may facilitate the rule-out of myocardial infarction in institutions without access to a high-sensitivity cardiac troponin platform. While novel, more sensitive point of care assays are in development, the encouraging findings of Body


CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method).We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users. LoxP sites in introns flanking critical exon/s or (less commonly) in intergenic regions or flanking regulatory regions such as promoters and enhancers. When crossed with a Cre recombinase-expressing driver mouse, the Cre enzyme recognizes LoxP sequences and removes the intervening sequence. This leads to functional inactivation of the targeted gene in only the cells where the Cre is expressed and capable of targeting the DNA . However, as well as single nucleotide proportions, we also include dinucleotide proportions . We do this for each of the sequences  from Additional file With the labels and feature matrix, we then trained a random forest model using scikit-learn . We traiTo quantify the model’s performance, we take advantage of the out-of-bag (OOB) error property of random forests. This value is generated during training by evaluating each “tree” in the random forest model using the samples that were not included for training that tree (through bootstrapping). We observe an OOB error of 0.222. Finally, we can identify important features using the “feature_importances_” property of the random forest model.https://gist.github.com/aydun1/932f526867f7f8139b8e8eae7c76e866 and Zenodo doi: 10.5281/zenodo.3339039 under the MIT license.Source code is available on GitHub repository U test. A generalized linear model calculation was performed with success of the two-donor floxing method as a response. Predictive variables were efficiency of the sgRNA; probability of LoxP insertions in 5′ and 3′ (5′_LoxP and 3′_LoxP); simultaneous insertion of the two LoxP sites (interaction between 5′_Loxp and 3′_LoxP); Cas9 mRNA, protein, plasmid, and ssODN concentrations; and distance between the distal and proximal target sites. Variance for each predictor was determined by calculating the diagonal of the variance-covariance matrix. Effect sizes and type II error were determined using Cohen effect size d statistics and power calculation. All statistical analyses were performed using Rstudio v1.1.423. The results were considered statistically significant at p < 0.05.To determine the statistical differences between the proportions, we performed a Fisher exact test or a Kruskal-Wallis sum-rank test or a Kolmogorov-Smirnov test. For means, we performed a Mann-Whitney Additional file 1: Table S1. The guide RNA and singlestranded Oligonucleotide DNA sequences, their concentrations and the length of genomic regions floxed (in bp) reported in this study. (XLSX 29 kb)Additional file 2: Table S2. The results of the two-donor floxing approach via microinjection for 49 unique loci. (XLSX 21 kb)Additional file 3: Table S3. The results of the two-donor floxing approach via electroporation for 7 unique loci. (XLSX 11 kb)Additional file 4: Table S4. Overall efficiency of the two-donor (2sgRNA-2ssODN) floxing method of generating the cKO alleles (XLSX 9 kb)Additional file 5: Table S5. The details and the results of the 330 unique loci attempted for ssODN knock-in projects (i.e. point mutation knock-in or short-tag insertion projects). (XLSX 14 kb)Additional file 6: Table S6. The details and the results of alternate methods of floxing, tested on 18 loci. (XLSX 23 kb)Additional file 7: Table S7. The Evaluation of the modified method of two-donor floxing method  reported in the Horii et al. 2017 report. (XLSX 11 kb)


Continuous positive airway pressure (CPAP) therapy is very often the treatment of choice for obstructive sleep apnoea (OSA). The association between intracranial hypertension and spontaneous cerebrospinal fluid (CSF) rhinorrhoea is being increasingly recognized among patients with OSA. However, spontaneous CSF rhinorrhoea following initiation of CPAP therapy for OSA is very rarely documented in the literature. In this report, we describe a 53‐year‐old woman with severe OSA who, while being evaluated for possible intracranial hypertension, developed spontaneous CSF rhinorrhoea and CSF aspiration pneumonitis as a complication of CPAP therapy. Magnetic resonance imaging confirmed fluid tracks at the skull base, and a nasal swab demonstrated positive β2‐transferrin. Computer tomography (CT) chest showed findings consistent with CSF aspiration pneumonitis. Resolution of both CSF leak and pneumonitis were noted following treatment with azetozolamide and curative endoscopic trans‐nasal surgery along with ventriculoperitoneal shunt. Continuous positive airway pressure (CPAP) therapy is very often the treatment of choice for moderate to severe obstructive sleep apnoea (OSA). The association between intracranial hypertension and spontaneous cerebrospinal fluid (CSF) rhinorrhoea is being increasingly recognized