Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.The online version contains supplementary material available at 10.1186/s12967-021-03214-5. GM-CSF impairs human EBI formation by decreasing CD163 adhesion molecule expression.GM-CSF decreases mouse BM erythroblasts and EBI macrophages as well as impairs EBI formation via decreasing surface expression of CD163 and Vcam1.Granulocyte–macrophage colony-stimulating factor (GM-CSF), a significant myelopoietic growth factor and pro-inflammatory cytokine, has been shown to be upregulated and has attracted increasing interest as a therapeutic target for many inflammatory diseases, including coronavirus disease 2019 . In genMarcel Bessis in 1958 ) in 1958 , was fun in 1958 , who fou in 1958 ). Recent in 1958 , 17, 18. in 1958 , 21. TheJoshua Tay et al. found that G-CSF reduces EBI frequency in the BM by more than 100 times [In previous studies, both G-CSF and GM-CSF were used to mobilize HSCs from the BM into the blood in order to harvest large quantities of HSCs for subsequent transplantation in humans , 23. Bot00 times . In contIn the present study, we demonstrate that GM-CSF significantly decreases human EBI formation in vitro. Bioinformatics analysis of RNA sequencing (RNA-seq) on GM-CSF-treated and control EBI macrophages further confirmed the impaired EBI formation, as evidenced by decreased adhesion molecule expression of CD163. In particular, GM-CSF injection into mice also significantly decreases BM erythroblasts as well as EBI numbers by decreasing both the number of EBI macrophages and adhesion molecule expression of CD163 and of Vcam1. Our study presents novel data that GM-CSF impairs erythropoiesis by disturbing EBI formation and proposes targeting EBI macrophages as a potential treatment option for AI.All antibodies related to this study are listed in Additional file About 30–50 μl of peripheral blood was collected through the orbital vein using an Eppendorf tube containing 1 μl of 0.5 M ethylenediaminetetraacetate  after anesthesia. Blood was diluted 1:10 in phosphate balanced saline (PBS) and analyzed via an advia120 hematology analyzer. The RBC numbers, hemoglobin, hematocrit (HCT), and reticulocytes of mice before and after GM-CSF injection were analyzed using GraphPad Prism 9.0 software.Under terminal anesthesia using isoflurane, mice were killed via cervical dislocation. The BM and spleen (SP) were collected and processed for single-cell preparation for flowcytometry. The single cell suspensions were prepared as previously described . In brie6 cells were blocked with 50 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with BV-421-Lin (1μL/5 × 106cells), BV-421-CD41(1μL/5 × 106cells), PE-Cy7-CD34(1μL/5 × 106cells), PE-Cy7-CD16/32(1μL/5 × 106 cells), APC-Cy7-Scal1(1μL/5 × 106 cells), APC-CD117(1μL/5 × 106 cells), and Percp-CD71(1μL/5 × 106 cells) for 30 min on ice in the dark. For murine erythroblast staining, 3 × 106cells were blocked with 25 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with anti-CD11b-APC-Cy7 (0.1 µg/106cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-CD45-APC-Cy7 (0.1 µg/106cells), anti-Ter119-PE (0.5 µg/106cells) or FITC-Ter119(0.5 µg/106cells), and anti-CD44-APC (0.2 µg/106cells) for 30 min on ice in the dark. For macrophage staining, 5 × 106 cells were blocked with 50 μL PBS + 0.5%BSA containing rat anti-mouse CD16/CD32 (dilution 1:100) for 15 min at 4℃, then stained with anti-CD11b-APC-Cy7 (0.1 µg/106cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-AF647-F4/80 (5 µg/106 cells), anti-Percpcy5.5-CD106 (0.3 µg/106 cells), anti-FITC-CD169 (4 µg/106 cells), anti-Percp-CD163 (0.2 µg/106 cells), anti-FITC-Timd4 (0.25 µg/106 cells), anti-PE-Cy7-Mertk (0.5 µg/106 cells), anti-PE-Cy7Axl (0.125 µg/106 cells), anti-PE-Cy7-MHC-II (0.5μL/5 × 106 cells), anti-FITC-CD206 (1μL/5 × 106 cells), anti-Percpcy5.5-CD14 (1μL/5 × 106 cells), anti-FITC-CD86 (1μL/5 × 106 cells), and anti-PE-Cy7-CD80 (1μL/5 × 106 cells) for 30 min on ice in the dark. After staining, the cells were washed once with PBS plus 0.5%BSA and 2 mM EDTA. DAPI were used to gate out dead cells. Then, cells were resuspended with PBS plus 0.5%BSA and 2 mM EDTA, and run on a BD Air III (BD bioscience). Flow Jo software (BD) was used to analyze the data.For burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E) staining, 5 × 10For P-STAT5 staining, the BD Transcription Factor Phospho Buffer Set (BD Cat#565575) was used. In short, the BM cells were starved for 4 h, and were stimulated with GM-CSF for 15 min. Then, the cells were fixed with TF Fix/Perm Buffer(1X) for 50 min at 4℃ and washed using 1 × TFP Perm/Wash Buffer. After fixation, cells were incubated with BD Phosflow™ Perm Buffer III, and the cells were washed once to remove Perm Buffer III using 1 × TFP Perm/Wash Buffer. Finally, cells were stained with CD11b, Gr-1, F4/80, and P-stat5  for 50 min on ice in the dark, washed using 1 × TFP Perm/Wash Buffer, and run on a BD Air III. Flow Jo software (BD) was used to analyze the data.Jessica A. Hamerman’s group [6cells), anti-Gr1-APC-Cy7 (0.1 µg/106cells), anti-AF647-F4/80 (5 µg/106 cells), and anti-FITC-Ter119 (0.5 µg/106cells) for 30 min on ice in the dark to detect cells that had phagocytosed RBCs. BD Air III was used to collect events and Flow Jo software (BD) was used to analyze the data.We followed the same method as ’s group . BM singEBIs in mouse BM were enriched and the numbers were quantified. Our EBI enrichment method matches that of previous studies , 21, 29.SP cells were obtained from both GM-CSF treated and control mice and immediately embedded into an optimal cutting temperature (OCT) compound. The tissues were kept in a − 80 ℃ freezer. Then, hematoxylin and eosin (HE) staining was performed as previously described .+ cells. The detailed cultured method was same as our previously described [+ cells were purified by CD34 MicroBeads  from human healthy donors at Zhengzhou University. The cell culture procedure was comprised of 3 phases. In the present, two phases of cell culture were used. Composition of the base culture medium was Iscove's Modified Dulbecco's Medium , 2% human peripheral blood plasma (Stem Cell Technologies), 3% human AB serum , 200 μg/mL Holo-human transferrin (Sigma Aldrich), 3 IU/mL heparin , and 10 μg/mL insulin . In the first phase (day 0 to day 6), CD34+ cells at a concentration of 105/mL were cultured in the presence of 10 ng/mL stem cell factor , 1 ng/mL IL-3(Stem Cell Technologies), and 3 IU/mL erythropoietin . In the second phase (day 7 to day 11), IL-3 was omitted from the culture medium. Then, the D11 erythroblasts were harvested for the next step of co-culture experiment. Human “EBI-like” macrophages were also derived from CD34+ cells. In the Day 0 to Day 7, CD34+ cells were cultured in IMDM containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 10 ng/mL SCF, 1 ng/mL IL-3, 100 ng/mL M-CSF and 50 ng/mL FLT3, 1 × penicillin–streptomycin. At day 7, the suspensions were removed and IMDM containing 2% human peripheral blood plasma, 3% human AB serum, 3 IU/mL heparin, 10 µg/mL insulin, 100 ng/mL M-CSF and 50 ng/mL FLT3 were added. Then, the adherent cells were cultured for another 4 days. At D11, all the adherent cells will differentiate into macrophages as our previously described [2+, and 5 mM Ca2+. 1 × 105 cells were collected for cytospin analysis.Erythroblasts and “EBI-like” macrophages were derived from cord blood CD34escribed , 17, 31.https://www.biosino.org/node) under accession number OEP002596.RNA-seq was prepared and analyzed as previously described , 31, 32.Absolute cell count was measured via flowcytometry using 123count eBeads™ Counting Beads (Cat#: 01–1234-42) according to the manufacturer’s protocol.+ macrophages were enriched via F4/80 microbeads . BM cells were incubated with F4/80 microbeads for 15 min on ice in the dark. The cells were washed with PBS plus 2% FBS and 2 mM EDTA, centrifugated at 300 g for 10 min, and resuspended with 2 mL PBS plus 2% FBS and 2 mM EDTA. Finally, the BM F4/80+ macrophages were enriched by Quadro MACS following the manufacturer's instructions. More than 90% purification was achieved, as previously described [Control and GM-CSF-treated mouse BM F4/80escribed . These cCD163, CD169, EMP and αV-integrin used in the present study are the same in our previous work [IL1-β, TNF-α, iNOS, TGF-β, IL-10 and Arg1 are listed in Additional file QRT-PCR, cytospins and Giemsa-Wright staining were performed as in our previous papers , 31, 32.ous work . The priP < 0.05 was considered to indicate a statistically significant difference.GraphPad Prism 9.0 software  was used to perform the statistical analysis. All the experiments were replicated at least three times. All data were reported as mean ± SEM. Comparisons between different groups were performed by Student’s t test. + cells as previously described [To begin to study the potential roles of GM-CSF on human EBI formation, we performed human EBI formation assay using “EBI-like” macrophages and late-stage erythroblasts derived from human cord blood CD34escribed . “EBI-liescribed , 14, 17.escribed , 36–38. To define transcriptional changes following GM-CSF treatment, we performed RNA-seq analysis. As expected, GM-CSF-treated human “EBI-like” macrophages clustered distinctly from control human “EBI-like” macrophages in principal component analysis  expressed by EBI macrophages. ChIP-X Enrichment Analysis Version 3 (ChEA3) indicates that several key molecules significant for the function of EBI macrophages such as CD163, VCAM1, HMOX1,MERTK,AXL, and IGF1 may be regulated by NR1H3 or MAF , II (CD117+CD71low/medi), and III  and M2  surface markers on mouse BM EBI macrophages using flowcytometry. Notably, GM-CSF significantly increased the expression of MHC-II Fig. A, F but GM-CSF is barely detectable in the peripheral blood of healthy people but significantly increases in the presence of inflammatory conditions, such as, COVID-19, SCD, and so on , 3, 9. IIn the present study, we tested the roles of GM-CSF in human and mouse EBI formation, finding that GM-CSF significantly decreases EBI formation both in vitro and in vivo. G-CSF also impairs EBI formation in vivo , 21. ConMarcel Bessis in 1958 [GM-CSFR (CSF2R) is not expressed by late-stage erythroblasts but is highly expressed in human EBI macrophages, suggesting that GM-CSF may be unable to directly signal erythroblasts. Alternatively, the low expression of GM-CSFR in early-stage erythroblasts (FPKM < 10) may be insufficient, raising questions about the indirect effects of GM-CSF on erythropoiesis in vitro and in vivo. EBI, first described by  in 1958  is compo in 1958 , 45. Usi in 1958 , we foun in 1958 , 37, 39.Because GM-CSF can stimulate numerous inflammatory processes and increase expression of pro-inflammatory chemokines, an anti-GM-CSF strategy might have broader effects than other immunomodulatory approaches in immunosuppressive therapy . Indeed,Primitive and definitive erythropoiesis in mouse embryos requires the signal transduction of GM-CSF . HoweverMertk−/− mice showed significantly decreased engulfment of pyrenocytes by EBI macrophages [Axl−/− mice showed decreased a 50% decrease in phagocytotic apoptotic cells [Phagocytosis of senescent RBCs represents another important role of EBI macrophages during erythropoiesis. In general, RBC clearance is thought to occur mainly in the spleen, where senescent RBCs are phagocytosed by splenic macrophages . Our prerophages . Macrophic cells . Phosphaic cells . As suchMany proinflammatory cytokines  are known to inhibit steady-state BM erythropoiesis . Heat-kiIn summary, we have identified