Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood–CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF. Streptococcus suis [Bacterial meningitis is a life-threatening disease  in humancus suis ,3. The icus suis . In addicus suis  that sercus suis ,7,8.S. suis two NET-evading nucleases have been described: EndAsuis and SsnA. The membrane-anchored EndAsuis only contributes to NET-evasion. The released SsnA has additionally been shown to efficiently reduce antimicrobial effects of NETs to improve survival of S. suis in the presence of NETs [S. suis meningitis [Various bacteria have evolved DNases as NET-evasion factors to escape from NET-entrapment and killing. For  of NETs ,10. Furtningitis . HoweverActinobacillus pleuropneumoniae and Haemophilus influenzae survive even better in the presence of neutrophils treated with DNases, because degraded NETs release nutrients as NAD, which enhance bacterial growth [Generally, the specific role of nucleases during bacterial infections is controversially discussed in the literature. A recent publication discussed the infusion of DNase 1 into the CSF  as a trel growth .S. suis meningitis in vivo and in vitro.In this study, we investigated the role of the bacterial nuclease SsnA and the role of the host DNase 1 during S. suis [S. suis meningitis, we conducted two animal experiments. The first was performed as a survival experiment to examine the influence of the DNase on mortality over a period of 13 days. The second experiment was performed to study the immune response, as well as bacterial load, during the onset of meningitis (early phase). In the survival experiment, slightly higher internal body temperatures occurred in the S. suis 10-infected group than in the 10ΔssnA-infected group . A similar morbidity as in the initial survival experiment was found. The internal body temperature of infected animals increased significantly in both groups after infection and a slightly higher body temperature was detected in the S. suis 10-infected group post-infection (6 to 3.7 × 107 CFU/mL). The brain lesion score in this experiment was comparable between both infection groups  post-infection. Indeed, we detected NETs in CSF in both infection groups -staining identified neutrophils in the choroid plexus and meninges only in animals with CNS disorders  [To confirm the influence of DNase 1, we have chosen a more sensitive in vitro system and performed a neutrophil killing assay with the phagocytosis-sensitive 0cpsΔEF) ,25. Usin0cpsΔEF) , but onlS. suis meningitis in vivo and in vitro. Bacteria developed nucleases in evolutionary biology for phage defense [Streptococcus mutans, Streptococcus pyogenes, Bacillus anthracis, Vibrio cholerae or Aeromonas hydophila [S. suis field isolates found in inner organs of pigs express SsnA [S. suis strains were re-isolated from different inner organs of piglets without CNS disorders  is a virulent wild-type serotype 2 strain  of multilocus sequence type (ST) 1 and has been used for infection studies before [s before ,46. The s before ,47.S. suis strain 10∆ssnA (10∆ssnA), a nuclease A-deficient in-frame deletion mutant [S. suis strain 10cpsΔEF (10cpsΔEF), a capsule-deficient isogenic mutant [In addition to the wild-type strain, two isogenic knock-out mutants were used: c mutant .Strains were cultivated out of frozen glycerol stocks on Columbia Agar with 7% Sheep Blood (Thermo Scientific™ PB5008A).2. The 40 mL portions of the suspension were centrifuged and after discarding the supernatant bacteria were suspended in 3 mL PBS, leading to a bacterial concentration of 4 × 109 CFU/mL. The exact CFU/mL was determined by plating serial dilutions on blood agar plates .For the infection of piglets, bacterial strains were grown to early stationary phase in tryptic soy broth (TSB) without dextrose  at 37 °C with 5% CO2. Aliquots with 15% Glycerol  were frozen in liquid nitrogen and stored at −80 °C.For in vitro assays, working cryostocks of bacterial strains were generated by growing the bacterial strains to early stationary phase in TSB without dextrose  at 37 °C with 5% COS. suis 10 but not free of S. suis in general. Handling and treatment of animals was in strict accordance with the principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, as well as the German Animal Protection Law. The animal experiments were approved by the Committee on Animal Experiments of the Lower Saxonian State Office for Consumer Protection and Food Safety (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) under permit numbers 33.14-42502-04-12/0965 (last update 2013) and 33.12-42502-04-12/0991 (last update 10/2017). Post-infection, animals were monitored every eight hours for internal body temperature, food uptake, behavior, breathing, posture, motion sequence and lameness.All pigs used in this study were castrated male German Landrace piglets from a herd known to be free of 9 CFU S. suis 10 or 10ΔssnA, as previous described [Eighteen 4–5-week-old pigs were divided into two groups. After pre-disposition with 1% acetic acid under anesthesia , ketamine 10 mg/kg bodyweight ), the animals in the respective groups were intranasally challenged with 1 × 10escribed . The heaescribed .9 CFU S. suis 10, 10ΔssnA or mock infected with PBS (uninfected) as described above. Thirty-nine 8-week-old piglets were divided into three groups and infected with 6 × 10S. suis ΔssnA infected group had to be sacrificed 72 h after infection and one animal of the S. suis 10 infected group 86 h after infection due to the clinical symptoms of meningitis for animal welfare reasons.During anesthesia, before infection, swabs of the tonsils, heparin blood and serum samples were collected. Piglets in the survival experiment were sacrificed as described above, 13 days post-infection at the latest. In the early phase experiment, we sacrificed the first half of each group  48 h post-infection, the second half of all animals  96 h post-infection. One animal of the In both experiments the animals were anesthetized for euthanasia as already described and heparin blood and serum samples were collected. After euthanasia with T61 i.v., CSF samples were taken by puncturing of the cisterna magna . During Streptococci . Isolated S. suis strains were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Isolation of the challenge strain was confirmed by multiplex PCR detecting mrp, epf, sly, arcA, gdh, cps1, cps2, cps7 and cps9 [ssnA gene, to verify the knock-out in the respective isolates. Primers used for ssnA detection are listed in Organ and swab samples collected during section of the animals were spread onto blood agar plates, with tonsils additionally on selective agar for 5 CFU either S. suis 10 or 10∆ssnA for 2 h. During infection, the samples were rotated on a rotator at 8 rounds per minute in an incubator at 37 °C. At time-points 0 h and 2 h the CFU was determined by plating serial dilutions on blood agar plates . The survival factor was calculated by dividing the number of CFUs after 2 h of incubation by the number of CFUs at 0 h. Survival in the blood was determined by infecting 0.5 mL porcine whole blood with 3 × 10®; Invitrogen, Carlsbad, CA, USA) was performed to determine free DNA in CSF and serum, as described previously [A Pico Green quantification assay  for a maximum of 72 h. The regions of the corpus striatum, hippocampus and cerebellum, the latter two containing the choroid plexus, were embedded in paraffin and cut into 2–4 µm sections for hematoxylin-eosin (HE) staining and immunofluorescence staining. ® slides . From all animals with CNS disorders, fever or altered CSF, cells in CSF were counted. The cell number was adjusted with HBSS to 1 × 104 to 1 × 105 cells/100 µL and cells were added to the CellView® slides. The slides were centrifuged at 370 g for 5 min at room temperature and fixed with a final concentration of 4% paraformaldehyde (PFA).At a maximum of 30 min after collection of CSF, 100 µL CSF of all non-infected animals and of animals without fever or altered CSF were transferred to CellViewS. suis: CSF was fixed with 4% paraformaldehyde . Staining was performed as previously described [S. suis [Staining of NETs and [S. suis  (1:500).S. suis: staining was performed as previously described [S. suis [Staining of NETs and intra- and extracellular escribed  using fi[S. suis  (1:500).S. suis staining, were performed as previously described [Slides of cell culture experiments and with isolated neutrophils: the co-staining of NETs and elastase, as well as the extracellular and intracellular escribed .Hematoxylin-Eosin (HE) Staining: Automated dying in LEICA ST 4040 with 0.1 % hematoxylin  and 1 % eosin .S. suis [Immunofluorescence Staining: The staining was performed as previously described  with theS. suis  (1:500) ®-GelStain  at 100 V for 30 min. Bands were detected with Biorad ChemiDoc MP  and the degradation of the calf thymus DNA was classified into 4 groups between no (0) and total (3) degradation, by eye  was added. The samples were incubated at 37 °C for 20 h. After incubation, samples were run on an 1% electrophoresis gel containing Roti, by eye .DNASE 1 ELISA Kit (pig) , PR-39 ELISA Kit , PMAP-23 ELISA Kit  and PMAP-37 ELISA Kit  were used to analyze CSF samples. The tests were performed following the manufacturers’ instructions.® ERS-2  voltmeter to control the barrier density. Some filters were infected with S. suis 10 for one hour in the blood compartment. Before adding the freshly isolated porcine neutrophils, medium in the blood compartment was changed in all filters. Transmigration of neutrophils was performed for 4 h at 37 °C and 5% CO2. Serial dilutions of transmigrated bacteria were plated on blood agar  to determine the CFU/mL and 250 µL medium of the CSF compartment was fixed with a final concentration of 4% PFA  for flow cytometric analysis. TEER was measured again. The experiment was repeated three times on independent days.Cell Culture Model of the Porcine Blood–CSF Barrier A: After 2 CFU S. suis 10. Thus ultimately yielded: non-infected wells, non-infected wells with DNase 1, infected wells and infected wells with DNase 1. In the upper compartment, 400 µL heparinized whole pig blood  under no. 33.9-42502-05-18A302, last update 07/2018) was added and incubated at 37 °C and 5% CO2 for 4 h. After incubation, serial dilutions of CSF were plated on blood agar plates  to determine the CFU/mL, and 250 µL CSF was fixed with a final concentration of 4% PFA  for flow cytometric analysis. TEER was measured again. The experiment was repeated five times on independent days. CSF was collected from healthy euthanized pigs. The euthanasia of these pigs was approved and registered by the local Animal Welfare Officer in accordance with the German Animal Welfare Law under number TiHo-T-2019-14.Cell Culture Model of the Porcine Blood–CSF Barrier Mimicking Physiological Conditions : FiltersPorcine choroid plexus epithelial cells (PCP-R)  were seeTM version 10.6.1  software by drawing a gate to exclude the debris.Two hundred microliters of PFA-fixed CSF samples from the cell culture experiments were measured in an Attune NxT Flow Cytometer  with forward and sideward scatter using a 488 nm laser. Data were analyzed with FlowJoTM separating solution 6115 , as described previously [Porcine neutrophil granulocytes were isolated from fresh heparinized blood  no. 33.9-42502-05-18A302) by density gradient centrifugation using Biocoll eviously . After r5 freshly isolated porcine neutrophils in RPMI were infected with 2 × 105 CFU 10cpsΔEF [2 serial dilutions were plated on blood agar plates  to determine the CFU/mL.In total, 2 × 1010cpsΔEF  (MOI 1) p-value has the same settings in all graphs .Data were analyzed using Excel 2010 and 2016 . Statistical analyses were performed with GraphPad Prism Version 8.0.1 . A detailed description of tests used can be found in the figure legends, but the