among patients with OSA 2O following a CPAP titration study.A 53‐year‐old woman presented to the emergency department with a 4‐week history of rhinorrhoea and post‐nasal drip, exacerbated by coughing and bending forward. Onset of rhinorrhoea was noted three days following the initiation of nasal CPAP therapy for severe OSA (apnoea hypopnea index (AHI) of 35/h, more severe during Rapid eye movement sleep, AHI 82/h). CPAP therapy was initiated at a pressure of 11 cm HThe patient's past medical history included poorly controlled type 2 diabetes and hypertension. The patient was also undergoing investigation for constant headaches for several months to years, and benign intracranial hypertension was being considered in the differential diagnosis. She also reported recent onset of dry cough during this presentation, which coincided with the onset of rhinorrhoea. There was no other significant past medical history; in particular, she did not report previous cranial or sinus trauma or cranio‐facial surgery. Clinical examination showed that she was febrile at presentation, and other vital signs were unremarkable, including oxygen saturation of 97% on room air. Fundoscopy demonstrated bilateral papilloedema. Respiratory examination showed clear breath sounds, with no crackles or signs of consolidation or pleural effusion. Systemic and neurological examinations were unremarkable; in particular, there were no signs of meningitis.2O. The CSF fluid cell count, glucose and proteins were within normal range. A blood test showed raised inflammatory markers with a C‐reactive protein (CRP) of 122 mg/L and raised white cell count of 22 × 109/L with neutrophilic predominance. Liver function test was normal. Connective tissue disease, vasculitis screening, and blood cultures were negative. Derangement of renal function and proteinuria were noted and were considered to be secondary to poorly controlled type 2 diabetes.Lumbar puncture showed an opening CSF pressure of 24 cm HRhinorrhoea was confirmed to be secondary to CSF leak by nasal swab, demonstrating positivity for β2‐transferrin. Magnetic resonance imaging (MRI) of the head demonstrated an “empty” sella turcica, enlargement of Meckel's cave bilaterally, and bilateral optic nerve sheath effusions, along with flattening of the posterior optic disc consistent with intracranial hypertension. There was also cortical thinning of the floor of the sella and cribriform plate. Fluid was also demonstrated within the frontal and sphenoid sinuses. A computer tomography venogram demonstrated absent left transverse sinus and sigmoid sinus, and the venous drainage was noted predominantly through right transverse sinus and sigmoid sinus. Dedicated T2 MRI scanning through the anterior cranial fossa, skull base, and paranasal sinuses was performed to identify the site of the CSF leak, which demonstrated multiple fluid tracks noted in the region of the cribriform plate Fig. A, B. CT The patient reported significant improvement in headache and rhinorrhoea following initiation of azetozolamide. Subsequently, the skull base was repaired via an endoscopic trans‐nasal surgery, and a ventriculoperitoneal shunt was placed for CSF diversion. During the follow‐up visits, she had no further headaches and was free of CSF leak. Her pulmonary symptoms improved spontaneously, and follow‐up chest CT showed complete resolution of the previously noted opacities within one week of resolution of CSF rhinorrhoea.The case presented here is a very rare and uncommonly reported complication of CPAP treatment for OSA (CSF rhinorrhoea and CSF aspiration pneumonitis). Previous studies have documented an association between OSA and spontaneous cerebrospinal fluid rhinorrhoea (SCSFR), and with a diagnosis of OSA, the odds of SCSFR is noted to be around 4.73 times higher than the control cohorts Repeated apnoea and hypoxia are thought to increase the intracranial pressure in patients with OSA Although rare, it is noted that the most frequently reported complications of CSF rhinorrhoea are meningitis and pneumocephalus We present this case to add to the limited number of reported cases. We also believe that this report may encourage other clinicians to report similar cases to better understand the mechanism, prevention, and early diagnosis and management of this condition. Nevertheless, a history of headaches and visual symptoms may need to be considered in patients diagnosed to have OSA prior to commencing CPAP therapy. If there is a history of intracranial hypertension or clinical symptoms of intra cranial hypertension or previous cranial or sinus trauma or cranio‐facial surgery, careful monitoring for any evidence of CSF leak following the commencement of CPAP therapy and CSF aspiration pneumonitis needs to be considered in the relevant clinical context. Accurate diagnosis and treatment with surgical intervention can be curative Appropriate written informed consent was obtained for publication of this case report and accompanying images.