previously unknown roles of proinflammatory cytokine GM-CSF in erythropoiesis. Based on our findings, we propose that GM-CSF significantly impairs erythropoiesis by affecting the functioning of EBI macrophages. This conclusion is supported by several lines of evidence. (1) GM-CSF impairs human and mouse EBI formation by decreasing the expression of adhesion molecule CD163. (2) GM-CSF reduces mouse BM erythroblast numbers by decreasing the numbers of EBI macrophages and EBI formation. (3) GM-CSF impairs phagocytosis of senescent RBCs by decreasing the engulfment-related expression of Mertk, Axl and Timd4. Although we cannot exclude the effect of GM-CSF on erythropoiesis through influencing on other myeloid cells, our findings nonetheless provide new insights into GM-CSF’s role in impairing terminal erythroid differentiation at least in part by affecting EBI macrophages, and our findings can help better understanding of elevated GM-CSF levels of inflammatory diseases. Targeting GM-CSF or GM-CSF inhibitors might be a novel option for treating AI. This conclusion must be confirmed in mice in which the GM-CSFR gene is specifically depleted by EBI macrophages.Additional file 1: Figure S1. Targeting gene expression regulated by transcription factors analyzed using ChIP-X Enrichment Analysis Version 3 (ChEA3).Additional file 2: Figure S2. The gating strategy of BFU-E and CFU-E: the gating strategy of lin-CD16-CD32CD41-CD34-Scal- cell populations.Additional file 3: Figure S3. The gating strategy of terminal erythroid cells: the gating strategy of CD11b-Gr1-CD45-Ter119+ cells.Additional file 4: Figure 4. Peripheral Blood Routine of control and GM-CSF-treated mice: (A) RBCs assessments of control and GM-CSF-treated mice. (B) Hemoglobin assessments of control and GM-CSF-treated mice. (C) Hematocrit assessments of control and GM-CSF-treated mice. (D) Reticulocyte assessments of control and GM-CSF-treated mice. N=6.Additional file 5: Figure 5. GM-CSF does not induce stress erythropoiesis in SP. (A) The SP weight index (mg/g body weight) of control and GM-CSF-treated mice. (B) Representative flowcytometry image of terminal erythroid cells in SP in control and GM-CSF-treated mice. (C) Representative HE staining image of SP in control and GM-CSF-treated mice(20X). (D) Representative HE staining image of SP in control and GM-CSF-treated mice(100X).Additional file 6: Figure 6. The gating strategy of macrophages: the gating strategy of the CD11b-Gr1-SSClow cell population.Additional file 7: Figure 7. (A) Representative flowcytometry image of terminal erythroid cells in liposome-control and clodronate-loaded liposomes treated mouse BM. (B) Quantitative analysis of erythroblast numbers in in liposome-control and clodronate-loaded liposomes treated mouse BM (N = 6).Additional file 8: Figure 8. GM-CSF increased the mRNA expression of IL-1β, TNF-α, and iNOS and decreased the mRNA expression of IL-10, TGF-β, and Arg1. (A) The relative mRNA expression of IL-1β, TNF-α, and iNOS among GM-CSF treatment enriched BM EBI macrophages. (B) The relative mRNA expression of IL-10, TGF-β, and Arg1 among GM-CSF treatment enriched BM EBI macrophages (N = 3).Additional file 9: Table S1. The list of antibodies used in this study.Additional file 10: Table S2. Preparation of EBI enrichment buffer with different concentrations.Additional file 11: Table S3. The list of primers used in this study.Additional file 12: Table S4. The differentially expressed genes between control and GM-CSF treated human macrophages.


Over the past decade, repeated studies demonstrated that the vertebrate brain had a specialized lymphatic transport pathway, which overturned the traditional concept of central nervous system (CNS) immune privilege. Despite the lack of lymphatic vessels, the glymphatic system and the meningeal lymphatic vessels provide a unique pathway for solutes transport and metabolites clearance in the brain. Sleep, circadian rhythm, arterial pulsation, and other physiological factors modulate this specialized lymphatic drainage pathway. It has also changed significantly under pathological conditions. These modulatory mechanisms may arise critical targets for the therapeutic of CNS disorders. This review highlights the latest research progress on the modulation of lymphatic transport in the CNS under physiological and pathological conditions. Furthermore, we examined the possible upstream and downstream relation networks between these regulatory mechanisms. The lymphatic transport system in vertebrates maintains body fluid homeostasis, immune surveillance, and lipid reabsorption in the peripheral organs; it also plays an essential role in the pathological process underlying inflammation, cardiovascular disease, tumor metastasis, and in the onset of other diseases For a long time, researchers have suggested that cerebrospinal fluid (CSF) may be the "sink" through which brain metabolites is cleared Numerous studies have focused on this specialized lymphatic transport system's physiological modulation and pathological changes Sleep is a naturally occurring physiological state of decreasing arousal. During non-rapid eye movement (NREM) sleep, more CSF flows into ISS, and the brain's metabolites clearance is significantly more efficient than that in awake NREM sleep electroencephalography (EEG) slow-wave activity  represents the principal marker of the process S during sleep Still, there are some unclear and even confusing issues. As a marker of neurons' spontaneous and rhythmic electrical activities, EEG has been a leading tool to study brain function in health and disease. However, much less is known about its content, given the complex relationship between EEG features and microcircuit structure Besides, some studies found that the glymphatic inflow decreased following sleep deprivation 217O to capture both paracellular flow and diffusive transcellular exchange of water showed a faster and more active glymphatic transport 217O tracer freely travels through the intracellular and extracellular pathways, extracellular volume fraction will also have a minor impact on the water transport of CSF Furthermore, current researches on glymphatic transport rely on tracers of larger molecular solutes to simulate CSF transport, which may underestimate the flow of CSF water into brain parenchyma. The classical tracer study measured AQP4-dependent paracellular flow of the liquid exchange of ISS confirmed that tracers inflow were related to molecular solute size During NREM sleep, the inhibition of the adrenergic system increases the area of the ISS; consequently, it reduces the fluid resistance and increases the solute transport efficiency -2 adrenergic receptors; this effect is consistent with the inhibition of norepinephrine release during natural sleep The sympathetic tone is reduced during natural sleep, whereas the parasympathetic tone is increased Summarizing these results, we speculate that the autonomic nervous system (ANS) has a wide range of modulatory effects on the glymphatic inflow. The ANS also regulates physiological parameters such as respiration, heart rate, and vascular pulsation. Some studies examined the impact of physiological parameters on GS, which were reviewed later in this paper.Endogenous sleep factors (ESF) such as adenosine, nitric oxide, and prostaglandin D2 have significant efficiency in regulating NREM sleep homeostasis (process S) The circadian rhythm controlled by the suprachiasmatic nucleus of the hypothalamus plays the role of a pacemaker in the two-process model of sleep regulation Many physiological factors, including intracranial pressure (ICP), blood pressure, heart rate, and respiratory, regulate the fluid transport of the cerebrovascular system and PVS in vivo two-photon imaging have demonstrated that aPVS was a flat, double tubular eccentric structure, 1.4 times (whereas fixation reduced this ratio to 0.14) the cross-sectional area of the adjacent artery with low fluid resistance 217O, a recent study revealed that the glymphatic transport was dramatically faster and more extensive than previously thought It is still controversial about the fluid transport mode in the ISS: convection or diffusion. Some modeling studies showed that arterial pulsation alone could not provide sufficient driving force to account for convective transport due to the narrow aPVS The flow of CSF in the ventricular system and subarachnoid spaces (SAS) is mainly regulated by respiration, while the contribution of cardiac pulsations on this process is low Decompressive craniectomy reduces ICP and impairs the glymphatic inflow, which cranioplasty reversed in mice Some researchers believe that the efficiency provided by arterial pulsation should not exceed 15-25%, and vasomotion is a more efficient driving force Mammalian aquaporins, transmembrane proteins that promote the bidirectional transport of water across the cell membrane, are the primary water homeostasis regulators and participate in molecular transport and membrane protein expression, cell adhesion, and cell volume regulation AQP4 also plays a vital role during brain edema. CNS edema is associated with increases in total AQP4 expression and AQP4 subcellular translocation after TBI, spinal cord injury (SCI), and stroke Is there any intrinsic relationship between brain edema and GS transport? The source of water for brain edema is also a controversial issue. A recent study showed that CSF flowed into the ischemic area through PVS (the same inflow pathway as the GS), driving acute tissue swelling Figure D 76. DoeAQP4 inhibitors may benefit from the reversible blockade of AQP4 within one week after brain injury CSF is mainly produced by the choroid plexus of the ventricular system and other tissues (such as the BBB) The direct measurement of CSF production in mice by blocking the aqueduct of Sylvius indicated that isoflurane caused a higher rate of CSF production than that of the K/X cocktail In contrast, although there is no direct research evidence, other studies have demonstrated that the increased CSF production was consistent with the increased glymphatic influx. The production of CSF increases during the mid-rest phase and the treatment with non-selective β-adrenergic receptor antagonists in mice We speculate that the regulation of CSF production under physiological conditions may be intrinsically related to the modulation of sleep, ANS, physiological parameters, and cerebrovascular, all of which ensure a stable and adequate brain clearance. Changes in CSF production during pathological conditions may be related to brain clearance disorder's compensatory or decompensated mechanism.CSF in the ventricles converges into the fourth ventricle through the interventricular foramen, the third ventricle, the midbrain aqueduct, and eventually flows into the cisterna magna circulate in SAS. CSF in the fourth ventricle can also flow into the central canal of the spinal cord. The drainage of CSF into the spinal cord is far less than that into the cranial cavity under physiological conditions The novel pathway, GS, elaborates that some CSF flow into the brain parenchyma before efflux out of the skull cavity In addition, these traditional and novel drainage pathways also have some outstanding open scientific issues that need to be further characterized. For example, is there an essential connection between the conventional drainage path of the perineural space and the MLVs located around these nerves? The exact contribution of these outflow pathways to CSF drainage remains clarified. What anatomical pathway does the ISF pass into the dorsal and base MLVs?Although the mechanism