To investigate differences in the tibial tubercle-trochlear groove (TT-TG) and patellar tendon-posterior cruciate ligament (PT-PCL) distances in symptomatic patients with non-contact internal knee derangements (IKD) and symptomatic patients with internally intact knees (control).t tests were used to determine differences in TT-TG distance between the internal derangement and control groups. Chi-square tests were used to compare categorical variables for distributional equality between study groups.A retrospective review of MRI studies was completed by comparing 78 patients with meniscal and ligamentous derangements of the knee to 63 internally intact knees . MRI findings were reviewed independently by two board-certified radiologists to assess for agreement. TT-TG and PT-PCL distances were measured on proton density-weighted axial images by two independent observers blinded to the MRI and arthroscopic findings. Independent p < 0.001). The mean PT-PCL distance similarly averaged across both raters was 20.6 mm  for the IKD group compared to 18.2 mm  for the control group (p < 0.001). Among the IKD group, there were 51 meniscal tears, 12 cruciate ligament tears, and 15 tears with a combination of meniscal and cruciate findings. IKD was significantly correlated with greater TT-TG distance (p < 0.001) and greater PT-PCL distance (p < 0.003) when compared with control.The mean TT-TG distance averaged across the two raters in the IKD group was 11.5 mm , compared to 8.3 mm  in the control group (Increased TT-TG distances and PT-PCL distances are associated with both cartilaginous and ligamentous internal knee injuries in the present study, with TT-TG distances greater than the 12 mm representing a new threshold for concern. The tibial tubercle-trochlear groove distance (TT-TG) and patellar tendon-posterior cruciate ligament (PT-PCL) distances are important measurements in the assessment of patellofemoral disorders –4. In caIn the context of a patellar dislocation, TT-TG values greater than 15 to 20 mm are widely considered to be abnormal –11, yet Thus, the goal of the present study was threefold as follows: to further define the normal TT-TG range in anatomically intact knees, to provide a more comprehensive stratification of the TT-TG measurement in the context of internal knee derangements, and to test the validity of these findings by measuring the PT-PCL distance given its consistency irrespective of knee positioning , 18. Cru. The age range of 13–50 years and non-contact mechanism of injury were both required for inclusion in the present study, with 13 years being chosen as the lower limit due to TT-TG measurement stabilization by this age [Institutional review board approval and waiver of informed consent were obtained for this retrospective case-control study prior to the start of the study. Investigators identified 280 consecutive knee MRI examinations from subjects referred to the medical center imaging department for non-traumatic symptomatic knee pain, between January 1, 2017, and July 15, 2018MRI images were obtained using a Siemens Avanto 1.5 Tesla scanner. The knees were imaged in an Invivo 8 channel receive only knee coil. The knees were slightly flexed up to 15°. The axial images used to measure the knee anatomy were fat-suppressed proton density fast spin echo sequence with a field of view of 160 mm, matrix 384 × 512, slice thickness of 3 mm, gap of 0.6 mm, bandwidth of 181 Hz/pixel, repetition time (TR) 2600 ms, and echo time (TE) 12 ms. The scans were analyzed on a Centricity PACS workstation . MRI-based diagnosis of each study subject was determined independently by two board-certified radiologists, and subjects were sorted into control and IKD groups when a diagnostic agreement was reached.TT-TG and PT-PCL measurements were performed independently on axial fat-suppressed PD TSE MR images by one board-certified radiologist with over 10 years of experience and one fourth-year medical student, who were both blinded to the MRI diagnostic findings. Measurements were performed following the methods described by Schoettle et al. for the TT-TG Fig. . The TT-d = 0.47 would require n = 73 per group or n = 146 overall subjects. A power of 79% was reached in the present study.The number of subjects needed in the present study to detect a statistically significant difference in TT-TG distance between groups was performed in a similar fashion to the method described by Saper . Saper eStudy data was imported into SPSSv25.0 (IBM Corp.) software. Subjects were classified based on presentation with internal knee derangement (IKD) or not (control). The primary outcome variable, TT to TG distance, was blindly measured by two independent raters and the two-way mixed-effects intraclass correlation model ) was used to measure the absolute agreement of the two raters. The ICC  with a 95% confidence interval fell within 0.75 to 1.00; thus, the two raters were deemed in agreement and their values were averaged to a single value. A similar procedure was performed for the secondary measure, PT-PCL distance, with ICC value indicating high reliability hence both rater measurements were also averaged into a single metric value.t test since the sample size is large enough to invoke the central limit theorem to justify normality. In the presence of overall significant differences, Tukey post hoc HSD tests were performed to compare IKD subgroups to control. Age was also compared between the two study groups and compared for mean equality. Gender was also compared for distributional equality between the two study groups via Pearson chi-square test. A significant gender imbalance resulted in the use of a multivariate regression model with factors for study group and gender. Thus, the primary analysis for inference purposes is the comparison between study groups after adjustment for gender.TT-TG and PT-PCL distance (mm) were compared between the two study groups, IKD and control, using an independent samples Student’s p = 0.007); however, mean age was comparable  as was knee laterality (p = 0.182). The two independent, blinded rater measurements of TT-TG and PT-PCL distances were assessed as having excellent reliability via a two-way ICC  mixed model for absolute mean agreement value of 0.985 (95%CI 0.979–0.990) and 0.945  for the TT-TG and PT-PCL measurements respectively and therefore averaged . A significant difference was also observed between the mean PT-PCL distance in the IKD group and the control group .The demographic characteristics for each group are shown in Table p = 0.165 and p = 0.233, respectively); however, each subgroup was significantly greater in TT-TG distance than control after Tukey HSD pairwise post hoc testing (p < 0.004 for each). Each IKD subgroup was significantly greater than control (p < 0.05 for each) in PT-PCL distance with the exception of the meniscal tear subgroup (p = 0.069).Among the IKD group, there were 51 meniscal tears, 12 cruciate ligament tears, and 15 tears with a combination of meniscal and cruciate findings (Table p < 0.001) and 2.600 mm of PT-PCL (p < 0.003) length associated with IKD. Further, after ROC analysis was applied using the maximum Youden’s J index to determine the optimal cut-off, it was determined that TT-TG distances above 12 mm were strongly associated with the presence of IKD, with only four out of 39 cases (10.3%) having an internally intact knee.A multivariate regression model was determined separately for average TT-TG and PT-PCL distance with predictive factors for gender and IKD. IKD was a significant factor in each model even after adjustment for gender with an additional 3.016 mm of TT-TG length . This falls within the lower limit of what has previously been reported , 12–15, p < 0.004) in the present study to the ACL deficient group  in the Saper study (the primary pathology accounted for in the study), the results closely approximate each other. These findings could be explained by the confounding presence of meniscal tears within the Saper study, leading to a type II error in the control group. With meniscal tears in their control group, the normal range was overestimated. By accounting for meniscal tears in the present study, the data reveal a greater difference (3.27 mm) between the internally deranged and anatomically intact knees than what was previously reported by Saper et al. (1.63 mm) [Further, in comparison to the normal range of 10.4 mm  reported by Saper,  where a 1.63 mm) . This dip < 0.069) in this meniscal tear subgroup. Changes in knee positioning or a relative decrease in interrater reliability due to the increased challenge of picking the same slice across observers while measuring the PT-PCL measurement (as compared to the TT-TG measurement) are both plausible reasons for this occurrence. Despite these results, the combination derangement subgroup (meniscus tears and ACL tears) had the highest mean across the TT-TG (13.0 mm) and PT-PCL (21.7 mm) measurements. Across the TT-TG and PT-PCL measurement, the meniscal tear subgroup had the lowest mean, followed by the cruciate subgroup, and finally by the combination derangement group which had the highest TT-TG and PT-PCL overall. Collectively and individually, these averages were well above the normal ranges for the TT-TG and PT-PCL measurements and well below the patellar dislocation rage of 15–20 mm for the TT-TG measurement, supportive of the study hypothesisHowever, within the context of the PT-PCL measurement in the present study, statistical significance was lost , an imbalance may arise between the stabilization of rotational vectors at work within the joint causing increased imbalance during static and dynamic rotation. It is hypothesized that over time, this imbalance is what may predispose subjects with increased TT-TG and PT-PCL distances to tears both within the menisci and cruciate ligaments. Once the lateralization reaches a critical threshold value of 15–20 mm; however, the risk of patellar dislocation exponentially increases and thus becomes the overarching pathology observed. While further studies are needed to explore this hypothesized difference between patients with normal and wide TT-TG and PT-PCL distances, the present study provides critical findings from which to investigate these relationships.There are several limitations to our study. The retrospective nature of this study limited our ability to explore the number of control patients that would eventually convert to the meniscal or cruciate injury groups as well as those who might eventually convert to a patellar dislocation. The post hoc power of 79% for our primary comparison was close to our minimal goal of 80%, and it was decided rather than introduce problems with multiplicity and potential inclusion biases to not expand enrollment. In the present study, it is also worth noting that while all subjects in the control group had anatomically normal MRIs, many received scanning due to complaints of knee pain or knee discomfort, for which no anatomical explanation was ultimately found. Although a future study with asymptomatic and anatomically normal patients would bolster our current findings, the cost-prohibitive and prospective nature of the study was beyond our scope, as was delineating which specific types of meniscal tears occurred more commonly, due to our relatively small study population.Another challenge of this study was found in choosing which MR slice best depicted the patellar tendon/tibial tubercle junction and the medial border of the PCL , with thGender imbalance within the IKD group  may also have affected our results. Given that females have an increased relative risk of ACL rupture in non-contact injuries,  likely dIn conclusion, this study adds to the literature regarding normative TT-TG values and additionally draws attention to a previously unreported association between increasing TT-TG and PT-PCL distances and internal knee derangements. While the TT-TG measurement has been scrutinized due to a dependence on knee positioning, the present study utilizes the PT-PCL measurement as a secondary measurement to the TT-TG, given its consistency irrespective of knee positioning. In the present study, the IKD group falls in a previously unidentified region between the anatomically normal control group cases and the previously reported range for patellar dislocation cases. Even after adjustment for gender, an additional 3.016 mm of TT-TG length and 2.600 mm of PT-PCL length was associated with IKD. Further, it was determined that TT-TG distances above 12 mm were strongly associated with the presence of IKD, with only four out of 39 cases (10.3%) having an internally intact knee. While the TT-TG is only one contributing factor, a distance greater than the 12 mm threshold appears to be statistically associated with internal knee injuries in this study group.