The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and treatment‐resistant malignancy. The lack of pathway‐informed biomarkers hampers the development of rational diagnostics or therapies. Recently, the protein abnormal spindle‐like microcephaly‐associated (ASPM) was identified as a novel Wnt and stemness regulator in PDAC, while the pathogenic roles of its protein isoforms remain unclarified. We developed novel isoform‐specific antibodies and genetic knockdown (KD) of putative ASPM isoforms, whereby we uncovered that the levels of ASPM isoform 1 (iI) and ASPM‐iII are variably upregulated in PDAC cells. ASPM isoforms show remarkably different subcellular locations; specifically, ASPM‐iI is exclusively localized to the cortical cytoplasm of PDAC cells, while ASPM‐iII is predominantly expressed in cell nuclei. Mechanistically, ASPM‐iI co‐localizes with disheveled‐2 and active β‐catenin as well as the stemness marker aldehyde dehydrogenase‐1 (ALDH‐1), and its expression is indispensable for the Wnt activity, stemness, and the tumorigenicity of PDAC cells. By contrast, ASPM‐iII selectively regulates the expression level of cyclin E and cell cycle progression in PDAC cells. The expression of ASPM‐iI and ASPM‐iII displays considerable intratumoral heterogeneity in PDAC tissues and only that of ASPM‐iI was prognostically significant; it outperformed ALDH‐1 staining and clinico‐pathological variables in a multivariant analysis. Collectively, the distinct expression patterns and biological functions of ASPM isoforms may illuminate novel molecular mechanisms and prognosticators in PDAC and may pave the way for the development of therapies targeting this novel oncoprotein. © 2019 The Authors.  Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment‐resistant malignancy and the third leading cause of cancer‐related death in the USA A prevailing new paradigm of tumorigenesis proposes that only a subset of tumor cells with stem‐like properties, termed ‘tumor‐initiating cells (TICs)’ or ‘cancer stem cells (CSCs)’, has the ability to self‐renew and sustain tumorigenesis ASPM transcripts, which encode the protein isoforms consisting of 3477 (isoform 1), 1892 (isoform 2), 1389 (isoform 3), and 1062 amino acids (isoform 4), respectively, may exist in normal and malignant human tissues Whilst panCSCs have been implicated in the aggressiveness and the progression of human PDAC, the molecular regulators driving their existence and stemness properties remain unclarified. Recently, a novel co‐regulator of canonical Wnt–β‐catenin signaling, abnormal spindle‐like microcephaly‐associated (ASPM), was shown to regulate panCSCs and their stemness properties and tumorigenic potential  isoform , 1892  were maintained in DMEM  supplemented with 10% fetal bovine serum and antibiotics. Human pancreatic ductal epithelial (HPDE) cells  were propagated on tissue culture plastics as previously described To probe the protein expression of all the putative ASPM isoforms Figure A, we raiIB analysis and co‐IP experiments were performed according to standard protocols. Antibodies used for IB experiments included anti‐dishevelled (Dvl)‐2 , anti‐cyclin E , and anti‐non‐phospho (active) β‐catenin (D13A1) and anti‐caspase‐3 . A goat anti‐rabbit IgG  was used in conjunction with the polyclonal antibodies raised for the immunodetection of ASPM isoforms as described above. Proteins were revealed following SDS/PAGE and immunoblotting with the indicated antibodies.ASPM transcript variant 1 ‐specific small hairpin RNA (shRNA). The specific KD of the expression of ASPM‐vII (NCBI RefSeq: NM_001206846) was carried out using three different RNAi target sequences that cross the splicing junction site connecting exons 17 and 19 (unique to ASPM‐vII). The construction of the lentiviral vector expressing the selected shRNA and the virus production procedure are described in supplementary material, Supplementary materials and methods. Nonspecific KD of all putative ASPM variants, including ASPM‐vI and ASPM‐vII, was achieved by lentivirus‐mediated RNAi using a commercial shRNA oligonucleotide (clone TRCN0000118905) in the lentivector pLKO.1‐puro .The specific knockdown (KD) of the expression of For luciferase reporter analysis, cells were transduced with Cignal Lenti TCF/LEF Reporter . Following stimulation of the cells with recombinant human Wnt‐3a  or vehicle, the reporter activity was measured by using the ONE‐Glo™ Luciferase Assay System .The ALDEFLUOR assay  was performed according to the manufacturer's recommendations. Flow cytometry used a FACSCanto™ II flow cytometer (BD Biosciences). The tumorsphere assay was performed as described previously IF staining of cells grown on culture plastics was performed using standard protocols as described in supplementary material, Supplementary materials and methods. Specific antibodies against ASPM‐iI or ASPM‐iII (described above), Dvl‐2 , active (non‐phospho) β‐catenin , and ALDH‐1  were used for the staining. For IHC analysis, we acquired formalin‐fixed, paraffin‐embedded tissues of PDAC from 50 patients who had received tumor resection at the National Cheng Kung University Hospital (NCKUH) during 2015–2018 in conformity with Institutional Review Board‐approved protocols. Informed consent was obtained from all the patients. The clinical characteristics of the patient cohort are presented in supplementary material, http://cran.r-project.org) and SPSS 10.0 software  were used to conduct the statistical analysis of our data. Survival curves were generated using the Kaplan–Meier method. The curves were plotted and compared using the log‐rank test in Prism 6.01 software . A cut‐off value that best discriminated between groups with respect to outcome was determined using the concordance index, where an index of 1.0 is perfect discrimination The statistical programming language R , which led to much reduced abundance levels of both