of how CSF is transported to MLVs is unclear, many studies showed that CSF primary outflowed cranial cavity through MLVs The degeneration of MLVs in aging mice and the blockade of the CSF outflow pathway does not increase brain water content and ISF pressure Moreover, the proliferation and remodeling of MLVs have both sides in CNS disorders. Structural and functional disorders of MLVs will impair the brain clearance system and reduce the immune-inflammatory response, which may be a potential therapeutic target for CNS autoimmune response such as multiple sclerosis 2 partial pressure; after that, through internal chemical regulation mechanisms, the cerebrovascular dilation and vascular compliance increases, along with the lowering of heart rate, which eventually increases the arterial pulsation amplitude In this review, we systematically review the physiological regulatory mechanisms of the lymphatic transport in the CNS based on the two-process model of sleep regulation. It is now clear that no single pathological feature can explain this complex modulatory process in isolation. These mechanisms are related and interact upstream and downstream Figure . Sleep iUnlike physiological regulation, pathological changes are often compensatory or decompensated physiological reactions occurring after disease onset. Like inflammation, which has both protective and adverse effects, we need to investigate the comprehensive impact of these physiological regulations on different diseases and stages after pathological CNS disorders and further identify the regulatory strategies that can be the most beneficial. These physiological parameters will also change during pathological stress after brain injury in different periods throughout the disease course. Many clinicians have been developing individualized and accurate therapeutic schemes by adjusting these physiological parameters to reduce secondary brain injury, and these therapeutics are also controversial. For example, which is a better therapy after severe TBI, the Lund concept, or the Brain Trauma Foundation guidelines Under physiological conditions, the rapid outflow of CSF reduces the glymphatic influx. Pathologically blocking MLVs does not increase cerebral water content and glymphatic influx. We speculate that the outflow pathway of CSF in the SAS may differ from that in the brain; other complementary routes may be hidden. Further studies that characterize the exact anatomical structure of these outflow pathways will help us find new therapeutic strategies that effectively regulate these pathways to counteract the brain damage caused by the changes of outflow pathways in pathological conditions.The lymphatic transport may be involved in most pathophysiological processes in the CNS, so these directions are far from enough. We can restudy any CNS disease by targeting the specialized lymphatic transport system with new information and insight.


We constructed DANDYISM images and evaluated the CSF area distribution with decreased motion-dephasing signal using a scoring method.This study examined ten subjects aged 25–58. We acquired DWIs on a 3T clinical scanner with The DANDYISM images showed statistically significant higher CSF scores in the ventral posterior fossa, suprasellar cistern, and Sylvian vallecula compared to the lateral ventricle and frontal and parietal CSF spaces, indicating greater CSF movement in the former areas.The results indicated prominent CSF motions in the ventral portion of the posterior fossa, suprasellar cistern, and Sylvian fissure but smaller motions in the lateral ventricles and parietal subarachnoid space. This method may provide information of CSF dynamics in the clinical settings within short imaging time. The classical theory of cerebrospinal fluid (CSF) dynamics, which was established in the early twentieth century , 2, suggb = 500 s/mm2 reflects changes in the CSF dynamics [b = 500 s/mm2 for CSF within the lateral ventricle showed a higher signal in the ventricle dilatation group than in the control group. However, a single b value such as b = 500 s/mm2 cannot provide the detailed distribution of the CSF dynamics. DWIs of various b values can visualize the degree of CSF motion. As shown above, although not quantitative, a DWI with a lower b value will show motion-related signal dephasing only in areas with larger CSF motion, whereas DWI with higher b value will show motion-related signal dephasing of CSF in a wider area with small to large CSF motion. We developed a composite imaging method using various low b values called “Diffusion ANalysis of fluid DYnamics with Incremental Strength of Motion proving gradient (DANDYISM)” for evaluating the distribution of areas with signal dephasing on DWIs of various b values.Diffusion-weighted images (DWIs), which visualize the motion of water molecules as a signal decreases due to a phase shift caused by the motion, can be used to study CSF dynamics. When the phase shift becomes larger than ± π or multiple velocities exist in a single voxel, the signal decrease in the DWI becomes prominent according to the b values . Thus, CSF motion causes notable signal decrease due to its large or non-uniform motion , 4. It hThe purpose of the current study is to evaluate CSF dynamics using DANDYISM, which is a composite imaging method using various low b value DWIs, and to evaluate the feasibility of this method to provide information of whole cranial CSF dynamics.b value = 0, 50, 100, 200, 300, 500, 700, 1000 s/mm2, 3-axis mixed, with fat suppression, no flow compensation, no motion correction, no distortion correction.This study on DWIs for CSF dynamics has been approved by the institutional review board of our institution. The subjects of this study were ten volunteers, aged 25–58 years, without any abnormal findings in a conventional MRI brain scan; the subjects provided written informed consent. DWIs Fig.  were acqb = 50 s/mm2, yellow: b = 100 s/mm2, light green: b = 200 s/mm2, green: b = 300 s/mm2, blue: b = 500 s/mm2, indigo: b = 700 s/mm2, purple: b = 1000 s/mm2  . The areb = 50 s/mm2 image, 6: signal drop on a b = 50 s/mm2 image exists in the area, 5: signal drop on a b = 100 s/mm2 image exists in the area, 4: as previously but for b = 200 s/mm2, 3: for b = 300 s/mm2, 2: for b = 500 s/mm2, 1: for b = 700 s/mm2, 0: for b = 1000 s/mm2). A higher score indicates higher CSF motion, and vice versa. Images were evaluated by two neuroradiologists with more than 20 years experience. Any discrepancies between the observers were resolved through discussion, until consensus was reached. Various areas in the cranium were evaluated: the foramen magnum , foramen Luschka, foramen Magendie, fourth ventricle , aqueduct, cerebellopontine angle, prepontine cistern , suprasellar cistern, ambient cistern, quadrigeminal cistern, Sylvian vallecula, Sylvian fissure , third ventricle , foramen of Monroe, lateral ventricle , interhemispheric fissure , subarachnoid space of the frontal lobe, and subarachnoid space of the parietal lobe. We performed a Tukey test to compare the scores of representative areas . We also conducted a rank Spearman’s test of the scores and ages in the areas indicated above. The threshold for significant difference was set at P < 0.05.We evaluated the distribution of the CSF area that shows signal drop due to motion-related dephasing using a scoring system  compared to other areas. The suprasellar cistern, ambient cistern, and Sylvian vallecula also show greater CSF movement, whereas the CSF within the lateral ventricle shows less motion (lower scores). Moreover, the subarachnoid space of the parietal region shows less motion.rs = 0.61) and posterior portions (rs = 0.81) of the third ventricle, indicating greater CSF motion in younger subjects  [In a DWI, a moving water molecule exhibits phase shift according to the velocity of the water molecule and length of motion-proving gradient, which can be expressed as  effect) , 4. For The dynamics of CSF have been discussed since a long time , 2, 8–12Although the information for CSF dynamics provided by DANDYISM is mainly on the fast and pulsatile motion of the CSF, the results of the current study provide evidence that casts some doubt on the classical model of CSF dynamics. The CSF motion in the trigon of the lateral ventricle, which is considered to be a site of CSF production in the classical model, exhibited less CSF motion. Regarding absorption, less CSF motion was observed in the subarachnoid spaces in the parietal or frontal area, which are close to the arachnoid granules. In contrast, greater movement of the CSF (higher scores) was observed in the ventral posterior fossa as compared to that in other areas. The suprasellar cistern, ambient cistern, and Sylvian vallecula also exhibited greater CSF movement. These areas contain large arteries including the vertebral arteries, basilar artery, and middle cerebral arteries. The arterial pulsation of these areas may influence the corresponding CSF movement. The findings of this study may, therefore, cast further doubt on the classical model of river-like CSF, which assumes CSF is produced by the choroid plexus in the lateral ventricle and absorbed in the arachnoid granules distributed in the parasagittal area, because the motion of the water molecules is slow in both the ventricle and the subarachnoid space in the frontal and parietal regions.There are several advantages of DANDYISM method compared to specially designed methods such as the time-spatial labeling inversion pulse (Time-SLIP) or 4D-flow methods. The short acquisition time of DANDYISM method is potentially a great advantage. The acquisition time of the sequence employed in this study was 4 min. As the 4D-flow method uses cardiac gating, the acquisition time tends to be long if attempting to cover the whole brain. Coverage of the whole brain is an additional advantage of this method. The Time-SLIP method can sample only one place within the brain for single evaluation. Therefore, the DANDYISM method is a potentially useful and convenient technique for evaluation of CSF dynamics. As a study for iNPH cases using 4D flow method applied in lower part of the cranium showed significant increase in the CSF stroke volumes at the foramen of Magendie and cerebral aqueduct , this DAD*) from the signal of b = 0 and low b-value diffusion images [The major disadvantage of this DANDYISM method is lack of quantitativeness. It might be possible to calculate the perfusion-related diffusion coefficient (n images . Howevern images . We usedThere are several limitations to the current study. First, as mentioned previously, the DANDYISM is not a quantitative method and, in addition, the chronological aspect cannot be evaluated. Considering that point, this method can be used as a screening method to evaluate the CSF dynamics. The current study did not include subjects with altered CSF dynamics, such as hydrocephalus cases. We will perform further study to apply the DANDYISM method in such cases, in which the results of the current study can be used as control data.In conclusion, distribution of motion-related signal dephasing by CSF was evaluated on DANDYISM method. The DANDYISM images indicated that CSF motions were prominent in the area including the ventral portion of the posterior fossa, suprasellar cistern, and Sylvian fissure, whereas they were small in the lateral ventricles and parietal subarachnoid space. It was also indicated that CSF motion is correlated with age in the third ventricle. The DANDYISM method may provide information of whole cranial CSF dynamics in the clinical settings within short imaging time.