Streptococcus pneumoniae, exceeds 50% in sub-Saharan African countries with high HIV prevalence. Underlying causes of high mortality are poorly understood. We examined the host and pathogen proteome in the CSF of adults with proven pneumococcal meningitis (PM), testing if there was an association between differentially expressed proteins and outcome.Mortality from bacterial meningitis, predominately caused by S. pneumoniae.CSF proteomes were analyzed by quantitative Mass-Spectrometry. Spectra were identified using the Swissprot human and TIGR4 pneumococcal protein libraries. Proteins were quantitated and analyzed against mortality. Unique proteins in PM were identified against published normal CSF proteome. Random-Forest models were used to test for protein signatures discriminating outcome. Proteins of interest were tested for their effects on growth and neutrophil opsonophagocytic killing of S. pneumoniae protein Elongation Factor Tu (EF-Tu) was significantly increased in CSF of non-survivors [False Discovery Rate (q) <0.001]. Expression of EF-Tu was negatively co-correlated against expression of Neutrophil defensin (r 0.4 p p < 0.002), but not against complement proteins C3 or Factor H. In vitro, addition of EF-Tu protein impaired S. pneumoniae neutrophil killing in CSF.CSF proteomes were available for 57 Adults with PM . Three hundred sixty individual human and 23 pneumococcal proteins were identified. Of the human protein hits, 30% were not expressed in normal CSF, and these were strongly associated with inflammation and primarily related to neutrophil activity. No human protein signature predicted outcome. However, expression of the essential S. pneumoniae EF-Tu protein in CSF was associated with reduced survival in meningitis in a high HIV prevalence population. We show EF-Tu may inhibit neutrophil mediated killing of S. pneumoniae in CSF. Further mechanistic work is required to better understand how S. pneumoniae avoids essential innate immune responses during PM through production of excess EF-Tu.Excessive  Streptococcus pneumoniae  in sub-Saharan Africa, where the combination of high HIV prevalence and high burden of nasopharyngeal carriage create a potent environment for PM to flourish in all age groups  is a leading cause of infectious mortality and morbidity worldwide; an estimated 2.8 million incident cases of community-acquired ABM were reported in 2016 occurring predominately in children and young people . There ie groups . Ambitioe groups . Howevere groups . In Afrie groups .S. pneumoniae by phagocytosis  kit from Fast-Track Diagnostics (FTD Luxemburg) according to the manufacturer’s instructions, bacterial loads were estimated from Ct values. Additionally, CSF was screened for Herpes viruses including EBV, CMV, and HSV1. We excluded patients with active viral co-infection in the CSF. We collected 2.0 ml of CSF for proteomics, stored on receipt in the laboratory (within 2 h of LP) at −80 degrees Celsius. In-hospital HIV testing was done on all patients by the clinical teams using point-of care Genie™ HIV1&2 test kits .Routine CSF microscopy, cell count, and CSF culture was done at the Malawi-Liverpool-Wellcome Trust Clinical Research Programme laboratory in Blantyre, Malawi as previously described . Cultureg and the pellet stored at −80°C until peptide extraction. Proteins were treated with the surfactant 0.1% (v/v) RapiGest™ (Waters) at 80°C for 10 min followed by reduction with diothiothreitol (DTT) at a final concentration of 3 mM (60°C for 10 min) and alkylation with iodoacetamide (IAA) at a final concentration of 50 mM . The enzyme trypsin  was added at an enzyme:substrate ratio of 1:50 and incubated overnight with agitation at 37°C. The surfactant was inactivated the following day by treatment with 0.1% trifluoroacetic acid (TFA) (37°C for 1 h) and peptides were recovered following centrifugation at 13,000 g.Protein concentration in all CSF samples was measured by nanodrop  and normalized to 200 µg/ml. Samples were centrifuged at 13,000 −1. Peptides were separated over linear chromatographic gradients composed of buffer A (2.5% ACN: 0.1% FA) and buffer B (90% ACN: 0.1% FA). Two gradients, 60 (3–50% buffer B in 40 min) and 180 min (3–60% buffer B in 140 min), were employed for analysis. Full scan MS spectra were acquired over the m/z range of 350–2,000 in positive polarity mode by the Orbitrap at a resolution of 30,000. A data-dependent Top20 collision induced dissociation (CID) data acquisition method was used. The ion-trap operated with CID MSMS on the 20 most intense ions .Resultant CSF peptides were separated by RPLC using a DIONEX UltiMate™ 3000LC chromatography system and MSMS analysis performed on an LTQ Orbitrap Velos using Xcalibur software v2.1 . Peptides (10 µl = ~500 ng) were injected onto the analytical column , which was maintained at 35°C and at a nanoflow rate of 0.3 µlminH. sapiens  and S. pneumoniae . A fixed carbamidomethyl modification for cysteine and variable oxidation modification for methionine were specified. A precursor mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.6 Da were applied. The results were then filtered to obtain a peptide false discovery rate of 1%. Protein inference and quantification was performed using the “Relative Quantitation using non-conflicting peptides” option in Progenesis. Protein quantification values are determined from all peptides but weighted according to peptide intensity. Proteins were annotated as differentially expressed if they achieved an FDR corrected q value of 0.05. Outcome prediction was performed by random forest using the cforest function of the “party” package in R. Pathways analysis of proteins was done using Innate DB.Data was initially mass recalibrated using the mzRefiner filter of the Proteowizard msconvert tool. The resulting files were then processed using Progenesis QI . Samples were aligned according to retention time using a combination of manual and automatic alignment. Default peak picking parameters were applied and features with charges from 1+ to 4+ featuring three or more isotope peaks were retained. Database searching was performed using Mascot . A Mascot Generic File, created by Progenesis QI, was searched against a merged database of the reviewed entries of the Uniprot reference proteome set of E. coli cells containing pEQ30_EF-Tu were kindly donated by Prof. Sven Hammerschmidt. E. coli was grown at 25°C in LB supplemented with 100 µg/ml ampicillin . EF-Tu was modified to contain a polyhistidine tag to aid purification and detection. EF-tu is insoluble when expressed in E. coli and precipitates in inclusion bodies. Firstly, the cells were pelleted by centrifugation at 3,250 × g, then the pellet was resuspended in 10 ml of 50 mM NaH2PO4, 300 mM NaCl (pH 8). This was followed firstly by sonication and then by centrifugation at 4,300 × g, for 30 min at 4°C. The pellet containing the inclusion bodies was then washed in 50 mM NaH2PO4, 300 mM NaCl (pH 8) and resuspended in 500 µl of 50 mM NaH2PO4, 300 mM NaCl, 4 M Urea (pH 8) to solubilize the inclusion bodies. This suspension was then centrifuged at 18,000 × g for 30 min at 4°C. The supernatant that contained EF-Tuf was then mixed with Ni-NTA  that had been resuspended in the same buffer as the protein. The column was then washed with 10 column volumes (CV) of 50 mM NaH2PO4, 300 mM NaCl, 4 M Urea (pH 8). The protein was eluted with 6 CV’s using 50 mM NaH2PO4, 300 mM NaCl, 4 M Urea, 250 mM immidazole (pH 8). Eluted EF-Tuf was refolded by dialysis in 20 mM Tris-HCl 5 mM MgCl2 pH 7.4. Finally, the protein was recovered, and the buffer exchanged into PBS during concentration. Presence of the protein was confirmed via an anti-His western blot. Identity of the protein of choice was confirmed by running an anti-His western blot of the cell lysates prepared in the presence and absence of IPTG. Non-induced cell extracts did not react with anti-his antibody  or thawed human cerebrospinal fluid (CSF). CSF was obtained from patients who underwent therapeutic lumbar puncture for idiopathic intracranial hypertension, all had biochemically normal, acellular CSF. CSF was kindly donated by Professor Diederik van de Beek . Four ug of purified EF-Tu, bovine serum albumin (BSA), NanA or PiaA or PBS control were added to five technical replicates.Strains of 1 ST5316  were gro1 ST5316 . BacteriGrowth for 24 h was detected by changes in optical density at 620 nm with shaking in a microplate reader (Tecan® USA) at 37°C.Bacterial killing by purified human neutrophils was done using a previously described opsonophagocytosis assay . BrieflyAll conventional statistical tests were two tailed, alpha <0.05 determined statistical significance. Ninety-five percent confidence intervals are presented for odds ratios. Logistic regression was used to model associations between clinical outcomes and risk factors while controlling for confounding factors.All proteomic data was submitted to the ProteomeXchange: Accession number:Submission Reference: 1-20200901-167440Submission Path:/nfs/pride/drop/pride-drop-003/philipjb_20200901_134730All participants or nominated guardians gave written informed consent for inclusion. Ethical approval for the transcriptomics study was granted by both the College of Medicine Research and Ethics Committee (COMREC), University of Malawi , and the Liverpool School of Tropical Medicine Research Ethics Committee, UK  Committee, Liverpool, UK.Fifty-seven patients with proven PM, whose CSF was stored within 4 h of presentation to hospital, were included in the study, 33 (57%) of whom had died within 6 weeks  Table 1.A total of 336 peptides matching Uniprot identifiers were found in the CSF , the manganese transporter lipoprotein PsaA (required for protection against oxidative stress), ABC transporter component GalT1  , and theTo investigate the hypothesis that lower GCS reflects worsening inflammation in the CNS, we used a Random Forest model to test for a protein signature to predict outcome in CSF. Principal component analysis of the CSF proteome in PM did not show any separation between outcome groups testing either peptides or proteins  and Elongation Factor Tu   (6 units/ml (Std 1.38 × 106)] compared to 1.64 × 105 units/ml CSF (Std 3.4 × 105) in survivors  compared to 2.4 × 107 (Std 4.2 × 107) in non-survivors (ANOVA p 0.008).To further investigate the possible effects of EF-Tu on the host-pathogen interaction in PM, we tested for statistical correlations within with host proteins from the 130 unique PM protein dataset. EF-Tu did not positively correlate with any human CSF proteins, but did negatively correlate with levels of neutrophil defensin , suggesting no significant effects of EF-Tu on autolysis in vitro   involved.Neutrophil defensins are multi-functional immunomodulatory proteins with direct anti-microbial activity . We testof EF-Tu . EF-Tu fS. pneumoniae culture in CSF in vitro (unpublished data). Finally, our data are from a single time point, and we cannot determine if dynamic changes in human or S. pneumoniae CSF protein predict outcome.Patients in our study were predominately HIV co-infected, and we were unable to discriminate