ASPM‐iI and ASPM‐iII being detected using their respective antibodies  expressional heterogeneity within the same tumor  and ASPM‐vII (encoding ASPM‐iII) in PDAC cells using lentivirus‐mediated and isoform‐specific RNA interference. We were able to specifically knock down the expression of ASPM‐vI to a level of approximately 25% of that of the cells infected with the control shRNA virus . Notwithstanding this technical difficulty, we were able to demonstrate that the specific KD of ASPM‐vI expression (ASPM‐vI KD) dramatically attenuated Wnt pathway activation in PDAC cells to an extent comparable to that induced by a pan‐ASPM‐variant KD  of PDAC cells  and a decrease in the cells in S phase , indicating a partial cell‐cycle arrest at the G1 phase, in PDAC cells . By contrast, the SI of ASPM‐iII or ALDH‐1 was not prognostically significant ; specifically, patients with tumors with a high ASPM‐iI SI had an approximately six‐fold increase in the long‐term risk of mortality than those with tumors with a low ASPM‐iI SI  ASPM gene  cohort of PDAC patients, and requires further verification in an independent and larger patient cohort than the present one. Similarly, whether or not the SI of ASPM‐iII remains prognostically insignificant also awaits further investigation.In keeping with the differential biological functions of ASPM isoforms in PDAC cells, we demonstrated that ASPM‐iI, but not ASPM‐iII, co‐localizes with active β‐catenin and the panCSC marker ALDH‐1 in human PDAC tissues. Notably and importantly, the expression pattern of ASPM‐iI, as reflected by the ASPM‐iI SI, significantly outperformed that of ALDH‐1 and standard clinico‐pathologic variables in the prognostic prediction of PDAC patients. This finding supports the idea that a knowledge‐based approach may offer an opportunity to identify best‐performing biomarkers or classifiers that additionally provide pathogenetic information in cancer patients. Moreover, a pathway‐ and biology‐informed biomarker like ASPM‐iI SI may fulfill the need of a diagnostic assay that can help clinicians or investigators prognostically classify patients, whereby clinical trials related to the specific cancer biology can be conducted in a patient‐tailored manner. It should be noted, however, that the prognostic role of ASPM‐iI was determined in a relatively small  of ASPM transcripts or ASPM‐vI expression does not induce apoptosis in PDAC cellsClick here for additional data file.Table S1. Clinical characteristics of the patients in the NCKUH cohortTable S2. Multivariate Cox regression model predicting overall survival by the ASPM‐iI Staining Index and clinico‐pathological criteria.Click here for additional data file.


Cardiovascular diseases are the number one cause of death globally contributing to 37% of all global deaths. A common complication of cardiovascular disease is heart failure, where, in such cases, the only solution would be to conduct a heart transplant. Every 10 minutes a new patient is added to the transplant waiting list. However, a shortage of human donors and the short window of time available to find a correct match and transplant the donors' heart to the recipient means that numerous challenges are faced by the patient even before the operation could be done, reducing their chances of living even further.TM) in improving the efficiency of heart storage based on journal articles obtained from PubMed, Elsevier Clinical Key, and Science Direct. This review aims to evaluate the application of the Organ Care System (OCS Studies have shown that OCS is capable of extending the ischemic time 120 minutes longer than conventional methods without any detrimental effect on the recipient nor donor's safety. Based on the PROTECT I and PROCEED II study, 93% of transplantation recipients using the OCS system passed through the 30-day mortality period. OCS is able to prolong the ischemic time of donors' hearts by perfusing the organ at 34°C in a beating state, potentially reducing the detrimental effect of cold storage and providing additional assessment options. Another clear advantage is the implanting surgeon can assess the quality of the donor heart before surgery as well as providing a time safety buffer in unanticipated circumstances that will reduce the mortality risk of transplant recipients. Cardiovascular disease is currently the number one cause of death globally contributing to 37% of all global deaths . A commoIn the UK, only 25% of all heart donor candidates are eligible for transplantation. This low percentage is mostly contributed by the donors' age or limited ischemic time. Donor hearts still experience low levels of anaerobic metabolism during cold ischemic time, depleting adenosine triphosphate (ATP) supply and increasing acidosis, marked by elevated lactate levels. Thus with increasing ischemic time, donor hearts are at an increased risk of primary graft failure (PGF). Currently, the maximum ischemic time of 4-6 hours is observed before the heart is classified unsafe for use [TM, TransMedics, Andover, United States), presents a new avenue in expanding the donor pool, reducing cold ischemic time while providing additional donor heart assessment options for clinicians.Every 10 minutes a new patient is added to the transplant waiting list. Ever increasing demands of heart transplant lists globally coupled with medically constant rates of donor organ procurement results in increased cardiac mortality. On average, 22 people die each day while waiting for a transplant . One of TM, TransMedics, Andover, United States) in improving the efficiency of donor heart storage.This review aims to evaluate the potential application of the Organ Care System , case-control, cohort study, case series, or case studies. In addition, studies were included if (1) they reported the use of OCS on subjects before or after circulatory death, (2) parameters related to cardiac function or