We calculated TT-mediated margins based on length of the longest axis of (i) the tumour prior to treatment and (ii) the maximum tissue deficit formed 7–14 days after TT treatment. We compared these TT-mediated margins for each tumour to two surgical approaches to MCT excision in general practise: modified proportional margins (with 2 cm upper limit) and 3 cm fixed margins. For most dogs, TT-mediated margins were less than half the length of the margins calculated for the two surgical approaches in removing the same tumour. There was a trend for TT-mediated margins to increase with increasing tumour volume. Nonetheless, even for the larger tumours in this study (>2 cm3 volume), 50% of TT-mediated margins were less than half the length of the two surgical margins. Eighteen cases were lower limb MCTs, sites often surgically challenging in veterinary practise. On these lower limbs, TT-mediated margins were less than half the length of the corresponding proportional margins in 56% of cases and larger than proportional margins in only two cases. This study suggests that, in many cases, smaller and more targeted margins could be expected when treating MCTs <10 cm3 volume with TT compared with surgical excision. TT-mediated margins are a novel approach to conceptualise tissue deficits after intratumoural TT treatment.Tigilanol tiglate (TT) is a novel small molecule registered as a veterinary pharmaceutical for intratumoural treatment of canine mast cell tumours (MCTs). The drug has a multifactorial mode of action resulting in rapid destruction of the treated tumour by haemorrhagic necrosis and subsequent slough of the necrotic tumour to reveal a tissue deficit that is left to heal by second intention with minimal to no veterinary intervention. Here we introduce the concept of TT-mediated margins, the calculated margin of tissue loss analogous to surgically applied margins to help clinicians conceptualise tissue deficits formed following tumour destruction by TT relative to surgical excision. We used data from 51 dogs that were recurrence-free 12 months after a single administered TT dose into a single target MCT <10 cm Tigilanol tiglate (TT) is a novel small molecule recently approved as a veterinary pharmaceutical (trade name Stelfonta®) in Europe, the United Kingdom, the United States, and Australia for local treatment of canine mast cell tumours (MCTs) –3. The dvia secondary, rather than primary, intention as is the case with most surgical excisions.In a previous paper we used data from a controlled, randomised clinical trial involving 123 client-owned dogs in the USA to (i) describe wound (tissue deficit) formation following intratumoural treatment of MCTs with TT, (ii) show that the area of individual tissue deficits was primarily related to pre-treatment tumour volume and that body location and cytologically diagnosed grade of the MCTs were unimportant in this respect, and (iii) demonstrate that time to healing  was dependent on the area of the tissue deficit and on body location . Here weThe aim of this study is to assist veterinarians in better conceptualising and understanding within a clinical context the tissue deficits associated with successful TT treatment by comparing them with two common approaches using surgical margins adopted for excision of the same MCT.3 of tumour volume, with a maximum volume of 10 cm3) to a single MCT on each dog. Data used in our analysis are for all dogs that were (a) available for assessment and (b) recurrence free at 12 months after a single TT treatment  individual treated tumours and (ii) the subsequent tissue deficits that formed at the treatment site that were collected by investigators as part of a US clinical trial evaluating efficacy and safety of TT that was administered intratumourally  and (b) two tumour locations  as have been used in our previous publications  three tumour volume classes  was used to generate descriptive statistics, regression analyses, Mood's median, and Wilcoxon signed rank non-parametric tests to analyse increasing tumour volume and treatment group effects. SigmaPlot  was used to generate Dunn's multiple pairwise comparison and polynomial regression analysis of TT-mediated margins (cm) compared with TT-mediated tissue deficits (cm2).Minitab 17 Statistical Software  of the multiple tissue deficits for each individual case and compared these to estimated areas of the tissue deficit associated with the different surgical margins around the tumour  and are likely associated with minor inaccuracies in the measurement of small tumours and/or small tissue deficits by the clinical investigators.There were small negative values for TT-mediated margins that were also associated with small tissue deficit areas. These results were obtained from nine dogs . The median TT-mediated margins increased across these tumour volume classes, with median margins and 95% confidence intervals of 0.3 , 0.8 , and 1.2  cm, respectively . Median values for TT-mediated margins were similar for both the lower limb (n = 18 dogs) and the body and upper limb (n = 33 dogs) locations in each of the three tumour volume classes .For the 51 dogs that had a single tissue deficit, the estimated TT-mediated margins after TT treatment are shown in 3 were healed, with 68% (19 out of 28) of tumours >0.5 cm3 healed. By 84 days, all but one of the tissue deficits were healed, this case being a lower limb MCT, which had reduced to 1% of the original size of the tissue deficit . The tissue deficits in all patients healed without complications and required no direct veterinary interventions  to aid the secondary intention healing process. Two out of the 51 cases did require Elizabethan-like collars to prevent the dogs from excessively licking at the treatment site during the initial period of tumour slough and early stages of subsequent healing, while oral antibiotics, in the absence of any diagnostic confirmation of infection, were prescribed prophylactically immediately after treatment at the discretion of the treating veterinarians in 20 cases.The time for tissue deficits to heal  varied with tumour volume and body location . Across n = 34) of dogs , TT-mediated margins were less than half the length of proportional and 3 cm margins in only 5 of the 10 dogs in this class . These results are consistent with our previously reported findings that used multivariable analyses of tumour features to identify the determinants of maximal area of tissue deficits following TT treatment and where only tumour volume was found to be important . While t(a) Differences between individual tumours in the occurrence and local invasiveness of the microscopic tendrils of tumour cells that typically extend out from the main mass of MCTs ;(b) Differences in the local immune context of the tumour in individual patients affecting both inflammatory response and immune cell recruitment that are associated with the mode of action of TT , 5, 8, 9(c) Anatomical location, especially in relation to the extent of the local inflammatory response and the effects on lymphatic drainage and the speed of resolution of oedema; and(d) Variability in dose administration with different “fanning” techniques and/or, in the case of smaller tumours, incorrect dose calculation and/or dose delivery.3 in volume, which comprised 80% of the tumours in the study population. This likely reduced “collateral damage” to healthy tissue surrounding the tumour mass associated with TT treatment may reflect the mode of action of TT in selectively targeting tumour vasculature  in this group in our study.For our comparison of TT-mediated margins with surgical margins that would have been theoretically applied to treatment of the same tumour, we selected (i) aggressive 3 cm margins because they are widely used in general veterinary practise , 22, 23 culature  and in iculature . For tumTumours on the lower extremities are often technically challenging in both general and specialist veterinary practise. The frequent proximity of tumours on the lower limbs to other vital structures and tissues, together with potential difficulties in achieving adequate surgical closure of tissue deficits due to tight skin, often preclude use of a surgical dose to achieve wide “clean margins” at these sites. In these situations, more complex treatment approaches are often adopted involving incomplete margins in combination with radiotherapy , 14, 19 3. These results are likely associated with minor inaccuracies in the measurement of these small tumours and/or small tissue deficits by the clinical investigators. While careful use of digital callipers alleviates much measurement error, it is difficult to completely negate the minor inaccuracies that were encountered in the clinic. Third, there were only a relatively small number of patients in the >2 cm3 tumour volume class, which limits a more definitive interpretation of comparisons for these larger tumours. Future studies would need to be mindful of these limitations during study design and planning, and in addition to the manual methods, consideration of other imaging modalities, such as ultrasonography for determining tumour dimensions and digital planimetry for healing of the tissue deficits, are recommended.We recognise three limitations in this study. First, the methodology of theoretical margin comparisons only considered the horizontal plane. The depth measurements of the resulting tissue deficits were not recorded in the pivotal study, and while there are no true definitions of a “fascial plane” in the medical literature , future 3 in volume. Further work is required to fully validate the concept and comparisons with surgical margins, including the aspect of deep margins in the vertical fascial plane, for larger MCTs, and for a wider range of tumour types seen in veterinary and human medicine. Equally, the concept of the “drug-induced” margin may have broader application for the conceptualisation of local effects of other intratumoural chemotherapeutics in veterinary and human medicine, especially to establish whether their effects are more targeted than surgery or radiotherapy and, consequentially, whether they cause less deleterious collateral damage to surrounding healthy tissue.In this study, we establish the principle behind TT-mediated margins and show that for TT, the mediated or “drug-induced” margins are, in general, smaller and more targeted compared with surgical excision when treating MCTs, especially those <2 cmtom.deridder@qbiotics.com.De-identified patient data supporting the conclusions of this article will be made available by the authors on request from qualified veterinarians and researchers. Requests to access the datasets should be