which inflammatory CSF proteins were directly related to HIV infection rather than PM due to the small number of HIV-negative participants. Neither control CSF nor validated CSF proteomic libraries from healthy individuals living with HIV were available . HoweverS. pneumoniae ribosomal protein and EF-Tu were found in non-survivor CSF, and in vitro EF-Tu may inhibit neutrophil killing of S. pneumoniae in CSF. To better understand the causes of mortality in PM, the role of EF-Tu requires further investigation, using models that accurately reflect conditions in during acute disease.The CSF proteome in PM is highly inflammatory with evidence of BBB breakdown, but we did not find a human protein signature correlated with clinical outcome. However, higher levels of the The proteomics data presented in this article is publicly available at the PRIDE repository under the accession number PXD021268.EW, GL, PB, VT, RB, AC, SG, RH, and JB conceived and designed the work. EW, GL, VT, MN, VM, TA, ER-S, and BD collected the data. EW, PB, BW, JB, RH, and VT analyzed and interpreted the data. EW drafted the article. EW, PB, GL, VT, VM, BD, MN, TA, ER-S, EC, AC, SG, DL, BW, RB, RH, and JB critically revised the article. EW, PB, GL, VT, VM, BD, MN, TA, ER-S, EC, AC, BG, DL, BW, RB, RH, and JB approved the final version to be published. All authors contributed to the article and approved the submitted version.This study was funded by a Clinical Lecturer Starter Grant from the Academy of Medical Sciences (UK), a UCL-Institutional Strategic Support Fund award to EW from both the Wellcome Trust and the Robin Weiss Foundation and by a PhD Fellowship in Global Health to EW from the Wellcome Trust (089671/B/09/Z). The Malawi-Liverpool-Wellcome Trust Clinical Research Programme is supported by a core grant from the Wellcome Trust (101113/Z/13/Z). The laboratory work was undertaken at LSTM and UCL Respiratory, which received a proportion of funding from the National Institute for Health Research University College London Hospitals Department of Health’s NIHR Biomedical Research Centre. JB is supported by the Centre’s funding scheme. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data and the final responsibility to submit for publication. JB, VT and BW acknowledge funding from Medical Research Council (MR/R00/1871/1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Flow cytometry, real-time PCR, transwell and Cell Counting Kit-8 (CCK-8) assays were used in this study. The results showed that both preventative and therapeutic G-CSF administration could significantly inhibit bleomycin-induced pulmonary fibrosis. G-CSF enhanced BMSC migration to lung tissues, but this effect could be alleviated by AMD3100, which blocked the SDF-1/CXCR4 axis. We also found that BMSCs could inhibit fibroblast proliferation and transdifferentiation into myofibroblasts through paracrine actions. In conclusion, G-CSF exerted antifibrotic effects in bleomycin-induced lung fibrosis, in part by promoting BMSC homing to injured lung tissues via SDF-1/CXCR4 chemotaxis.Bone marrow mesenchymal stem cells (BMSCs) have multi-lineage differentiation potential and play an important role in tissue repair. Studies have shown that BMSCs gather at the injured tissue site after granulocyte-colony stimulating factor (G-CSF) administration. In this study, we first investigated whether G-CSF could promote BMSC homing to damaged lung tissue induced by bleomycin (BLM) and then investigated whether SDF-1/CXCR4 chemotaxis might be involved in this process. Next, we further studied the potential inhibitory effect of G-CSF administration in mice with lung fibrosis induced by bleomycin. We examined both the antifibrotic effects of G-CSF in mice with bleomycin-induced pulmonary fibrosis  Currently, there are no specific and effective treatment methods, and the mortality rate of IPF is extremely high2. Therefore, searching for an effective treatment is urgently needed.Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by extensive proliferation of fibroblast (FB) and deposition of extracellular matrix resulting in the formation of fibroblastic foci, the signature morphological lesion of IPF3. Researchers have investigated that MSCs play a vital role in tissue repair by paracrine signals and directly differenting into substitute functional cells4. Mesenchymal stem cells have been applied to the study of clinical diseases such as the nervous system, cardiovascular system and respiratory system8. However, the administration of exogenous MSCs is limited due to various factors, including complex production techniques and high costs. Therefore, developing effective methods for promoting autologous MSC participation in tissue repair has become an alternative approach in stem cell research and is of great significance for improving clinical treatments.With the rapid development of stem cell biology, stem cell therapy has become a popular area of research in the medical field. Mesenchymal stem cells (MSCs) are multipotent stem cells, and their highly plastic and differentiation abilities hold extensive promise for clinical applicationin vivo, and they play an important role in tissue repair. G-CSF is a strong bone marrow stem cell mobilizer. Several studies have shown that BMSCs, but not haemopoietic stem cells (HSCs), mobilized by G-CSF promote the repair of various damaged tissue types10. Although its potent of mobilizing bone marrow stem cells is known, whether G-CSF participates in tissue repair via other mechanisms has not yet been fully elucidated.BMSCs are an important source of MSCs 12. In locally damaged tissue, SDF-1 expression is upregulated, which causes the BMSCs to migrate along the concentration gradient of SDF-1 via their surface receptor CXCR4 to the injured tissue14. Furthermore, that up-regulation of the expression of CXCR4 on the surface of BMSCs also promote more BMSCs to migrate to damaged tissues, and will be more conducive to tissue damage repair.Stromal cell-derived factor-1 (SDF-1), a chemokine of the CXC family. Studies have found that SDF-1 and its specific receptor CXCR4 play an important role in stem cell mobilization, chemotaxis, homing and colonization of damaged myocardial tissueAt present, the potential therapeutic effect of G-CSF mobilizes autologous BMSCs in the treatment of pulmonary fibrosis remains unclear. The aim of our study was to investigate whether mobilization of autologous BMSCs by G-CSF could inhibit pulmonary fibrosis, and further explore the mechanism of action. Our results provided the first evidence that G-CSF could promote the migration of BMSCs to damaged lung tissue through upregulating the expression of CXCR4 on BMSCs, which could effectively alleviate pulmonary fibrosis.Female C57BL/6 mice were injected intratracheally with BLM (5 mg/kg) on day 0. Two schedules were designed to better evaluate the effects on pulmonary fibrosis. For preventative treatment, the mice were treated with G-CSF (40 and 60 µg/kg) for 3 consecutive days starting on day 1 after BLM was injected Fig. . The obvTo identify the mechanism underlying the antifibrotic effect of G-CSF, we used flow cytometry to measure the number of MSCs in lung tissue Fig. . The datin vivo and play an important role in tissue repair. To determine whether an incline of MSCs in the lung is associated with an increase in BMSCs, we first observed the direct effect of G-CSF on the homing activities of BMSCs. BMSCs were phenotypically identified by a lack of expression of the immune markers CD31 and CD34 and positive expression of CD29, CD44 and Sca-1 on the cell surface, as determined by FCM , Intercellular cell adhesion molecule-1 (ICAM-1), and Very late antigen-4 (VLA-4) as well as the CXC chemokine receptors-7 (CXCR7) and CXCR4 on the surface of the BMSCs were investigated. The results showed that G-CSF treatment affected neither the expression of the adhesion molecules mentioned above nor the expression of CXCR7. However, G-CSF treatment obviously increased the mRNA and protein level of CXCR4, which plays vital roles in the chemotactic activity of various cell types Fig. . In our In recent years, studies have shown that BMSC paracrine functions play critical roles in tissue repair. Therefore, in this study, the effects of BMSC-conditioned medium on the proliferation and transdifferentiation of FBs were investigated. The proliferation of NIH3T3 cells was significantly lower in BMSC-conditioned medium than in standard or FB-conditioned medium Fig. . Regardi16. Therefore, two schedules were designed to better evaluate the treatment effects on pulmonary fibrosis. The results showed that both the preventative and therapeutic administration of G-CSF could significantly inhibit BLM-induced pulmonary fibrosis, fully confirming effects of G-CSF in antifibrosis.BLM-induced pulmonary fibrosis in mice has been widely used as a model to study the pathophysiological mechanisms of IPF. Typical morphologic features of lung fibrosis were observed on day 14 in BLM-induced pulmonary injury10. Although its ability to mobilize bone marrow stem cells such as HSCs, BMSCs and endothelial progenitor cells (EPCs) is known, the mechanism of G-CSF involved in tissue repair remains unclear. Since HSCs can significantly aggravate lung injury, we focus on the therapeutic effects of BMSCs mobilized by G-CSF on pulmonary fibrosis, and the role of G-CSF-mobilized EPCs in improving pulmonary fibrosis will be further studied in subsequent experiments.Several studies have shown that BMSCs but not HSCs mobilized by G-CSF repair of many damaged tissue typesin vivo and play an important role in tissue repair. To determine whether an increase in MSCs in the lungs is associated with an increase in BMSCs, we studied the direct effect of G-CSF on the homing activities of BMSCs. The results showed that more G-CSF-pretreated BMSCs than nontreated BMSCs entered the lung tissue. Therefore, we propose that G-CSF promoted BMSCs homing to the lung tissue.In this study, the effect of G-CSF on BMSCs homing to damaged tissues was investigated. Our data showed that the G-CSF treatment group has a significantly higher level of MSCs than the BLM group. BMSCs are an important source of MSCs 19. In addition, cells in the blood circulation with increased surface adhesion molecule and chemokine receptor expression can also promote cell migration to damaged tissues23. In this study, expression of several major adhesion molecules, VCAM-1, ICAM-1, and VLA-4, and chemokine receptors, CXCR7 and CXCR4, was detected on the surface of BMSCs before or after G-CSF treatment. G-CSF treatment did not affect the expression of the adhesion molecules or CXCR7, but it did significantly upregulate the expression of CXCR4. Furthermore, G-CSF pretreatment could promote the chemotactic migration of BMSCs to lung tissues, as shown in Fig. Studies have shown that a variety of factors in damaged tissues could affect the migration of cells in the peripheral circulation, such as chemokines released from local injury sites, increased vascular permeability due to endothelial damage, and upregulated expression of vascular endothelial adhesion moleculeset