length of stay or survival rate or transplant rejection were reported, (3) studies compare OCS use versus routine care (cold storage). The rest were excluded from this review.Our initial search resulted in 397 articles found, of which 32 were excluded due to duplicate citations. Two-hundred and sixty four articles were then excluded on the basis of title and abstract; most were studies concerning the use of OCS or ex vivo perfusion not on heart but in lung and renal transplant patients. Of the remaining 101, 91 were excluded because they did not meet the inclusion criteria. Finally, 10 articles were found that fulfilled all of our inclusion criteria, 8 of which were clinical studies with 2 experimental studies – 1 in a porcine model and another in juvenile landrace pigs .A total of 423 subjects were found from all the studies: 408 from clinical studies and 15 from experimental studies. The majority of studies were performed on standard donor criteria with 2 studies using extended donor criteria. However, there were 3 studies, 1 on humans and 2 on experimental models examining OCS use in heart transplant after circulatory death which is of great interest .OCS is able to prolong the ischemic time of donors' hearts by perfusing the organ at 34°C in a beating state, potentially reducing the detrimental effect of cold storage and providing additional metabolic (lactate levels) and functional  assessment options. OCS consists of two main components, portable platforms and specific organ perfusion sets, which operate together as one integrated technology  Figure .A heart perfusion platform including oxygen supply and pumps are used to maintain warm, oxygenated, and nutrient-rich blood flow to the heart. Pulsatile flow is maintained with the use of a diaphragmatic pump while a heating plate ensures the maintenance of normothermic conditions. A wireless monitor simultaneously controls the perfusion rate and assists in the evaluation of the donor heart by providing vital information such as aortic pressure, coronary flow rate, temperature, oxygen saturation, and haematocrit levels. The perfusion module is designed to allow for ultrasound assessment and blood sampling for further off-line analysis. The perfusate consists of insulin, methylprednisolone, sodium bicarbonate, antibiotics, multivitamins, and fresh donor blood , 18.The use of OCS in heart transplantations is able to potentially reduce cold ischemic time while prolonging cardiac graft preservation. It has been well-documented that prolonged cold ischemia results in greater risks of complications for transplanted patients. OCS use has been found to provide longer ischemic time for donor hearts without adversely affecting patient outcome . One stuChan et al. reported similar results with no significant differences found in the 2-year survival rate, MACE, and vasculopathy incidence of OCS and CS patients . The lonWith prolonged ischemic time, the donor heart can now travel for longer distances, expanding the list of potential recipients and increasing the chances of gaining a matching donor heart. This reduced ischemic time is especially beneficial in patients with previous cardiac surgery or in redo transplantation, giving the surgeons additional time to safely lyse all the adhesions and prepare the cuffs before arresting the heart in the OCS, thus allowing better prognostic outcomes.In addition, the urgency and risk associated with ischemic time often results in the need of a dangerous high-speed trip for the transplant team, which increases the risk of serious injury and death on the road. In the United States, 122 accidents and 68 injuries were reported by transplant teams from the period of 1990-2007 in which only 16% of respondents felt safe during the transportation of a donor heart to the recipient hospital [Another advantage for the ex vivo perfusion of donor hearts is the expansion of the donor pool and acceptance of marginal donors. The inclusion of donor hearts with a high risk of an adverse donor-recipient profile or the so-called “extended criteria” shows great promise in increasing the supply of donor hearts available today , 21. CurA study conducted by Saez et al. evaluating the use of OCS in extended criteria donor hearts (n=26) found a 100% 30-day survival rate, preserved cardiac function in 92% of patients with a mean duration on inotropic support of 113 ± 85 hours, and a length of ICU stay of 6 days . In a suThe use of donated after circulatory death (DCD) donor hearts also present a new avenue in increasing organ supply . Using Dst successful DCD human heart transplant in St. Vincent's Hospital [In an animal study conducted by Saez et al., donor hearts from nonheparinized donation after cardiac death (DCD) porcine donors were found to be successfully resuscitated using the application of OCS . SimilarHospital .The OCS device also enables the implanting surgeon to assess the quality of the donor heart before any irreversible steps are undertaken on the recipient. Therefore, in any unanticipated circumstances, a time safety buffer is available, thus reducing the mortality risk in transplant patients. Alongside a standard hemodynamic assessment of the donor heart, further examination such as echocardiography, lactate profile, serum creatine kinase MB levels, and other examinations for suspected coronary artery disease (CAD) can be done to ensure the quality of donor hearts before transplantation. CAD can be suspected in the presence of persistent aortic pressure unresponsive to increased adenosine infusion or increased lactate profiles (more than 5 mmol/l) .In the OCS PROCEED II trial, 79 donor hearts were included in the study initially out of which 3 were eventually excluded based on lactate profile. A confirmatory histopathological examination of these hearts further revealed significant myocardial contusion in two of the donor hearts and undiagnosed