directed to Institutional Animal Ethics was not required for this study as it was under a United States Centre for Veterinary Medicine — Food and Drug Administration Protocol — Investigational New Animal Drug (INAD) No. I-012436 . Written informed consent was obtained from the owners for the participation of their animals in this study.TD and PR were responsible for data compilation, analysis, and manuscript preparation. All authors contributed to the article and approved the submitted version.The authors gratefully acknowledge the clinicians and other veterinary staff who gathered the case data at each study site. Triveritas Limited provided independent VICH GCP study management for the clinical efficacy study. Sheryl Pacchiardi provided administrative support and formatted and proof-read the manuscript.https://www.triveritas.com/). Upon study closure, Triveritas cleaned and locked the data prior to providing the final dataset to QBiotics Group for analysis and publication.TD, PR, PJ, GB, and JC are all employed by QBiotics Group Ltd. This study was funded by QBiotics Group Ltd and the company owns the intellectual property and patents associated with tigilanol tiglate (Stelfonta). QBiotics Group also funded the pivotal US study which provided the data for this study, the veterinary trial sites received compensation for participating in the study on a set fee per case enrolled, the monitoring and conduct of the study was through a third party research contract organisation Triveritas (All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pseudomonas aeruginosa infections in the UK. We report a case of post-operative meningitis caused by a New-Delhi MBL-producing (NDM-1) strain, outlining the microbiological and management challenges.Carbapenem resistance due to MBL production is relatively uncommon in P. aeruginosa, along with blood cultures and CSF. Susceptibility testing as per EUCAST methodology indicated the isolate was resistant to all β-lactams, aminoglycosides and quinolones but susceptible to colistin. Susceptibility to cefiderocol by disc diffusion was in the area of technical uncertainty at 21 mm (S≥22 mm). Immunochromatographic flow testing (CARBA5 NG Biotech) detected an NDM carbapenemase. WGS (Illumina) confirmed the presence of blaNDM-1 and a plethora of AMR genes responsible for the XDR phenotype {cephalosporins , aminoglycosides [aph(3')-VIa], quinolones (crpP), macrolides , phenicols (catB7) and fosfomycin (fosA)}. No mutations in PBPs associated with resistance to β-lactams or iron acquisition systems involved in uptake of siderophore conjugated antibiotics were identified. The isolate belonged to ST1047, serotype 07, previously identified as a high-risk epidemic clone originating in Myanmar.The patient was admitted to intensive care after craniotomy and evacuation for an intracerebral haemorrhage. Three weeks later she was febrile due to nosocomial meningitis. Purulent material evacuated from extradural and subdural spaces cultured Treatment was commenced with IV and intrathecal colistin as well as IV cefiderocol Figure ; a novelThe first problem encountered was a lack of evidence for the CNS penetration of cefiderocol and efficacy in the treatment of meningitis. The external ventricular drain required to administer intrathecal colistin enabled us to monitor CSF cefiderocol levels. Five CSF samples were shipped at −80°C for analysis in Germany by mass spectrometry. CSF cefiderocol levels of 1.22 μg/mL, 1.34 μg/mL, 2.39 μg/mL, 3.53 μg/mL and 3.90 μg/mL were disappointingly close to the pharmacodynamic breakpoint of 2 μg/mL and only available after treatment withdrawal. Dosing of cefiderocol was initially cautious starting at 1 g/8 h due to a creatinine clearance of 53  mL/min but later increased to 1.5 g/8 h. Further dose increases guided by serum and CSF monitoring could feasibly lead to therapeutic CSF cefiderocol levels. Clinical and pharmacological studies are needed in this area. To our knowledge this is the first attempt to treat a case of XDR neurosurgical meningitis using cefiderocol with precision treatment and achieving microbiological cure.


With the ongoing opioid epidemic in the US, there has been an increase in the proportion of deceased organ donors with a history of injection drug use (IDU), raising concern for additional infectious risks to transplantation.We sought to determine how recent IDU among deceased organ donors impacted donor culture results.Candida. Multivariable logistic regression was used to determine the association between recent IDU and donor culture positivity. Secondarily, the association between donor IDU and isolation of (1) a multidrug-resistant organism (MDRO) on culture, (2) Staphylococcus aureus on culture, (3) Candida on non-respiratory culture, and (4) bacteria or Candida on blood culture were determined. A retrospective cohort study was conducted at four transplant centers in Philadelphia between 1/1/2015 and 6/30/2016. All deceased organ donors who donated ≥ 1 organ to one of the centers were included. Exposed donors were those with a recent history of IDU (defined by use in the prior 12 months based on donor chart review). Unexposed donors were those with no recent history of IDU. The primary outcome was any positive donor culture  for bacteria or P=0.04) and Candida on non-respiratory culture . However, recent IDU was not significantly associated with increased odds of MDRO on culture , S. aureus on culture , or positive blood culture .Of 394 total donors, 66 (17%) had a history of recent IDU and 343 (87%) had at least one positive donor culture. On multivariable analysis, recent IDU was associated with significantly increased odds of having at least one positive donor culture ) Emily Blumberg, MD, Amplyx )Hologic (Research Grant or Support)Merck Takeda  Jennifer Han, MD, MSCE, GlaxoSmithKline  Judith A. Anesi, MD, MSCE, Nothing to disclose


External ventricular drain (EVD)-related infections (EVDIs) are feared complications that are difficult to rapidly and correctly diagnose, which can lead to unnecessary treatment with broad-spectrum antibiotics. No readily available diagnostic parameters have been identified to reliably predict or identify EVDIs. Moreover, intraventricular hemorrhage is common and affect cerebrospinal fluid (CSF) cellularity. The relationship between leukocytes and erythrocytes is often used to identify suspected infection and triggers the use of antibiotics pending results of cultures, which may take days. Cell count based surveillance diagnostics assumes a homogeneous distribution of cells in the CSF. Given the intraventricular sedimentation of erythrocytes on computed tomography scans this assumption may be erroneous and could affect diagnostics.To evaluate the consistency of cell counts in serially sampled CSF from EVDs, with and without patient repositioning, to assess the effect on infection diagnostics.We performed a prospective single-center study where routine CSF sampling was followed by a second sample after 10 min, allocated around a standard patient repositioning, or not. Changes in absolute and pairwise cell counts and ratios were analyzed, including mixed regression models.Data from 51 patients and 162 paired samples were analyzed. We observed substantial changes in CSF cellularity as the result of both resampling and repositioning, with repositioning found to be an independent predictor of bidirectional cellular change. Glucose and lactate levels were affected, however clinically non-significant. No positive CSF cultures were seen during the study. Thirty percent (30%) of patients changed suspected EVDI status, as defined by the cell component of local and national guidelines, when resampling after repositioning.CSF cell counts are not consistent and are affected by patient movement suggesting a heterogeneity in the intraventricular space. The relationship between leukocytes and erythrocytes was less affected than absolute changes. Importantly, cell changes are found to increase with increased cellularity, often leading to changes in suspected EVDI status. Faster and more precise diagnostics are needed, and methods such as emerging next generation sequencing techniques my provide tools to more timely and accurately guide antibiotic treatment.Trial Registration NCT04736407, Clinicaltrials.gov, retrospectively registered 2nd February 2021.The online version contains supplementary material available at 10.1186/s13054-021-03715-1. The external ventricular drain (EVD) is an essential device in the treatment of the neurocritically ill, however, it is associated with a number of complications including EVD related infections (EVDIs) . EVDIs aIn the literature, the reported incidence of EVDIs ranges between 1 and 35% of inserted drains –12. ThisThe cell index has been suggested as a method to adjust for confounding caused by IVH in EVDI surveillance diagnostics, where the ratio of leukocytes to erythrocytes in CSF (LE ratio) is compared to the ratio in peripheral blood , 19. HowIn summary, due to the pathogens involved, patient characteristics, and confounding factors, early identification of EVDIs have proven difficult and no CSF parameter, by itself or in aggregate, have shown to reliably predict or identify EVDIs , 27, 28.We hypothesize that a heterogeneous distribution of blood components in the CSF may affect the variability of routinely analyzed CSF parameters. The aim of this study was to test this hypothesis by testing the reproducibility of serially drawn CSF samples and to evaluate if resampling and changes in a patient’s body position affects CSF parameters.This prospective observational single-center study with allocation, included adult (18 or older) patients treated with EVDs at the neurointensive care unit (NICU) at the Karolinska University Hospital in Stockholm. Exclusion criteria were patients admitted with bacterial or viral CNS infections. The data collection process is shown in Fig. Two CSF samples (paired samples) were collected biweekly or during workup due to suspected infection as per routine. The first eight patients were considered burn-in patients in order to familiarize the staff with the study and to identify any problems related to the sampling routine. All paired samples were allocated individually to two serially drawn samples performed before and after a standard clinical repositioning , or a 10-min wait period between two serially drawn samples . As such, individual patients could have both R-samples and C-samples. For R-samples, patients were positioned in a lateral side position two hours prior to sample 1. After the first sample, patients were repositioned to the contralateral side after which the second sample was collected. For C-samples, patients were stationary 2 h prior to sample 1 and 10 min separated the two samples, corresponding to the average time between R-samples, as identified during the burn-in period. Non-sedated and compliant patients were asked to lie in a lateral side position prior to the first sample in applicable cases. Open drains were closed 5 min prior to the first sample and remained so between samples. 