al. found that G-CSF mobilized HSCs into the blood by directly downregulating the expression of SDF-1 in the bone marrow, and CXCR4 expression in HSCs was upregulated indirectly24. Because the expression of SDF-1 is obviously increased in injured tissue, this might be the mechanism underlying the rapid homing of HSCs to damaged tissue after entering the blood. Our research showed that G-CSF directly upregulated the expression of CXCR4 on BMSCs, and this might be an explanation for its promotion of BMSCs homing to damaged lung tissue by activating the SDF-1/CXCR4 axis.Few studies have been conducted regarding the effect of CXCR4 on bone marrow stem cells induced by G-CSF. Petit 26. In recent years, studies have shown that BMSCs paracrine functions may play critical roles in tissue repair. In this study, the effects of BMSC-conditioned medium on the proliferation and transdifferentiation of FBs were investigated. The results showed that BMSC-conditioned medium could significantly inhibit the proliferation of FBs and prevent TGF-β-induced FBs from differentiating into myofibroblasts. In addition, we found that when G-CSF increased the homing activity of BMSCs, it could not enhance the effects of BMSCs on inhibiting the proliferation and transdifferentiation of FBs. Therefore, we indicated that G-CSF mainly increased the number of BMSCs in the lung by facilitating the migration of endogenous BMSC to lung damaged tissues, thus promoting the effect of endogenous BMSC on the tissue repair. This conclusion has also been supported in other part of our studies, in which we pretreated BMSCs with G-CSF in vitro, and then injected them into mice via tail vein. In that study, the results showed that pretreatment of BMSCs with G-CSF promotes the homing of exogenous BMSCs to the lung, leading to a marked increase in the antifibrotic effects of exogenous BMSCs27.BMSCs promote tissue repair by directly differentiating into substitute functional cells or by paracrine actions29. Adachi K et al. (2003) showed that G-CSF (100 µg/kg/day) exacerbates the lung injury induced by intratracheal administration of bleomycin . The exacerbating effects of G-CSF seem to be associated not only with the marked infiltration of activated neutrophils but also with the severity of the underlying lung injury28. Zhang F et al. (2011) demonstrated that the mobilization of bone marrow-derived cells by G-CSF (50 µg/kg/day) has a protective effect against bleomycin-induced lung injury and fibrosis29. Based on these experimental results, we hypothesized that the effects of G-CSF on BLM-induced lung fibrosis may be related to the dose of G-CSF administered. In our results, we found that 40 µg/kg G-CSF had obvilously therapeutic effects on bleomycin-induced pulmonary fibrosis. However, when the dose of G-CSF was up to 60 µg/kg, no significant inhibitory effect on BLM-induced lung fibrosis was observed in the treatment groups  and were approved by the Ethics Committee of Central South University .Eighty of the female C57BL/6 mice were captured from JingDa Laboratory Animal Company . After being anaesthetized by pentobarbital sodium, the mice were injected intratracheally with 50 μL of BLM (5 mg/kg)  on day 0. To investigate the antifibrotic effects of G-CSF , C57BL/6 mice were assigned randomly to one of the following groups: (1) the control group, intratracheal saline plus subcutaneous saline; (2) the G-CSF group, intratracheal saline plus subcutaneous G-CSF; (3) the BLM group, intratracheal BLM and subcutaneous saline; or (4) the BLM  +  G-CSF group, intratracheal BLM plus subcutaneous G-CSF. For preventative treatment, G-CSF (40 μg/kg/d) was administered subcutaneously from day 1 to day 3, and on day 21 lung tissues were harvested. For therapeutic treatment, G-CSF (40 μg/kg/d) was administered subcutaneously from day 14 to day 16, and lung tissues were harvested on day 21.33, with the average score of two investigators deemed the fibrosis score of each animal .Lung tissues were fixed with a 4% paraformaldehyde solution and then embedded in paraffin to prepare tissue sections for pathological examination. The sections were examined after staining with haematoxylin and eosin (H&E) and Masson’s trichrome. Two investigators blinded to group assignments analyzed the samples and determined the severity of fibrosis was assessed semiquantitatively according to the method described by AshcroftThe content of collagen was examined with a hydroxyproline (HYP) kit  performed according to the manufacturer’s instructions.Total RNA was extracted from lung tissues and BMSCs with RNAiso Plus , and cDNA synthesis was performed using a First Strand cDNA Synthesis Kit. SYBR Green and a Bio-Rad CFX96 real-time PCR detection system were used for detection. The primer sequences for RT-PCR are shown in Table 27.The mice were anaesthetized, while the pulmonary circulatory system was perfused with PBS to flush the cells in the blood. Lung tissues were removed and minced mechanically. The methods for obtaining MSCs in lung tissue have been described in our previous study27.GFP-labelled BMSCs were purchased from Cyagen Biosciences Incorporated , and BMSCs were extracted from the femurs of young C57BL/6 mice (4 weeks). The methods for extraction, culture and purification of BMSCs have been described in our previous study®647 rat anti-mouse CD34, PE-CyTM7 rat anti-mouse CD44 and PE rat anti-mouse Sca-1 . The BMSCs were stained with an anti-CXCR4 antibody to detect CXCR4 cell surface expression. Antibodies of the corresponding isotype served as the isotype controls.Flow cytometry was used to identify the phenotypes of MSCs isolated from the bone marrow or lungs. The cultured BMSCs or single lung cells were incubated with FITC hamster anti-mouse CD29, BV421 rat anti-mouse CD31, Alexa Fluor6 in 100 μL) were injected into the tail veins of mice in control group or BLM group. At 36 hours after GFP-labelled BMSC infusion, the mice were sacrificed, and the lungs were harvested. BMSCs were quantified as the number of GFP-labelled cells by FCM.First, GFP-labelled BMSCs were treated or untreated with rmG-CSF (30 ng/mL)  in DMEM/F12 medium supplemented with 5% FBS for 24 hours, and these cells were used as rmG-CSF-pretreated BMSCs and nontreated BMSCs, respectively. Then, the nontreated or rmG-CSF (30 ng/mL)-pretreated BMSCs (1 × 104 in 200 μL) or G-CSF (30 ng/mL)-pretreated BMSCs into the upper transwell compartment in 24-well plates with or without AMD3100 (100 µg/mL). The control lungs or BLM-treated lungs were cut into small pieces (1 mm3) and placed in the lower chamber. After migration of the GFP-labelled BMSCs for 18 h, and washed the non-migrated BMSCs on the upper side of the membrane were removed with a swab. The migrated GFP-labelled BMSCs were captured and counted.First, GFP-labelled BMSCs were treated with or without rmG-CSF (30 ng/mL) in DMEM/F12 medium supplemented with 5% FBS for 24 hours, and these cells were used as rmG-CSF-pretreated BMSCs and nontreated BMSCs, respectively. Then, we seeded the nontreated BMSCs  were acquired from the National Genetics Key Laboratory . For the various treatment conditions, After plated in 96 well plates NIH3T3 cells were then cultured for 24–72 h. NIH3T3 cell proliferation was tested using a CCK-8  according to the instructions.For two groups, the data were analyzed using Student’s t-test. For multiple groups, comparisons among multiple groups were analyzed with one-way ANOVA, followed by Tukey’s Multiple Comparison Test using GraphPad Prism 5.0. Data are shown as mean ± SD. Differences between groups with P values less than 0.05 were considered significant.Supplementary Information.


We present an extremely rare case of inguinal hernia coexisting with testicular ectopia in a child. Male infant 9.5 month old presented with an empty scrotum and the ipsilateral intravaginal testis lying in a high iliac crest position. When crying a moving right inguinal bulge appeared on clinical examination. This grew bigger in moments of increased abdominal pressure and seemed to move upwards towards the right ileac crest. No abdominal wall defect could be palpated. At operation a large hernia sac fixed in the area of the right iliac crest was identified. Adjacent was the fixation point of the gubernaculum and the testis was found in an ectopic location. We removed the large sac after separating the vas and vessels and the testis and we strengthened the dorsal inguinal wall and fixed the testis in a subdartos scrotal pouch. No postoperative complications happened. An undescended testis may present as an iliac crest ectopy, coexisting with moving inguinal hernia. In our case we propose that the higher position of the aponeurosis of the external oblique in combination with ectopia of gubernacular fixation in the ipsilateral scrotum may have caused the ectopic fixation of the sac in the ipsilateral inguinal crest. The cause of indirect hernia is the presence of a protruding peritoneal sac  at the deep inguinal ring. The deep inguinal ring is formed basically by aponeurotic bars of the transversalis fascia sling, with the inferior border of the ring formed by the iliopubic tract, its superior border formed by the transversus abdominis arch and its median border formed by the inferior epigastric vein Male infant 9.5 month old was examined in the emergency department of our hospital because of an empty scrotum and the ipsilateral testis lying in a high iliac crest position. With the increase in intrabdominal pressure [crying etc.] there was an ever-increasing swelling with spiral course from the groin to ipsilateral iliac crest. On clinical examination, a reducible mass was present in the right ileac crest . The swest phase, the transabdominal phase (8-15 weeks of gestation) is controlled by enlargement of the gubernaculum and regression of the cranial ligament. Insulin-like hormone 3 is the primary regulator of the 1st phase, possibly assisted by mullerian inhibiting substance/antimullerian hormone (MIS/AMH) and by regression of the cranial suspensory ligament induced by testosterone. The second phase (25-35 weeks of gestation), the inguinoscrotal phase, requires migration of the gubernaculums from the groin into the scrotum and its migration is guided by calcitonin gene-related peptide released by the genitofemoral nerve. The inguinoscrotal phase of testicular descent is regulated by androgens and by calcitonin gene-related peptide release by the sensory nucleus of the genitofemoral nerve [An undescended testis may present as an iliac crest ectopy. This is an uncommon presentation compared to undescended testis. There are also reports of “intraparietal hernia” and “abdominal wall ectopic testis”. Ectopic testes are thought to be greater at risk of trauma, testicular torsion, sub-fertility and malignancy -7. Our pal nerve -11. The al nerve . In our In conclusion perhaps the most common operative procedures in infants and young children involve the inguinal area for the repair of hernia, hydrocele and undescended testicle. Many of the unexpected findings in such operations are peculiar to children . An awarThe authors declare no competing interests.