left ventricular hypertrophy in the other. Thus, an elevated lactate profile can potentially serve as an important tool in assessing the viability of donor hearts before any irreversible steps are taken .This study was further confirmed by Tsui et al. in a study at Papworth Hospital and UCLA on the potential assessment capabilities of OCS involving 14 donor hearts. Donor hearts were assessed based on hemodynamic, echocardiography, and lactate profiles and evaluated for the presence of palpable coronary disease. Based on these parameters, 12 donor hearts were eventually accepted for transplantation while 2 were turned down based on end lactate profile despite adequate coronary flow. Further histopathological examination of one of the rejected donor hearts revealed widespread triple vessel coronary artery disease with >95% stenosis. The second donor displayed significant left and right ventricle wall myocardial contusion upon further investigation .Other avenues that could be explored on the ex vivo assessment capability of OCS include intravascular ultrasound and coronary angiography for the detection of occult coronary artery disease as well as contrast echocardiography to ensure myocardial perfusion , 28. TheAdditional resources such as support personnel, equipment, appropriate transport, and the collection of donor blood are required for the application of OCS thus making it a more costly endeavour compared to conventional cold storage preservation. NICE estimated a £30,758 cost per transplant by using OCS alone, not taking into account additional costs incurred during the hospital stay whereas cold storage of organs was found to cost only an estimated £118,80 per transplant .However, the long-term benefits of potentially making more donor hearts available for transplantation must be taken into consideration as well as the reduced medical costs incurred with a lower risk primary graft failure (PGF). Forty-three percent of patient deaths in the first 30 days after transplantation are a result of PGF. One of the main reasons for the costliness of heart transplantation includes the fact that when a recipient develops PGF thus requiring additional circulatory support and prolonged intensive care stay. Compared to the potential long-term costs from PGF, both financially and in quality of life, the use of OCS regardless of its cost can be financially justified.TM, TransMedics, Andover, United States), presents a new avenue in expanding the donor pool, reducing cold ischemic time while providing additional donor heart assessment options for clinicians. Intercollaborative effort between government, medical institutions, and clinicians as well as the publication of a clear set of guidelines regarding the usage of OCS would be helpful in increasing the number OCS use. Further studies could be done regarding the usage of OCS in an extended donor pool, DCD hearts, evaluation of its donor heart assessment capabilities, and the link between donor lactate levels with outcomes in the future.The use of an ex vivo normothermic perfusion machine, for example, the Organ Care System (OCS


Cell-based therapies with bone marrow-derived progenitor cells (BMDPC) lead to an improved clinical outcome in animal sepsis models. In the present study we evaluated the ability of granulocyte macrophage-colony stimulating factor (GM-CSF) to mobilize BMDPC in a lipopolysaccharide (LPS)-induced sepsis model and thereby its potential as a novel treatment strategy.in vitro.Male Wistar rats received LPS (25μg/kg/h for 4 days) intravenously and were subsequently treated with GM-CSF 12.5μg/kg . As control groups, rats were infused with sodium chloride or GM-CSF only. Clinical and laboratory parameters, proinflammatory plasma cytokines as well as BMDPC counts were analyzed. Cytokine release by isolated peripheral blood mononuclear cells from rat spleen upon incubation with LPS, GM-CSF and a combination of both were investigated In vivo, rats receiving both LPS and GM-CSF, showed a reduced weight loss and increased mobilization of BMDPC. At the same time, this regime resulted in an increased release of proinflammatory cytokines  and a significantly increased mortality. In vitro, the combination of LPS and GM-CSF showed a significantly increased IL-6 release upon incubation compared to incubation with LPS or GM-CSF alone.GM-CSF did not have a beneficial effect on the clinical course in our LPS-induced sepsis model. It synergistically promoted inflammation with LPS and probably thereby impaired survival. Septic shock with multiple organ failure is the leading cause of death in intensive care units and remains a major health problem. Despite extensive research and well-conducted clinical trials, no specific novel treatment strategy exists so far ,2. Since+/AC133+/KDR+), recently also referred to as proangiogenic hematopoietic cells (PHC) , proinflammatory cytokines and flowcytometry.The concentrations of interleukin (IL)-6, IL-8 and vascular endothelial growth factor (VEGF) were assessed in serum using enzyme-linked immunosorbent assay kits  in triplicate samples. Enzyme-linked immunosorbent assays were performed according to the manufacturer’s instructions.