1.5 mL of CSF was drawn and discarded prior to each sample. After the burn-in period of eight patients with only R-samples, all patients were allocated to R-samples or C-samples for each sample session. CSF was analyzed for cell counts, lactate, glucose, and albumin by the Karolinska University Hospital Laboratory. The laboratory utilizes machine cell counting using the Sysmex XN10 system. Sampling was performed by a registered nurse. Information on primary diagnosis, length of stay, sex, Glascow Coma Scale (GCS), GCS motor response, and antibiotic treatment were collected. The intraventricular blood volume was measured from CT scans using the MPR volume tool in the Sectra IDS7 system (version 21.1.1.1997) as well as estimated using the intraventricular hemorrhage score (IVHS) by Hallevi et al. . The shoThe cell count components of the Karolinska University Hospital  EVDI definitions were used to denote suspected EVDI status for each individual sample as suspected EVDI (SI) or no EVDI (NI) to identify clinical implications of any observed CSF cell count variability. The cell count component of our local guidelines is one of several metrics used to identify suspected infections and guide antibiotic treatment in wait of culture results. The cell count component of the new current Swedish national guidelines was also tested for the same purpose and is included below .CSF cellular constitution consistent with EVDI as follows: CSF-Leukocytes x|).Derived variables are defined as below. Clustered Wilcoxon Signed Rank test was used to compare the median location shift for each CSF parameter between sample 1 and 2 [Fligner-Killeen test was used to compare the variance of the derived variables between R-samples and C-samples. Mixed effects linear regression analysis was used to identify correlations between CT-derived variables and the pair-wise differences in a univariate setting with Patient ID as a random effect with random intercept, but not slope. Mixed effects logistic regression analysis was used to evaluate the effect of repositioning on pair-wise differences and to identify variables that were correlated to changes in suspected EVDI status between samples, also with Patient ID as random intercept but not slope. Outliers were identified based on Cook’s distance and removed. Fifteen R-samples and 5 C-samples had CSF cell counts below the functional sensitivity of the Sysmex XN10 system and were excluded from the mixed effects regression analyses and LE ratio calculations. Thus, 104 R-samples and 38 C-samples had the LE ratio calculated and were included in the mixed effects regression analyses.Continuous variables were described as median, interquartile range (IQR), and percentage. The LE ratio was calculated for samples with cell count levels above the lower limit of the Sysmex XN10 system  and erythrocytes  was significantly greater  between paired samples, 14 R-samples from 13 unique patients changed EVDI diagnostic group, corresponding to 30% of all patients with collected R-samples in our cohort. Eleven R-samples changed from NI to SI, and 3 R-samples from SI to NI. One patient had changes in both directions with one paired sample that changed from NI to SI, and one that changed from SI to NI. No changes in suspected EVDI status were seen for any C-samples. Two patients were initiated on broad-spectrum antibiotics due to significant pleocytosis in the second sample. No patient had a positive CSF culture confirmed EVDI in this study.Repositioning as a cause of suspected EVDI group change could not computationally be evaluated with logistic regression as all positive events occurred solely for R-samples. Repositioning was, however, significantly correlated with suspected EVDI group change in a chi-squared test , changes were generally bidirectional. Bidirectional changes make theoretical sense, as blood can be shifted both away and toward the catheter in the intraventricular space. Repositioning, and thus patient movement, was also found significantly related to changes in cell count, further compounding that of serial sampling. Moreover, changes, and direction of changes, of cell levels could not be readily predicted from our CT-derived variables, further suggesting that an initial heterogeneity of the sample space leads to random direction of change. In aggregate, it is clear from this study that a basic assumption of EVDI surveillance diagnostics that cells are homogeneously distributed in the CSF is erroneous and that resampling and a simple routine patient repositioning both result in changes in sampled CSF cell counts, impacting early identification of EDVIs.This study provides insight to the assumption of homogeneity. We found substantial cell variability between serially drawn CSF samples with the largest difference observed being -3505*10The potential impact of variable CSF cell counts on EVDI surveillance diagnostics is readily seen in this study where 30% of repositioned patients changed EVDI diagnostic status in the course of 10 min based on the cell count criteria for suspected infection. We also noted that variability increased with increasing cell counts levels (heteroscedasticity) such that large changes are found near to diagnostic cutoff levels. This heteroscedasticity, as shown in Fig. The LE ratio, or variations thereof based on the rationale that the number of leukocytes is related to the amount of blood in the non-infected state, is one of the methods used in EVDI surveillance diagnostics. We observed significant changes of the LE ratio when resampling. The changes in cell counts were bidirectional and independent between cell types. Repositioning exacerbated changes in the LE ratio, but we were not able to predict neither the magnitude nor the direction of the change which makes any variability difficult to adjust for in a clinical setting. While changes in the LE ratio were more stable compared to individual cell types, the variability increased here also with higher ratios in the first sample Fig.  . ApproprHistorically, suspected EVDI and verified EVDI have been difficult to separate prior to CSF bacterial culture results based on common diagnostic EVDI surveillance criteria, in which cell counts are central, resulting in frequent over-treatment with antibiotics , 27, 28.There are several limitations to this study. This is a single-center clinical trial and the external validity of this study may need further validation. As different centers will have different treatment regimes, routines, and algorithms concerning EVD care and EVDI surveillance diagnostics, findings may be to some extent be population and location dependent. Moreover, we have not studied the time component of cell change, where the aseptic inflammation component might be expected to increase over time, and heterogeneity might itself be related to the inflammatory process. However, as the risk of true infection also increases over time, late variability would also be expected to impact diagnostics. Additionally, our study cohort is 80% SAH patients, and power will not permit if there is a disease-related component to variability. As, we have no positive cultures we provide no information on if heterogeneity is different in true infection. Finally, our sample groups are unbalanced, mainly due to a burn-in period where all patient samples were allocated around repositioning, with by chance long ICU stays. We have attempted to account for this using mixed model analyses.Current methods of adjusting for IVH in EVDI surveillance diagnostics are based on the assumption that blood is homogeneously distributed in the CSF and that CSF sampling provides a fair representation of intraventricular blood volumes and granulocyte counts. Our findings in this prospective observational study with allocation to repositioning or not suggest that this assumption is erroneous. CSF cell counts varied greatly in serially drawn samples with repositioning also significantly contributing to change in mixed effect models. This change was seen to be in both directions and is larger at higher counts, confounding EVDI surveillance diagnostics. Importantly, one-third of the patients in our repositioned group changed suspected EVDI status in the time frame of 10 min between CSF samplings based on the cell count criteria from local and national EVDI diagnostic guidelines. Two patients who would otherwise not have been initiated on broad-spectrum antibiotics received treatment due to significant pleocytosis in the second sample. Consequently, analysis of current CSF parameters may not be adequate to distinguish between natural aseptic inflammation and EVDIs in neurocritically ill patients. Our findings add yet more complexity into identifying and defining EVDIs prior to bacterial cultures and may explain why no routinely analyzed CSF parameter, singularly or in aggregate, have been found to reliably predict or identify EVDIs.Faster and more precise diagnostics are needed, and methods such as emerging next-generation sequencing techniques may provide tools to more timely and accurately exclude EVDIs and guide antibiotic treatment.Additional file 1: Sysmex XN10 machine cell counting.docx: Information regarding Sysmex XN10 machine cell counting and the term functional sensitivity.Additional file 2: Intraventricular Hemorrhage Score (IVHS).docx: Information regarding IVHS calculation.Additional file 3: Correlation of paired sample differences and cell types.PNG: Scatter plots comparing the granulocyte Δ with the erythrocyte Δ and the granulocyte Δ with the monocyte Δ, respectively. Granulocytes and monocytes exhibit a stronger correlation  vs. granulocytes and erythrocytes  suggesting that a greater concordance is seen for cells of similar types. Δ: pair-wise difference.Additional file 4: Computed tomography (CT)-related predictors of change between pair samples.PNG: Table with univariate mixed model linear regression analyses. The results of p < .01 are bolded. CT parameters appear most correlated with changes in erythrocytes, but not with direction of change. IVH volume, sediment depth, catheter distance, and IVHS are defined in the methods section. Δ: pair-wise difference, including absolute and relative derivatives. IVHS: intraventricular hemorrhage score. IVH: intraventricular hemorrhage.