Objective: This study describes the pharmacokinetics of parent pimobendan (PIM) and its active metabolite, o-desmethyl-pimobendan (ODMP), after oral and rectal administration of pimobendan to healthy dogs.Animals: A total of eight healthy privately owned dogs were used in this study.Procedures: The dogs received a single dose (0.5 mg/kg) of a commercially available pimobendan tablet per os (PO). Twelve blood samples were collected over a 12-h period for pharmacokinetic analysis. After a 24-h washout period, the dogs received the same dose of pimobendan solution per rectum (PR), and samples were obtained at the same time for analysis.Results: For PIM, PO vs. PR, respectively, the mean maximum plasma concentration  was 49.1 ± 28.7 vs. 10.1 ± 2, the time to reach a maximum concentration  was 2.1 ± 0.9 vs. 1 ± 0.4, the disappearance half-life  was 1.8 ± 0.8 vs. 2.2 ± 0.6, and the area under the concentration–time curve  was 148.4 ± 71.6 vs. 31.1 ± 11.9, with relative bioavailability  of 25 ± 8. For ODMP, PO vs. PR, respectively, Cmax was 30.9 ± 10.4 vs. 8.8 ± 4.8, Tmax was 3.2 ± 1.6 vs. 1.7 ± 1.1, and t1/2 was 5.0 ± 2.7 vs. 8.3 ± 4.8, with AUC of 167.8 ± 36.2 vs. 50.1 ± 19.2 and F of 28 ± 6. The differences between PO and PR were significant (P < 0.03) for AUC and Cmax for both PIM and ODMP.Conclusions and Clinical Relevance: The pharmacokinetics of PIM and ODMP were described following PO and PR administration. The findings suggest that pimobendan PR might achieve effective concentrations and, as such, warrant future studies of clinical effectiveness in treating dogs with congestive heart failure and which are unable to receive medication PO. Pimobendan, a benzimidazole-pyridazinone derivative, is known as an inodilator that possesses the unique combination of a positive inotrope and a vasodilator . It actsper os. As such, oral administration may not be possible. For those scenarios, injectable pimobendan has been developed and used in multiple countries including Australia, Japan, and the United Kingdom. When given intravenously, injectable pimobendan provides a rapid inotropic effect and decreases left ventricular end diastolic pressure in healthy dogs  –11. The The purpose of this study was to describe the pharmacokinetic characteristics of parent pimobendan (PIM) and ODMP following a single dose (0.5 mg/kg) of oral or rectal administration in healthy dogs to explore the prospect of its clinical application. Our goal was to identify a therapeutic dose of pimobendan PR for dogs. This study aimed to obtain the relative bioavailability with a hypothesis that when pimobendan is administered rectally, pimobendan and ODMP will achieve plasma concentrations previously demonstrated to be therapeutic based on orally administered drug and that the increase in dose necessary to increase these concentrations will be <3-fold of the oral dose . FinallyCmax) and 90% confidence level, it was determined that eight dogs were necessary to describe a confidence interval that ranged 30% about the mean. The dogs included in the study were 3.7 ± 2.5 years of age and weighing 26.8 ± 5.8 kg. All dogs were determined to be healthy based on benign medical history, normal physical examination, systolic blood pressure measured via Doppler technique, bloodwork (complete blood count and chemistry panel), and urinalysis. All dogs did not receive any drugs prior to and during the experiments other than the preventive medication. During the study period, all the dogs were housed individually in a ward designated for research within a veterinary teaching hospital. All the dogs were observed for adverse effects for a period of 12 h after drug administration and daily throughout the study period. All study protocols were approved by the institutional laboratory animal care and use committee, and informed consent was obtained from all the owners before enrollment into the study.Eight privately owned healthy adult dogs  were included in the study. The sample size was designed to be able to consider a 40% difference in area of the curve being significantly different between the two routes (PO and PR), given a variability of 40% around the mean difference. Based on the maximum plasma concentration . As such, for this study, the dogs received pimobendan orally at 0.5 mg/kg to the nearest tablet size , with the rectal dose being the same (mg) as the oral dose for each dog. Oral dosing was immediately followed by the administration of 5 ml water by syringe over the base of the tongue for complete swallowing. For the rectal administration, pimobendan suspensions were prepared a maximum of 30 min prior to the scheduled administration. The calculated dose of the pimobendan tablet for each animal was finely crushed to assure adequate disintegration. Then, the crushed tablet for each dog was prepared as a solution in 0.9% NaCl  by using two 20-ml Luer Lock syringes connected to a three-way stopcock to yield a pimobendan solution of 1 mg/ml. After mixing the entire suspension, thus the entire dose for each animal and only for that animal, the solution was pulled up into a syringe, and the total dose was administered ~10 to 15 cm into the rectum in a 20-ml syringe, with a 16-in. (41 cm), 10 French red rubber tube . To assure that the entire dose was administered, the tube in which the dose was mixed was washed with a final 5 ml of 0.9% NaCl and this was used to flush the tube, thus assuring that the entire calculated dose was administered to each animal. After the administration, the anus was digitally held closed for ~3 min to prevent the early expulsion of the drug.g, 10 min) and frozen separately at −70°C until analysis.For all the dogs, a 19 g, 16- or 25-cm central-line catheter  was placed in the saphenous vein for blood sampling, as described elsewhere (r2) was at least 0.99 and the predicted concentrations were within 10% of the actual concentrations.Plasma PIM and ODMP were quantified using high-performance liquid chromatography (HPLC) with ultraviolet detection as previously reported but with modifications. The HPLC system consisted of a Waters 717 plus auto-sampler, Waters Binary pump 600 controller, and a 2,487 UV–visible detector  . The mobThe linear correlation coefficient for the PIM and the ODMP standard curves was 0.998. The limit of detection for PIM and ODMP in canine plasma was 0.5 and 1 ng/ml, respectively. The lower limit of quantification for PIM and ODMP in canine plasma was 1 and 2 ng/ml, respectively. The precision (CV %) for PIM in canine plasma at 2, 14, 50, and 76 ng/ml was 2.49, 1.80, 2.74, and 3.90%, respectively. The accuracy (% recovery) for PIM in canine plasma at 2, 14, 50, and 76 ng/ml was 100.10, 103.43, 105.86, and 105.81%, respectively. The precision (CV %) for ODMP in canine plasma at 2, 14, 50, and 76 ng/ml was 4.41, 2.26, 2.39, and 4.23%, respectively. The accuracy (% recovery) for ODMP in canine plasma at 2, 14, 50, and 76 ng/ml was 101.40, 104.75, 103.09, and 104.45%, respectively.Cmax occurring at time to maximum concentration (Tmax) was recorded. Concentration at 12 h (C12) and at the last time point collected (Cmin) was also recorded. The slope of the terminal component of the drug–elimination time curve was based on non-linear regression. Because pimobendan was not given intravenously, the terminal component could not be confirmed to have been eliminated and thus both the terminal rate constant and the corresponding half-lives were reported as disappearance; half-life (t1/2) was reported as harmonic mean ± pseudo-standard deviation. Furthermore, neither clearance (CL) nor volume of distribution (Vd) could be determined and both are reported as the ratio of either to bioavailability (Vd/F or CL/F). Other parameters included mean residence time and the percent of the AUC that was extrapolated from the terminal component of the curve. The relative bioavailability of PIM and ODMP was estimated by AUCPR/AUCPO. The metabolite–parent AUC ratio was estimated by AUCODMP/AUCPIM.Plasma PIM and ODMP concentration vs. time data were subjected to non-compartmental analysis using computer software . Area under the concentration-vs.-time curve from zero to the last time point (AUC) was determined using the log-linear trapezoidal method. The actual t-test: Cmax, Tmax, and AUC. A t-test was used to compare the metabolite–parent AUC ratio between PO and PR. Values were considered significantly different at P < 0.05.Statistical analyses were conducted by a commercially available spreadsheet . The pharmacokinetic parameters were reported out as mean ± standard deviation. Kolmogorov–Smirnov test was performed to evaluate the normality of parameters. The following comparisons were made between PO and PR routes, using a two-tailed paired Pimobendan was well tolerated in all dogs receiving both routes. The authors did not observe the expulsion of pimobendan solution after the rectal administration. No adverse effects were noted with both PR and PO pimobendan.The mean of the actual doses administered was 0.51 (range 0.5–0.52) mg/kg for PO and 0.5 mg/kg for PR. One dog removed the central line catheter after completion of the PR trial and thus was excluded from the PO trial; the PR data from this dog were retained. Data on the mean ± SD log plasma drug concentration vs. the time plots of PIM and ODMP for either PO or PR are displayed in Cmax, Tmax, and AUC of PIM and ODMP were observed between PO and PR  than pimobendan PO . Pimobendan was more rapidly absorbed via PR (Tmax 1 ± 0.4 h) than PO (Tmax 2.1 ± 0.9 h) (P = 0.01). The relative bioavailability (relative F = AUCPR/AUCPO) of PIM and ODMP was 25 ± 8 and 28 ± 6, respectively, indicating that AUCPR was significantly lower than AUCPO (P < 0.05).Plasma PIM and ODMP concentrations from pimobendan PR were found to be comparatively low at all timepoints compared to pimobendan PO. Significant differences in t1/2 of PIM was shorter than that of ODMP in both routes. The concentration–time profile of ODMP lagged slightly behind that of PIM in both routes. The AUCODMP tended to be higher than the AUCPIM in both routes. The metabolite–parent ratio (AUCODMP/AUCPIM) was 1.46 ± 68 and 1.34 ± 1.25 for PO and PR, respectively. No statistical difference between PO and PR routes was detected (P > 0.05).Pimobendan was rapidly converted to ODMP after both PO and PR administrations because ODMP was detected in plasma within minutes . The t1/ODMP/AUCPIM) compared to its oral administration. Therefore, the findings in this study suggest that the use of pimobendan PR may achieve therapeutic plasma concentrations similar to pimobendan PO.To the best of the authors' knowledge, this study is the first to describe the pharmacokinetic profiles of PIM and its active metabolite ODMP following a rectal administration in dogs. In the authors' experiences, dogs presenting with CHF and requiring emergency treatment can be in severe respiratory distress, which may make the oral administration of pimobendan challenging in the United States where tablet is the only form available. The size of pimobendan tablets is relatively larger compared to other tablets, which makes it even more difficult to administer pimobendan PO especially to those patients. Furthermore, there are scenarios when dogs in respiratory distress vomit after medication has been administered PO, which makes the absorption to achieve a therapeutic concentration questionable . For thoCmax, and AUC compared to those of oral administration . Furthermore, in order to have sufficient data points to determine the relative bioavailability of pimobendan PR, the authors determined that 0.5 mg/kg dosing was necessary to be able to have sufficiently high plasma drug concentrations to allow quantitation. Pimobendan 0.5 mg/kg PR was well tolerated in this study, with no obvious signs of gastrointestinal discomfort or other physiologic changes. This study is not designed to evaluate the potential adverse effects of the long-term administration of pimobendan at a dose of 0.5 mg/kg because the PR route will benefit dogs that received a single dose during initial stabilization.To determine the pharmacokinetic profile and relative bioavailability of