+/CD133+-cells have been described and identified as BMDPC in the literature. In brief, one hundred microliters of PBMC (containing 1 x 106 cells) were incubated at 4°C for 30 mins with 10 μL of goat CD34 polyclonal antibodies  and 10 μL of rabbit CD133 polyclonal antibodies . The cells were washed three times to remove unbound antibodies and incubated at 4°C for 30 mins with the secondary antibodies swine anti-rabbit IgG- Fluorescein isothiocyanate isomer 1 (FITC)  and donkey anti-goat IgG (H+L)-Phycoerythrin (PE) . The cells were again washed three times to remove unbound antibodies and finally resuspended in 500 μL of BD FACS lysing solution . Flowcytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed using WinMDI 2•8 software . A minimum of 300,000 events were collected. Flowcytometry analysis of each probe was performed in triplicate. The frequency of BMDPC in peripheral blood was determined by a two-dimensional side-scatter/fluorescence dot-plot analysis of the samples after appropriate gating. BMDPC counts are expressed as percentage of total PBMC in each rat.Peripheral blood mononuclear cells (PBMC) were prepared by gradient centrifugation using Ficoll-Hypaque . The expression of cell-surface antigens was determined by two-color immunofluorescence staining using antibodies against CD34 –a marker protein for hematopoietic stem cells- and CD133 –a marker for progenitor cells. CD34The PBMC isolated from rat spleen were prepared by gradient centrifugation using Ficoll-Hypaque . Five specific pathogen free male Wistar rats weighting 450-500g served as organ donors. The rat spleen was first reduced to small pieces and then homogenized using a mesh . The obtained spleen cells were then diluted with phosphate buffer saline 1:4 and pipetted onto the Ficoll solution. Centrifugation was performed at 20°C with 1500 rpm for 30 minutes. After that, the interphase was extracted and purified by 3 successive centrifugation steps . The PBMC were then seeded in 24-well plates in growth medium  and stimulated with GM-CSF  in different concentrations , LPS (0.25μg/ml)  and the combination of both. After 24 hours, IL-6 and cytokine-induced neutrophil chemoattractant-1  were measured in the supernatant using ELISA kits  in triplicate samples. ELISA was performed according to the manufacturer’s instructions.All data are presented as mean ± SEM. The statistical comparisons were carried out by means of one-way ANOVA followed by Holm-Sidak’s post-hoc test as required. The survival rates determined according to Kaplan-Meier were compared with the log rank test. The corrections for multiple comparisons were ensured by the Holm-Sidak test. We considered p<0.05 to be statistically significant. Statistical analyses were performed using SigmaPlot 11.0 .2/FiO2 ratio  was not impaired in any group. On the contrary pH was reduced in the LPS+GMCSF group compared to 0h (p<0.001) and the LPS group (p = 0.006). Ventilation was not compromised, as PaCO2  was not altered. Rather we found a decreased base excess in the LPS (p<0.016) and LPS+GMCSF group p<0.001) compared to 0h. Hemoglobin was reduced in all intervention groups  compared to 0h. There was a also significant difference in hemoglobin between LPS and LPS+GMCSF (p = 0.002).Weight changes were assessed and blood gas analysis was performed at the beginning of the experiment (0h) and at the end (96h). The results are displayed in After 96h we found a statistical significant increase for ASAT (p<0.001), creatinine (p<0.001), urea (p<0.001) and creatine kinase (p<0.001) in the LPS+GMCSF group compared to 0h . In the We used ELISA to detect serum concentrations of IL-6, IL-8 and VEGF in the serum of rats in all the groups after 96 hours . For IL-+/CD133+-BMDPC in the peripheral blood of rats in the sham (n = 2), LPS (n = 3), GM-CSF (n = 3) and LPS+GMCSF group at two time points (0 and 96 h). Our results showed significant differences in the groups  .in vivo-model resulted in increased inflammation markers and mortality, we set out to assess the effect of GM-CSF and LPS alone and in combination on cytokine release in vitro. We isolated PBMC from rat spleen and performed ELISA with the supernatant after 24 hour incubation. The addition of GM-CSF in three ascending dosages to the medium resulted in a moderate increase of IL-6 concentration in the supernatant, although this increase did not reach statistical significance in any of the tested GM-CSF concentrations , LPS (281±24.2pg/ml) and the three different tested concentrations of LPS+GMCSF  compared to the medium (4.34±0.9pg/ml) . However, there has been no statistical difference between the three different concentrations of LPS+ .In vitro, we demonstrated that incubation of isolated PBMC from rat spleen with LPS and GM-CSF simultaneously results in a significantly increased IL-6 release.In previous studies, we and others, have demonstrated that progenitor cells are increasingly mobilized from the bone marrow into the circulation in patients with sepsis  and acutAlready 25 years ago, the use of G-CSF or GM-CSF treatment in animal models of bacteraemia was investigated ,18. Basein vitro by incubating PBMC from rat spleen with LPS and GM-CSF, which has also lead to an increased release of proinflammatory cytokines. It has been shown in the past, that GM-CSF does induce proinflammatory cytokine release, especially IL-6 and IL-8, in macrophages [In contradiction to these results, we have found an increased mortality by GM-CSF application in our sepsis-model. We could demonstrate that animals, which received LPS and GM-CSF, released increasing amounts of pro-inflammatory cytokines and confirmed these results rophages ,20. AlthWith regard to ARDS, there are conflicting results on the effect of GM-CSF. Goodman et al. have demonstrated that plasma from atients with ARDS enhanced PBMC viability, while blocking the GM-CSF receptor significantly reduced PBMC viability in ARDS plasma, but not in normal plasma . They suIn our present study, GM-CSF treatment resulted in an increased mortality of animals. Although BMDPC were increasingly mobilized in response to GM-CSF administration, they were not able to prevent the severe pro-inflammatory effects of the GM-CSF treatment. In line with these observations, levels of G-CSF mRNA correlated with severity of shock, infiltration of polymorphonuclear leukocytes (PMN), pulmonary edema and hypoxia in a rat model of hemorrhagic shock ,25. The Conflicting results in regard to the effect of G-CSF/GM-CSF have also been described for sepsis. In pre-clinical models of sepsis in rats, GM-CSF correlated positively with the