The highly significant differences between CSF and paired peripheral blood lymphoid phenotype (p<0.0001) confirms the existence of an active mechanism of lymphoid migration through the meninges.Cerebrospinal fluid (CSF) flow cytometry has a crucial role in the diagnosis of leptomeningeal disease in onco-hematology. This report describes the flow cytometry characterization of 138 CSF samples from patients affected by non-Hodgkin lymphoma, negative for disease infiltration. The aim was to focus on the CSF non-neoplastic population, to compare the cellular composition of the CSF with paired peripheral blood samples and to document the feasibility of flow cytometry in hypocellular samples. Despite the extremely low cell count  the study was successfully conducted in 95% of the samples. T lymphocytes were the most abundant subset in CSF  with a predominance of CD4-positive over CD8-positive T cells (CD4/CD8 ratio = 2) together with a minority of monocytes . No B cells were identified in 90% of samples. Of relevance, a normal, non-clonal B-cell population was documented in 5/7 (71%) patients with primary central nervous system lymphoma at diagnosis ( Neoplastic meningitis is a dramatic complication in cancer patients and the diagnosis of leptomeningeal metastasis represents one of the greatest challenges in neuro-oncology. Cerebrospinal fluid (CSF) analysis has a key role in routine clinical practice however conventional cytology, the gold standard for cell type identification, has considerable limitations regarding sensitivity and specificity, with a reported false-negative rate of up to 40% , 2.In recent years, several studies have demonstrated that CSF flow cytometry is superior to conventional cytology for detection of CNS involvement in non-Hodgkin lymphomas, acute leukemia and multiple myeloma –11. TherHowever, cancer cells represent only a proportion, often a minority, of the CSF population in neoplastic meningitis. A significant presence of lymphocytes has been documented, together with floating malignant cells, in CSF samples from patients with non-Hodgkin lymphomas and breast cancer leptomeningeal metastasis , 19; an Focusing on non-neoplastic populations, we report here the immunophenotype of the CSF leukocytes of patients affected by non-Hodgkin lymphomas without leptomeningeal involvement. The aim was to document the feasibility of flow cytometry in normal, thereafter, extremely hypocellular samples, to document the immunophenotype of CSF non-neoplastic population in non-Hodgkin lymphoma, to compare the cellular composition of the CSF with paired peripheral blood samples. Moreover, a possible correlation between the CSF lymphocyte subpopulations and diagnosis was evaluated.From March 2010 to December 2015 a cohort of 138 samples with non-Hodgkin lymphoma who underwent diagnostic lumbar puncture according to the routine clinical practice entered the study . All PCNth 2020.All CSF samples were analyzed by cytology and flow cytometry and had no evidence of infiltration. Patients with a positive diagnostic lumbar puncture due to CSF infiltration by pathological cells were excluded from this analysis. The Central Ethical Committee IRCCS Lazio, Section I.F.O. approved this retrospective study. Protocol n° 0009524, July 27CSF was collected in a tube without any transport medium or anticoagulant and processed within 1 to 3 h to minimize cell loss. To avoid peripheral blood contamination, the first 0.2 to 0.4 ml (four to eight drops) of CSF were discarded before sample collection. A standard cell count was performed using the Turk reagent and a Nageotte chamber. CSF was spun at 1,400 rpm for 5 min, the supernatant fluid was discarded and the cell pellet was suspended in 500 µl of phosphate buffered saline (PBS): 100 µl of cell suspension was used for cytomorphology and 100 µl/tube for the flow cytometric study.Cytospins were prepared using a Shandon CytoSpin cytocentrifuge. Morphological examination was performed by expert cytopathologists using May–Grünwald–Giemsa staining. All cases were morphologically negative for CNS localization.CSF samples were processed and stained using a 6-color monoclonal antibodies panel, 5 µl of each, and the “Duo-lyse” program of the Becton Dickinson Bioscience (BDB) Lyse-Wash-Assistant according to the following combinations: tube 1) CD3Fitc , CD56Pe , CD45PerCP-Cy5.5 , CD4PE-Cy7 , CD19APC  and CD8APC-Cy7 ; tube 2) CD5Fitc , CD10Pe , CD45PerCP-Cy5.5, CD2PE-Cy7 , CD79bAPC  and CD20APC-Cy7 ; tube 3) anti-Lambda Fitc , CD45PerCP-Cy5.5, CD34PE-Cy7 , CD22APC  and CD14APC-Cy7 . All antibodies were from BDB. Prior to sample acquisition, a flow cell cleaning with FACS flow (for 1 to 2 min run) was performed to avoid any event carry over. The whole volume of sample was acquired and analyzed using the FACSCanto II 2L flow cytometer and the FACSDiva software Version 6.1.3 (BDB). Single-stained cellular controls, BD FACS™ 7-color setup beads and BD FACSDiva CS&T IVD Beads have been used to adjust detector voltage, to set fluorescence compensation and to monitor instrument performance.Data are presented as the percentage of positive cells evaluated on the CD45-positive population. Lymphocytes were identified by CD45-strong/side scatter (SSC)-low. The CD4 and CD8 subsets were evaluated as a percentage of CD3-positive T lymphocytes. Monocytes were identified using the CD4-weak or CD14 staining. Neutrophils as CD45/SSC-high. Surface immunoglobulins (Ig) kappa and lambda light chain expression was evaluated on CD22-positive B cells. In agreement with the recommendations for the analysis of rare events, a cluster of 10 events was considered to define a positive result and identify a leukocyte subpopulation , was conducted in 104 paired cases.p-value of <0.05 indicating a statistically significant difference. All statistical analyses were performed using SPSS (version 21.0).Qualitative items were reported as absolute and percentage counts, while quantitative variables were summarized using mean and standard deviation, median and range. The difference in distribution between CSF and PB lymphoid subpopulations was assessed by Wilcoxon rank-sum test. Association between variables was evaluated with the Spearman’s ρ coefficient. The test was two-sided with a A cohort of 138 samples from 127 non-Hodgkin lymphoma patients, all negative for CNS disease involvement, entered the study Table 1.The study focuses on 107 cases; 24 cases (17%) were not included in analysis due to peripheral blood contamination of the CSF documented by the identification of red blood cells in the cytospin assessed morphologically as well as a population of CD45/SSC high  positive neutrophils. In seven cases the flow cytometry analysis was not evaluable due to the absence of clustered events.A median volume of 4.0 ml (range 2.0–12.0) of CSF was available for flow cytometry analysis. The CSF cell count was extremely low ; in 9 cases (8%) the CSF cell count was higher than the normal reference value of 4 cell/µl, with a median value of 22 cells/µl (5.0–35). Despite the low absolute cell number, flow cytometry characterization was successfully conducted in 95% of cases (131/138).p<0.001)  events were acquired and analyzed. A positive correlation was found between the volume (ml) of CSF and the number of events analyzed by flow cytometry  Figure 1The CSF population was represented by lymphocytes  together with a minor population of monocytes .The CSF lymphoid population was represented by CD2 CD3 CD5-positive T cells  with a prevalence of CD4-positive lymphocytes (CD4/CD8 ratio = 2). A minority of CD56-positive cells were also documented . No B cells (< 10 clustered events) were identified in 90% of cases Table 2.p<0.0001).In 11 patients a subpopulation of CD19 CD20 CD22 CD79b-positive B lymphocytes , with a normal/balanced Ig kappa/lambda ratio evaluated on the CD22-positive population, was identified: 5 PCNSL, 5 diffuse large B-cell lymphoma (DLBCL), 1 follicular lymphoma Table 3 p<0.0001)  and compared to the corresponding CSF lymphoid subpopulations. The absolute number of lymphocytes was 1100 cell/µl (range 70–3300), the median percentage was 19% (range 2–76). The analysis documented a population of CD3-positive cells  with a CD4/CD8 ratio of 1.4, CD56-positive  and CD19-positive  lymphocytes. A different distribution of CD3 CD56 and CD19 lymphoid subpopulations was observed between CSF and corresponding peripheral blood samples (<0.0001) Table 2 Leptomeningeal metastasis represents one of the greatest challenges in neuro-oncology and CSF is one of the most promising diagnostic tissues utilized in routine clinical practice , 24. In The normal cell count of the CSF in adults is up to 4 cells/μL and CSF cell count is a diagnostic criterion for leptomeningeal infiltration. The median cellularity in this cohort of patients was extremely low , but nevertheless flow cytometry was successfully conducted in 95% of cases, confirming its role of a highly sensitive and specific technique for detection of rare cell sub-populations, even in samples with as few as 1 leukocyte per µl of sample. Of note, in 8% of samples (n=9) the CSF cell count was significantly higher than the normal reference value (median 22 cell/µl). In these cases, unequivocal identification of the cell population was mandatory to exclude false positive interpretation: therefore, in addition to cytology, flow cytometry becomes essential. Moreover, the role of an increased number of lymphocytes in the CSF of non-Hodgkin lymphomas patients deserves to be investigated.p < 0.001) and taking into account the potentially extremely low cell count of the CSF, as well as some cell loss related to the staining technique, we recommend the withdrawal of not less than 4 ml of sample to ensure an adequate number of events for a reliable identification of minimally represented sub-populations. Moreover, peripheral blood contamination of the CSF, due to difficulty in the execution of the lumbar puncture, can occur. False positive results represent the major pitfall in all cases with peripheral blood infiltration by leukemic/lymphoma cells and blood contamination of the CSF (author statement manuscript in preparation) (High sensitivity has been reported utilizing a volume of 2.0 ml of CSF for flow cytometry characterization . The prearation) . Thereafp < 0.0001). This different distribution documents that the brain barrier actively selects a sub-population of T lymphocytes, supporting the involvement of the meningeal lymphatic network in lymphoid cell migration into the meninges as a potential alternative route to the cardiovascular system. This finding documents the existence of an active mechanism of lymphocyte localization and provides a promising rationale for the investigation of cellular immunotherapy in brain diseases.Studies conducted on normal CSF observed that the vast majority of leucocytes is represented by central memory T lymphocytes with significantly higher percentage compared to blood. The proportion of CD56-positive cells is low while B cells are almost absent (<1%) –30. Thisratio, evaluated on the CD22-positive population, was crucial for reporting the flow cytometry as negative for infiltration by clonal B cells. A possible correlation between the CSF lymphocyte subpopulations and diagnosis was evaluated. A normal, non-clonal B-cell subpopulation was identified at diagnosis in 71% (5/7) CSF samples of patients with PCNSL (p < 0.0001). In contrast with its low frequency in normal CSF and systemic non-Hodgkin lymphomas, the identification of a subpopulation of B cells in the CSF samples of PCNSL cases raises the question of a possible role of blood-brain barrier cell permeability in the pathogenesis of cerebral B-cell lymphomas. Due to the small number of PCNSL cases evaluated, validation on a larger cohort of patients is warranted to confirm this finding and to investigate the role of the CSF B cells in the pathogenesis and diagnosis of the B-cell lymphomas of the brain. A small clonal B‐cell population has been described in the CSF of patients with B‐cell lymphoproliferative disorders and multiple sclerosis suggesting that this finding is not diagnostic of clinically significant involvement of the CNS by lymphoid malignancy  T lymphocytes, monocytes represent the second most common leukocyte population of the CSF . Origin and turnover of this medium-size population, well represented in the CSF, is still largely unexplored. The role of monocytes regarding the CNS cellular immune surveillance and their involvement in onco-hematological meningitis deserves in-depth studies and attention.The cellular composition of the CSF in non-Hodgkin lymphoma patients negative for leptomeningeal involvement differs profoundly from peripheral blood regarding all major lymphocyte subpopulation. CSF cells are represented by T lymphocytes, in prevalence CD4-positive, and monocytes. B cells are rare and this analysis reveals a possible link with PCNSL. This real-life study confirms the critical role of flow cytometry in routine clinical practice for unequivocal characterization of CSF populations, even in samples with an extremely low cell count. The identification of clusters of normal T cells in the CSF represent a reliable internal quality control of the technique and the significant difference between CSF and paired peripheral blood lymphoid phenotype provides evidence of an independent cerebral lymphatic system. CSF is not an immune-privileged site anymore but a virtual secondary lymphoid organ. An in-depth knowledge of the function and role of the CSF immunological sanctuary is highly needed and has the potential to revolutionize the management of CNS diseases.https://gbox.garr.it/garrbox/index.php/s/6BY66PX6n3LaYZP).The datasets analyzed for this study can be found in the GARRbox (The Central Ethical Committee IRCCS Lazio, Section I.F.O. approved this retrospective study. Protocol no 0009524, July 27, 2020. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.IC: designed the study and draft the initial manuscript. SM and APas: flow cytometry studies. IC and SM: analysis, data collection, and quality control. DG: performed the statistical analysis and manuscript revision. MM: histopathological diagnosis and manuscript revision. ST, APac, AM, LC, EP, and PF: patient’s clinical management and critical manuscript revision. 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.