pimobendan PR, this study used a dose that was two times greater (0.5 mg/kg) than that routinely used in oral administration (0.25 mg/kg) . Pimobenstration , 23, 24.stration , 13, 24.In this study, the authors determined that 0.9% NaCl was appropriate for drug delivery because of its accessibility in clinical practice in comparison to specifically formulated suppositories. In addition, the dogs in the present study did not receive an enema or manual evacuation of fecal material prior to the PR administration of pimobendan solution. These methods that we used can be followed by any veterinarian with no specialized equipment. The solutions tend to be absorbed more quickly through the rectum, produce effects more rapidly, and may not avoid first-pass metabolism compared to other formula , 13, 24.Cmax of 38.1 ± 18.3 ng/ml (Cmax of 18.6 ng/ml (6.1–25.3), yet 0.3 mg/kg resulted in a Cmax of 7.3 ± 2.7 ng/ml  can achieve a plasma concentration of PIM and ODMP in healthy dogs which is higher than those provided in the package insert . Provide.7 ng/ml . This diTmax, which was significantly shorter for PR for both PIM and ODMP  compared to the PO route (P = 0.01 for both PIM and ODMP). The more rapid absorption following rectal administration has the potential to be advantageous over oral administration as it reduces the lag time from treatment to effect in dogs with severe respiratory distress. In addition, pimobendan was more rapidly converted into its metabolite ODMP. When administered PO, pimobendan undergoes the first-pass hepatic metabolism via oxidation and gets converted into ODMP  following pimobendan PR was similar to that of pimobendan PO. However, the dogs' rectums are ~5-cm long, and 2.5 cm of the rectum drains into the systemic circulation  pimobendan achieved presumably therapeutic plasma concentration in dogs based on the package insert. Given the rapid absorption to achieve maximum concentration in conjunction with the metabolite–parent AUC ratio similar to oral administration, the rectal administration of pimobendan may be a suitable option for the immediate stabilization of dogs with CHF. Additional studies need to be performed to determine the recommended therapeutic target range of plasma concentration of PIM and ODMP in dogs. Further studies are warranted to describe the pharmacokinetics of pimobendan following repeated dosing in conjunction with pharmacodynamics investigation for dosing recommendation for this route.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Auburn University Institutional Animal Care and Use Committee. Written informed consent was obtained from the owners for the participation of their animals in this study.JH contributed to study design, sample collection, data analysis, and manuscript preparation. KK read and approved the final manuscript, contributed to study design, and sample collection. RW contributed to study design and approved the final manuscript. CC-E contributed to manuscript preparation, sample collection, and data analysis. LB contributed to study design and read and approved the final manuscript. DB contributed to study design, sample collection, data analysis, and manuscript review and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ObjectiveCerebrospinal fluid (CSF) analysis is a common diagnostic tool used to evaluate diseases of the central nervous system (CNS). We sought to determine whether there is a difference between the composition of CSF sampled from an external ventricular drain (EVD) and lumbar drain (LD) and whether this made a difference in guiding therapeutic decisions.Patients and MethodsThis study was a retrospective analysis from a single neurosurgery service between the dates of January 2011 and April 2019. A total of 12,134 patients were screened. Inclusion criteria were ages 18-80 and the presence of both an EVD and LD. Exclusion criteria were not having both routes of CSF sampling and the inability to determine which samples originated from which compartment.ResultsSix patients underwent simultaneous spinal and ventricular routine CSF sampling <24 hours apart and were analyzed for their compositions. There were 42 samples, but only 20 paired EVD-LD samples that could be analyzed. When comparing the EVD and LD sample compositions, there were statistically significant differences in white blood cells , total protein (p = 0.042), and glucose (p = 0.043). Red blood cells  and polymorphonuclear leukocytes  were not statistically significant. We found a statistically significant correlation between cranial and spinal CSF WBC , protein , and glucose . We also found that there was a significant correlation between CSF and serum glucose . There was no statistically significant correlation between RBCs .ConclusionOur results demonstrate a correlation between the cranial and spinal CSF samples, except for RBCs, with statistically significant differences in WBC, glucose, and protein values between the two sites. This confirms that sampling CSF via lumbar puncture, which carries less risk than a ventriculostomy and provides accurate data to help establish a diagnosis for intracranial pathologies. Analysis of cerebrospinal fluid (CSF) is a common diagnostic tool used to evaluate various pathologies of the central nervous system (CNS). CSF sampling requires performing invasive procedures such as lumbar puncture, lumbar drain (LD), and ventriculostomy placement that can introduce the patient to many potential complications, including infections such as meningitis .Controversy exists as to whether there is a consistent significant difference between compositions of CSF in the ventricular system compared to the spinal compartment that is sampled via lumbar puncture. A review of the literature shows that, particularly in infectious processes, there may exist a discrepancy between ventricular and lumbar CSF sample composition. On the other hand, for some pathologies, cases illustrating that there is no statistically significant difference between the CSF composition of malignant cells in these two regions have similarly been reported . This raIn this study, we sought to determine whether there is a significant difference between the composition of CSF sampled from an external ventricular drain (EVD) and LD and whether this made a difference in guiding therapeutic decisions.This study was a retrospective analysis of a prospectively collected database from a single neurosurgery service between the dates of January 2011 and April 2019. A total of 12,134 patients were screened from our institution’s neurosurgical database based on a query for “EVD & LD.” Twelve patients were included in this study  with ages ranging from 23 to 65 years old. Inclusion criteria were patients ages 18-80 and the simultaneous presence of both an EVD and LD. Exclusion criteria were not having both routes of CSF sampling in place at the same time and the inability to determine which samples originated from which compartment. We also excluded individuals who had samples drawn within 24 hours of any surgical intervention.The placement of both the EVD and LD was performed at the bedside or in the operating room using standard sterile technique. Ventricular and lumbar CSF were collected through their respective sampling ports of the catheter either at the bedside or in the operating room and tested within four hours of collection. Samples were collected simultaneously to compare the composition of white blood cells (WBCs), red blood cells (RBCs), protein, glucose, lactate (when available), and polymorphonuclear leukocyte (PMN) percentages from both the EVD and LD CSF. We also collected infectious markers such as Gram stain and culture for the CSF.All data analysis was completed using the Statistical Package for Social Sciences . Paired sample T-tests were utilized for assessing differences between EVD and LD group means, and Pearson correlations were used to determine any relationship between integral variables. A statistical significance level of less than 5% was used.Twelve patients were found who had a simultaneous EVD and LD in place. Seven patients had intracranial hemorrhage, three had an infection, one presented with a tumor, and one had a vascular malformation. A total of six patients  underwent spinal and ventricular routine CSF sampling <24 hours apart, for prophylactic purposes, and were analyzed for their compositions. Of the six patients who met all inclusion criteria, there were 42 total CSF analyses, but only 20 paired EVD-LD samples that could be used for analysis. The average ages were 40 years of age for the female and 40.33 ± 13.08 years for males (p = 0.975). When comparing the EVD and LD sample compositions, there were statistically significant differences in WBC (p = 0.040), total protein (p = 0.042), and glucose (p = 0.043). Differences in RBC (p = 0.104) and PMN (p = 0.544) were not statistically significant. Of the six patients, who were included within the study, four individuals had at least one positive culture  plus Escherichia coli). Only one of the four patients who had a positive culture had positive cultures within two simultaneous EVD and LD samples. This positive culture grew K. pneumoniae. The individual patient lab values can be found in , protein , and glucose . We also found that there was a significant correlation between CSF and serum glucose . There was no statistically significant correlation between RBCs .Our study aimed to determine whether there is a significant difference between the composition of cranial and spinal CSF by simultaneously sampling CSF from both an EVD and LD catheter under standardized conditions. This is particularly relevant in patients with a suspected CNS infection. In contrast to previous studies by Kakadia et al. and Sommer et al. which evaluated for any differences between EVD and lumbar puncture, we sought to use an LD in order to allow us to do serial sampling at various times in each patient to evaluate for temporal differences ,5. CSF iOur results demonstrated significant differences in WBC, protein, and glucose between cranial and spinal CSF samples. The trends appear to show a larger concentration of WBC and protein within the lumbar compartment, whereas glucose tends to be higher within the ventricles. Our results demonstrate that sampling the lumbar spinal fluid would likely over-estimate the presence of true infections, but remains an overall safer diagnostic modality compared to ventriculostomies. Additionally, there was a clear correlation between cranial and spinal CSF WBC, protein, and glucose counts. One potential explanation for the lack of RBC correlation is that LD or puncture procedures frequently can elicit “traumatic taps,” which occur in 10-30% of these procedures ,12. ThisSimilarly, we found a moderate correlation between CSF and serum glucose values. This is an expected finding, as studies such as Hegen et al. demonstrated that CSF glucose is typically about 60% of the serum glucose levels . Of partThe retrospective nature of this study introduces an inherent selection bias. It may also contribute to inconsistent analysis for serum protein and lactate levels, but the CSF parameters can be utilized to inform a prospective study. A second limitation of this study was the low sample size. Unfortunately, many samples were unable to be incorporated into statistical analysis because they were inadequately labeled within the electronic medical record, making it impossible to determine which samples were ventricular versus lumbar.CSF sampling comes with inherent risks to patients, but is necessary for diagnostic and therapeutic purposes. Our results demonstrate a strong correlation between the cranial and spinal CSF samples, except for RBCs, with statistically significant differences in WBC, glucose, and protein values between the two compartments. Despite the differences between ventricular and spinal compartments, these CSF components trend similarly. It further affirms that the practice of sampling CSF via lumbar puncture, which carries significantly less risk than a ventriculostomy, provides accurate trends to help establish a diagnosis for intracranial pathologies.