survival outcome . A meta-in vivo studies, we also have to discuss the limitations of our experimental set-up. Our endotoxemia model induced by LPS is obviously not identical with an infection caused by a pathogen, in which LPS leads to an activation of the innate immune system to eliminate the pathogen. Also, the survival rate of the animals in the LPS group was hundred percent, which already shows, that the endotoxemia did only cause a mild sepsis. We can only speculate that GM-CSF application could lead to different results in a model of severe sepsis. Another limitation of our set-up relates to the application of GM-CSF simultaneously to the induction of sepsis, which does not reflect the clinical setting, where an application is only possible after sepsis has been manifested. A respective prolonged set-up might have revealed different result. Also, we have not tested a treatment strategy based on G-CSF, which could have also led to a different outcome, since previous studies showed a differing impact by GM-CSF and G-CSF [The limitations of this study are the limited number of experiments and the lack of GM-CSF receptor blockade studies. The results from this study remain therefore associative and loosely mechanistic. To interpret the results of our study within a clinical context and with respect to the design of future nd G-CSF . Anotherin vitro. We have demonstrated that GM-CSF administration not only results in an increased mobilization of BMDPC, but surprisingly impairs survival significantly in our experimental model. There is indication that GM-CSF administration amplifies the LPS-induced release of proinflammatory cytokines, which could also be confirmed


Oviductus Ranae is a nutritional product for both medicine and food. Its quality is uneven due to multiple factors. An efficient method was established to evaluate the quality of Oviductus Ranae using fingerprint techniques and chemometric methods based on the analysis of polyunsaturated fatty acids (PUFAs) in petroleum ether extract by high performance liquid chromatography (HPLC). During this process, 27 batches of Oviductus Ranae were analyzed by HPLC and two types of chromatographic fingerprints were established. The fingerprint that was obtained by matching six known peaks was used for the quantification of six PUFAs. Another fingerprint was obtained by matching sixteen peaks with a peak area ratio greater than 0.5% and it was used to classify the different qualities of Oviductus Ranae by further combining three different chemometric models. The 27 batches of Oviductus Ranae were divided into four categories, which was consistent with the analysis results of six PUFAs contents. The two high-quality samples with significantly higher contents were classified into one category, and samples with medium contents were divided into two categories, including eight and thirteen samples, respectively. The four inferior samples with lower contents were classified into one category. The results indicated that the newly developed method has potential application prospects for the quality evaluation of Oviductus Ranae. Oviductus Ranae, which is the dried oviduct of female Rana temporaria chensinensis David, is mainly produced in the Changbai Mountains area of Jilin Province, China ) and 24.19% (R2X[2]), respectively, and the cumulative variance contribution rate was 72.86%. This showed that the two principal components contain most information of all the variables, and the original data were fully reflected. The score chart was drawn while using the two principal components  to visualize the results  = 0.786, R2Y(cum) = 0.591, Q2(cum) = 0.313). The PLS-DA scores showed consistent with the results of HCA and PCA. The four predetermined categories each presented a tightly distributed state, in which the high-quality samples S12 and S14 were separately divided, and the four inferior samples  were divided on the leftmost side of As a supervisory recognition model, PLS-DA can maximize the differences between different groups and help to filter the markers that are responsible for class separation. In the PLS-DA model, the 27 Oviductus Ranae.The variable importance for the projection (VIP) diagram b was conOviductus Ranae in the main producing areas of Changbai Mountains. Two types of chromatographic fingerprints were established with six common peaks of six high content of PUFAs and sixteen common chromatographic peaks with peak area > 0.5%, respectively, which could provide reference for the consistency evaluation of Oviductus Ranae. The chromatographic fingerprint that was established by matching six known PUFAs peaks was used for the quantitative. In order to further classify the Oviductus Ranae, two unsupervised pattern recognition models (PCA and HCA) and a supervised pattern recognition model (PLS-DA) were established by using the chemometric methods to analyze the fingerprint of sixteen chromatographic peaks with a peak area > 0.5%. All three chemometric models obtained consistent results. The Oviductus Ranae samples with different fatty acid content were successfully divided into four categories, among which the samples  with significant higher content were divided into one group and those  with significantly lower content were clustered into one group. The results showed that the method has potential application value in the quality evaluation of Oviductus Ranae.In this study, HPLC was used to analyze the petroleum ether extracts of 27 batches of Oviductus Ranae while using HPLC fingerprint combined with chemometrics. It can provide reference for the establishment of quality criteria for regulatory authorities, increase people’s confidence in the effectiveness and safety of related nutritious products, and provide a reliable basis for the wide application of Oviductus Ranae.In conclusion, the study developed a feasible method for the evaluation of the quality of