Xenopus laevis frog is an ideal research platform upon which to study the roles of distinct frog leukocyte populations during FV3 infections. Frog macrophages (MΦs) are integrally involved during FV3 infection, as they facilitate viral dissemination and persistence but also participate in immune defense against this pathogen. In turn, MΦ differentiation and functionality depend on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by CSF-1 and iterleukin-34 (IL-34) cytokines. Our past work indicated that X. laevis CSF-1 and IL-34 give rise to morphologically and functionally distinct frog MΦ subsets, and that these CSF-1- and IL-34-MΦs respectively confer susceptibility and antiviral resistance to FV3. Because FV3 targets the frog kidneys and establishes chronic infections therein, presently we examined the roles of the frog CSF-1- and IL-34-MΦs in seeding and maintaining these chronic kidney infections. Our findings indicate that the frog CSF-1-MΦs result in more prominent kidney FV3 infections, which develop into greater reservoirs of lingering FV3 marked by infiltrating leukocytes, fibrosis, and overall immunosuppressive states. Moreover, the antiviral effects of IL-34-MΦs are short-lived and are lost as FV3 infections progress.Infections by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to global amphibian decline. The  Iridoviridae) are contributing to global amphibian population declines [The diseases and population die-offs associated with amphibian infections by Frog Virus 3 (FV3) and other members of the genus Ranavirus  were purchased as tadpoles from Xenopus1  and reared in-house. Animals were housed in an XR5 aquatic housing unit (IWAKI) with flow-through filtered (carbon and sediment filtration) and conditioned water at 21 °C. All infection studies were performed in, and all infected animals were housed in a designated quarantine/infection facility at 21 °C. Infected animals were housed in carbon- and sediment-filtered standing water in groups according to treatment. All animals were housed and handled under strict laboratory and IACUC regulations .X. laevis recombinant (r)CSF-1 and rIL-34 were produced as previously described [escribed . BrieflyThe recombinant control (r-ctrl) was generated by transfecting Sf9 cells with the empty pMIB/V5 His A expression vector (Invitrogen), grown and processed in parallel with rCSF-1 and rIL-34 production.2 for 5 days. The FV3-containing supernatants were pelleted through 30% sucrose by ultracentrifugation, re-suspended in saline, and viral titers were measured by plaque assays using BHK-21 cells.FV3 was generated following previously described methods . Brieflyn = 15 for infection studies or n = 6 for gene expression studies) were intraperitoneally injected with 2.5 μg of rCSF-1 or rIL-34 in APBS or equal volumes of recombinant control (r-ctrl). After 3 days . Animal mortalities were monitored, frogs were sacrificed after 3-, 7-, 14-, 21-, or 28-days post infection (dpi), and their kidney tissues were collected for histology and DNA or RNA isolation.MΦ enrichments and FV3 infections were performed as follows. Adult frogs , theFor all experiments, kidney tissues were collected in Trizol reagent (Invitrogen) and stored at –80 °C until RNA and DNA isolation. Kidney tissues were homogenized by passage through progressively higher gauge needles and RNA was isolated according to the manufacturer’s instructions. DNA isolation was performed subsequent to RNA isolation. The DNA-containing layers were mixed with back extraction buffer , followed by centrifugation to collect DNA containing aqueous layer. The DNA was precipitated with isopropanol overnight at −20 °C. The next day, the DNA was pelleted by centrifugation, washed with 75% ice cold ethanol, and re-suspended in a TE  buffer. The DNA was then purified by phenol:chloroform extraction and re-suspended in molecular grade water. Both RNA and DNA concentrations were measured using NanoDrop .gapdh endogenous control and normalized against the lowest observed expression.Quantitative analysis of gene expression and FV3 DNA copy number has been previously described ,25,26. I1–108) FV3 vDNA Pol fragment-containing plasmid , using 500 ng of isolated DNA from respective samples.Absolute qPCR analysis of FV3 DNA copy number was performed in comparison to serially diluted . All primers used in this experiment were validated before use and all primer sequences are listed in 5 PFU/frog; n = 5 per treatment group, per time point). After 7 and 21 days post infection (dpi), the animals were sacrificed, their kidneys were ascetically removed, homogenized in DMEM, serially-diluted and examined for FV3 loads by plaque assays, as described above.Adult frogs were stimulated with rCSF-1, rIL-34 (2.5 μg/frog) or equal volumes of r-ctrl and three days later infected with FV3  test via GraphPad Prism 7.0 software, and found to be not statistically significant.Statistical analyses of the gene expression were conducted using one-way analyses of variance (ANOVA) with a post-hoc Tukey’s test. All statistical analysis was performed using R Statistical Software (version 2.14.0) or GraphPad Prism 7.0 software and X. laevis frogs may be enriched for CSF-1- or IL-34-MΦs by injecting these animals with recombinant (r)CSF-1 or rIL-34, respectively [X. laevis) tend to succumb to FV3 infections, adult frogs generally clear FV3 infections without extensive mortalities [We previously established that ectively . Whereasectively . Notablytalities ,32,33,34talities , we wereCSF-1- and IL-34-MΦ enrichments affect a number of frog tissues, chiefly amongst them the frog kidneys ,22,24, wOur plaque assay analysis of MΦ-enriched, FV3-infected frog kidneys supported the above findings. Notably, at seven dpi, CSF-1-MΦ-enriched animals possessed significantly greater infectious FV3 particles than the r-ctrl-injected animals, whereas the IL-34-MΦ-enriched frogs had significantly lower FV3 loads in their kidneys B. After icp18), delayed early (rad2) and late (mcp) genes than the kidneys of CSF-1-MΦ-enriched frogs at three dpi and the loss of this protection with infection time  receptor gene , proinflammatory (tnf) and immunosuppressive  cytokine genes  cytokine genes . ChieflCCL19 and CCL21, which were also upregulated in chronically infected CSF-1-MΦ-enriched frogs, are particularly known for homing of T cells and dendritic cells to lymphoid tissues, but also perform several other functions including regulatory and memory T cell function . It is pWe observed extensive infiltration of both granulocytes and MΦs into the kidneys of CSF-1-MΦ-enriched, FV3 infected frogs, which coincided with more extensive pathology therein, as compared to the kidneys of control- and IL-34-MΦ-enriched animals. This is consistent with what has been observed across other vertebrates, where both granulocyte- and MΦ-lineage cells may invade infected tissues, exacerbating tissue damage . NotablyMany elements of these studies are specific to amphibian innate and antiviral immunity and provide insight into the roles of frog MΦ subsets during chronic ranavirus infections. It is, however, notable that the dichotomy of CSF-1- and IL-34-MΦ functionality appears to be evolutionarily conserved ,48. As s


Firstly, we would like to thank Drs. Keil and Fludder for their interest in our study.In Denmark, many children consult a chiropractor within their first year of life . We are not sure how this relates to the first statement.BK and CF states “BK and CF mention an area of interest that is not covered in the present publications: which joints/areas were treated. We agree that this is a very interesting secondary analysis, and we hope to publish that in a subsequent publication.The conclusion applies only to chiropractors without recognized post-graduate paediatric training.” That is both correct and intentional. All chiropractors in Denmark are licensed to treat infants based on their university-based education. Although many of these chiropractors have an interest in paediatrics and have attended several seminars etc., only few have formalized post-graduate training. Therefore, we believe the choice of chiropractors increases the relevance to current practice and thus disagree with the statement that the study “has minimal relevance to current practice and training”. The situation might be different in Australia, and if there are specialized chiropractors broadly available across the country, we would welcome an opportunity to replicate the study among those.Finally, they point out that “