Ataxia telangiectasia (A-T) is a devastating multi-system disorder characterized by progressive cerebellar ataxia and immunodeficiency. The neurological decline may be caused by multiple factors of which ongoing inflammation and oxidative stress may play a dominant role. The objective of the present investigation was to determine cerebrospinal fluid (CSF) proteins and possible low-grade inflammation and its relation to age and neurological deterioration. In the present study, we investigated 15 patients with A-T from 2 to 16 years. Our investigation included blood and CSF tests, clinical neurological examination, A-T score, and MRI findings. The albumin ratio (AR) was analyzed to determine the blood–brain-barrier function. In addition, inflammatory cytokines  were measured by the multiplex cytometric bead array. We compared the results with those from an age-matched control group. Three of the A-T patients were analyzed separately . Patient had significantly more moderate and severe side effects due to CSF puncture  compared with healthy controls. Total protein, albumin, and the AR increased with age indicating a disturbed blood barrier function in older children. There were no differences for cytokines in serum and CSF with the exception of IL-2, which was significantly higher in controls in serum. The AR is significantly altered in A-T patients, but low-grade inflammation is not detectable in serum and CSF. Ataxia telangiectasia (A-T) is a devastating human autosomal recessive disorder with genetic instability. A-T is characterized by cerebellar degeneration and oculocutaneous telangiectasia, accompanied by immunodeficiency, recurrent respiratory infections, growth retardation, and cancer predisposition –4. The pDisease progression of A-T is demonstrable at different organ levels of which neurological decline is the most prominent feature of disease , 6. MostThe neuropathological changes underlying the cerebellar motor findings in A-T show a macroscopically visible cerebellar atrophy with a thinning of cerebellar hemispheric foliae and cerebellar vermis, which result from a reduction in Purkinje cells, granular cells and basket cells , 16, 17.ClinicalTrials.gov (NCT02285348).In the period between July 2010 and March 2013, 15 patients with A-T were recruited for the study. All patients were clinically and/or genetically diagnosed with A-T according to recent WHO recommendations . The stuDiagnosis of A-T Age < 18 yearsAgreement to take the samples by blood sampling and lumbar punctureNo clinical or laboratory evidence of severe infection with central nervous system (CNS) involvementLeukocytes < 12,000/μLCRP < 2 mg/ dLNo recent CNS surgeryNo other CNS damageNo CNS malignancyNo stem cell transplantationInclusion criteria for A-T patients:Three of the 15 patients with an A-T were excluded from the evaluation of the so uncomplicated A-T group due to the exclusion criteria mentioned earlier. In detail, there was one patient after resection of a cerebral meningioma, one suffered from seizures after accidental irradiation of the brain suffering from acute leukemia since the diagnosis of A-T was not established at that time, and one patient who was recently treated by stem cell transplantation. All three patients (A-T identification number 13–15) showed morphologic clear cerebral structural changes at the MRI, which would have falsified the measurements for the uncomplicated A-T group Table . To analThe control group consisted of non-A-T patients receiving lumbar puncture due to clinical indication. The indications consisted of the exclusion of meningitis, therapeutic punctures in pseudotumor cerebri, diagnosis of facial nerve paralysis, suspected Lyme disease, headache and seizures. There were 12 patients; 8 were male and 4 female. The age distribution was an average of 9 years and 8 months. Again, there was a consecutive numbering with increasing age (identification number K-01–K-12). Table The lumbar punctures were performed according to the present clinical standard and as far as possible with atraumatic puncture needles (SPROTTE® 21G needles). In the applied puncture technique, the muscular layer is first pierced above the spinal canal by means of an introducer, via which the a traumatic CSF needle is then inserted. This reduces the complication rate of CSF , 26.In all cases, due to the existing psychomotor deficit and strong anxiety of the patients, the lumbar punctures were performed in deep conscious analgesic sedation using low doses of midazolam and Es-ketamine. The patients were monitored during the study by ECG, blood pressure measurement, and oxygen saturation. The procedure was performed in the presence of a pediatric intensive care physician. In eight cases, the lumbar puncture was performed following a routine cMRI in deep analgesic sedation. In these cases, analgesic sedation and circulatory monitoring was performed by the colleagues of the Anesthesiology Clinic of the Goethe University Frankfurt.In general, 2–3 ml CSF transferred to three tubes was taken regardless of age or body weight. One part of the CSF was analyzed routinely for cellular content, albumin, protein, lactate, and glucose; two vials were stored and frozen at – 80 °C until final measurement.No side effectsMild headache, nausea, vomitingModerate to severe headache requiring analgesia, nausea or vomiting with the need for anti-emesis treatmentAll patients were bed-rested for 12–24 h after lumbar puncture and received crystalloid infusion solutions as well as pain and anti-emetic agents as needed. Possible side effects were recorded as follows:Alpha Feto-protein (AFP), C-reactive protein (CRP), total protein, albumin, albumin CSF/serum ratio (AR), lactate, and glucose were determined from the blood serum and CSF in patients and controls. Since albumin is produced only outside the brain, it is a good marker for other proteins that leak from the blood into the cerebrospinal fluid, and thus for blood–brain-barrier integrity. After measuring the albumin in serum and CSF, the AR was calculated, in which the influence of the individual protein concentration in the serum on the CSF concentration is mathematically compensated. Higher AR values indicate a disturbed blood–brain-barrier function.The concentrations of inflammatory cytokines  were analyzed via CBA  using an enhanced sensitivity setting with detection of cytokines at femtogram levels, as described .The A-T patients were also examined by a pediatric neurologist as recently described , 20. DisPatients underwent standardized MRI analysis of the brain, including T1-, T2-, and fluid-attenuated inversion recovery (FLAIR) sequences. An evaluation of cerebellar atrophy, brain stem, and basal ganglia as well as cervical was done by an experienced pediatric neurologist. Atrophy was scored semi-quantitatively as non-discernible (−), obvious (+), moderate (++), and severe (+++) as described recently  /diameter of cerebrum (c)) was formed to obtain a comparability of the size ratios.t test was applied. Correlations were analyzed by Spearman’s or Pearson’s correlation coefficient. P values ≤ 0.05 were considered significant.For statistical analysis, GraphPad Prism 5.01 (GraphPad Software) and Microsoft Excel were used. Values are presented as arithmetic means with SDs. For comparisons between the two study groups, Student’s unpaired p < 0.001); as expected, more older patients were bound to wheelchair.As shown in Table p = 0.001). In addition, older patients more often showed moderate to severe side effects of lumbar puncture . The high rate of side effects was the reason that the study was terminated earlier than planned and no adult patients were included.In the present study, side effects of CSF puncture occurred in 9 of 12 (75%) A-T patients in the sense of a post-puncture syndrome, and 6 (50%) of them even moderate to severe side effects. In the control group, there were only three (25%) cases of mild side effects in terms of headache and nausea. Three among the 12 patients showed no symptoms due to the lumbar puncture  elevated in CSF compared with healthy controls as shown in Table Serum levels of CRP, AFP, lactate, and glucose are shown in Table p = 0.004).The AR of the A-T patients was significantly elevated (median 4.69 ± 2.66) compared with controls (median 3.07 ± 0.91). The AR increased with age, which correlated well with the protein in the CSF . In addition, CRP levels of controls and patients showed a significant correlation to IL-6 levels in CSF .In the control group, two CSF samples were found, with cell counts slightly above the limit of 10 cells/μL, but in each case, meningitis was ruled out. The standard value for pediatric patients is no more than 6–10 cells/μL in CSF (28). However, there was a trend for higher CRP levels of controls compared with A-T patients  were too small.In order to image the neurodegeneration pattern, MRIs of the patients were examined. Differences between patients below versus above 10 years of age are shown in Table ded Fig. . HoweverCSF puncture was not well tolerated in A-T patients.Total protein, albumin, and AR were elevated in CSF, indicating a disturbed blood–brain barrier and/or ongoing cell degeneration, as recently described by our group .There were no differences for cytokines in serum and CSF between A-T and controls of sufficient significance to withstand a statistical correction for multiple testing, such as Bonferroni.A-T is a devastating systemic disease characterized by neurodegeneration, increased risk for cancer, immunodeficiency, and failure to thrive. Disease progression and neurological decline are related to dysfunctional control of ROS and ongoing low-grade inflammation observed in the absence of the ATM disease protein –10, 12. The lumbar punctures were performed according to the present clinical standard with atraumatic puncture needles to reduce the rate of unwanted side effects. However, it should be noted that the overall complication rate in pediatric patients is higher than in adults , 28, 29.Although intravenous rehydration treatment was used in all patients after lumbar puncture to reduce these symptoms, there was a high rate of mild to moderate side effects in patients with A-T (A-T 75% vs. controls 25%). It was of great concern that 50% of the older patients had severe and long-lasting symptoms despite intravenous rehydration and anti-emetic treatment. This prompted us to terminate the study earlier than planned, although serious complications such as meningitis, nerve injuries, or seizures did not occur in any of the patients studied.The very high rate of side effects was unexpected and is difficult to explain. One contributing factor may be the marked reduced nutritional state present in elderly patients with A-T, which is caused by the progressive neurological manifestations of dysarthria, muscular hypotonia, and swallowing difficulties , 4.Another relevant point may lie in the fact that volume loss followed by rehydrating infusions would trigger a reduction of colloidal osmotic pressure within the brain ventricles. It is already known that hypo-osmotic as well as hyper-osmotic cell stress activates the ATM protein –32, thusThe increase of CSF total protein, albumin, and albumin ratio would normally be interpreted as evidence for blood–brain-barrier impairment, as often observed during inflammatory processes or meningeal carcinomatosis, given that albumin is not synthesized in brain but imported after synthesis in peripheral organs such as liver . HoweverAtm-deficient mice and individuals with A-T show elevated ROS and ongoing low-grade inflammation as a key component of A-T pathology [athology –15. Furtathology . In addiathology , 52.However, our data in serum and CSF cannot confirm recent reports of ongoing mild inflammation in patients with A-T –13, 15. The present study has some limitations. The number of patients who underwent CSF punction was very small since the study was earlier terminated due to severe side effects. In addition, no adults were included, thus more advanced disease stages were not investigated.It can be criticized that our controls suffered from suspected meningitis and therefore the cytokine levels were measured not in healthy children. This view is in accordance with slightly higher TNF values in CSF and CRP levels in controls than A-T patients. However, it was ethically not possible to sample CSF from healthy children. Consequently, it is extremely difficult to establish normal values of CSF. Therefore, the question has to be addressed how to define a normal control CSF. In order to achieve an almost “normal” CSF, we used CSF for further analyses only of those controls who had no significant elevation of leukocytes and CRP.This is the first study which showed that A-T patients had significantly more moderate and severe side effects due to CSF puncture compared with healthy controls. In CSF total protein, albumin and the albumin ratio (AR) significantly increased with age. There were no differences for cytokines in serum and CSF, most likely why our patient group was small and only few older patients were included.