Cryptococci from CSF, and survival at 2 weeks. The key components of this protective immune-response were interleukin (IL)-6 and interferon-γ, IL-4, IL-10 and IL-17 levels also made a modest positive contribution to the PC1 score. A second component of co-correlated chemokines was identified by PCA, consisting primarily of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). High CSF chemokine concentrations were associated with low peripheral CD4 cell counts and CSF lymphocyte counts and were predictive of immune reconstitution inflammatory syndrome (IRIS). In conclusion CSF cytokine and chemokine profiles predict risk of early mortality and IRIS in HIV-associated CM. We speculate that the presence of even minimal Cryptococcus-specific Th1-type CD4+ T-cell responses lead to increased recruitment of circulating lymphocytes and monocytes into the central nervous system (CNS), more effective activation of CNS macrophages and microglial cells, and faster organism clearance; while high CNS chemokine levels may predispose to over recruitment or inappropriate recruitment of immune cells to the CNS and IRIS following peripheral immune reconstitution with ART. These results provide a rational basis for future studies of immune modulation in CM, and demonstrate the potential of baseline immune profiling to identify CM patients most at risk of mortality and subsequent IRIS.Understanding the host immune response during cryptococcal meningitis (CM) is of critical importance for the development of immunomodulatory therapies. We profiled the cerebrospinal fluid (CSF) immune-response in ninety patients with HIV-associated CM, and examined associations between immune phenotype and clinical outcome. CSF cytokine, chemokine, and macrophage activation marker concentrations were assayed at disease presentation, and associations between these parameters and microbiological and clinical outcomes were examined using principal component analysis (PCA). PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting primarily of Th1, Th2, and Th17-type cytokines. The presence of this CSF cytokine response was associated with evidence of increased macrophage activation, more rapid clearance of  Cryptococcal meningitis is a severe opportunistic infection, estimated to kill several hundred thousand HIV-infected individuals each year. One of the factors contributing to this high death toll is the inadequacy of antifungal treatments. As few novel antifungal drugs are being developed, several groups have started to investigate the potential of immune modulation, with treatments designed to change the patient’s immune response to infection. However, our understanding of the immune response to cryptococcal infection in HIV-infected patients, and how these responses impact on clinical outcomes, is limited. In this study, we took advantage of the fact that we can sample cerebrospinal fluid (CSF) from the site of the infection in patients when they develop cryptococcal meningitis. We undertook a detailed analysis measuring levels of immune response parameters in the CSF of these patients, and demonstrated that there were several distinct components of the immune response. Variations in these responses were associated with both the rate at which patients cleared their infection during treatment, and with mortality. Our results provide a basis for the development of future immunomodulatory therapies, and may allow identification of patients most at risk of dying, enabling more intensive treatments to be given to those at highest risk. Cryptococcal meningitis (CM) is a leading cause of mortality in HIV-infected patients in low-resource settings , 2. CurrCryptococcus is derived from in-vitro and animal experiments. In these model systems effective immunity is dependent on robust CD4+ T-cell immune responses, the production of Th1-type cytokines such as interferon-γ (IFNγ), and “classical” activation of effector cells such as macrophages [+ T-cell responses and cytokines appear to play a protective role [Much of our understanding of the immune response to rophages –17, leadive role –21, whilive role –26.+ T-cell mediated immunity in the control of cryptococcal infection [+ T-cell response to Cryptococcus neoformans influences the outcome of CM [Human data are very limited. Epidemiological evidence clearly points to the importance of adequate CD4nfection , 28, andme of CM , 29. Theme of CM , and in me of CM , for effCryptococcus during HIV-associated CM we measured cytokine concentrations, chemokine concentrations, and levels of macrophage activation markers in the cerebrospinal fluid (CSF) of ninety patients with CM enrolled in a clinical trial investigating the utility of short-course adjuvant IFNγ therapy [To characterize the immune response to  therapy . The phe+-cell counts performed at study enrollment . Patients were started on antiretroviral therapy consisting of stavudine/lamivudine/efavirenz at 2 to 4 weeks post commencement of antifungal therapy, and followed for one year. Mortality outcomes and the occurrence of IRIS (diagnosed according to a standardized definition [Ninety HIV-positive adults (age ≥ 21 years) with a first episode of cryptococcal meningitis, diagnosed by CSF India ink or cryptococcal antigen testing , were enrolled sequentially between July 2007 and May 2010 into a clinical trial examining the effect of two different schedules of short-course adjuvant interferon-γ immunotherapy on the treatment of HIV-associated cryptococcal meningitis in Cape Town, South Africa . The stufinition ) were reCSF samples collected at day 1, 3, 7 and 14 were centrifuged, and the supernatant frozen at -80°C for subsequent quantification of cytokine concentrations. CSF IFNγ, tumor necrosis factor-α (TNFα), interleukin (IL)-2, IL-4, IL-6, IL-8 (chemokine (C-X-C motif) ligand 8), IL-10, IL-12p70, IL-17, IL-21, IL-22, IL-23, monocyte chemotactic protein-1  ligand 2), macrophage inflammatory protein-1α  ligand 3), RANTES (chemokine (C-C motif) ligand 5), granulocyte-macrophage colony-stimulating factor , and vascular endothelial growth factor (VEGF) concentrations were measured in all patients using the Luminex multianalyte platform (Luminex) and Bio-Rad cytokine kits (Bio-Rad) . The mac+-cell count, CSF lymphocyte count, baseline fungal burden, rate of clearance of infection, and macrophage activation marker concentrations were examined using Pearson’s correlation coefficient. Associations between PCs and rate of clearance of infection (but not variables recorded at baseline) were adjusted for treatment group. Differences in PC scores between patients who survived and died, and those who did and did not develop IRIS, were examined using a linear regression model adjusting for treatment group, CD4+-cell count, and the previously described risk factors for mortality, baseline fungal burden and altered mental status [2 tests, or Fisher’s Exact tests as appropriate. Comparisons of paired groups were made using the Wilcoxon matched pairs test. Permutation tests, with 5000 permutations, were used to control the family wise-error rate (FWER) for the fifteen correlations / partial correlations between the cytokines IL-4, IL-10, IL-17, MCP-1 and baseline and outcome variables. Permutation tests used the max statistic for adjusting the p-values to ensure FWER of 0.05 while allowing for the potentially strong correlations between variables. Unadjusted p-values for those associations that maintained a FWER<0.05 across the multiple comparisons are reported. Support vector machine (SVM) learning and decision tree analyses were used to assess the capacity for baseline CSF cytokine measurements to predict mortality or IRIS. The support vector machines algorithm was implemented in R v2.15.1 using the kernlab package and ksvm function with a linear kernel (vanilladot), cost 1 and leave one out cross validation. Decision tree classification was performed using Waikato Environment for Knowledge Analysis (WEKA) v 3.6.9 using different decision tree classifiers  with leave one out cross validation. For the purposes of all analyses the two IFNγ treatment groups were considered as a single “IFNγ treated” group. Statistical significance was defined as p≤0.05.Data were analysed using Stata version 13.0 , R version 3.0.2 , and Graphpad Prism version 6 . Baseline CSF cytokine and chemokine concentrations were analysed using principal component analysis (PCA), a method of reducing complex correlated datasets into a series of linear, non-correlated “principal components” (PCs) and avoiding multiple comparisons. The first principal component accounts for as much of the variability in the data as possible, and each subsequent component accounts for the highest variance possible under the constraint that it is uncorrelated with preceding components , 35. Forl status . A sensiThe study was approved by the Human Research Ethics Committee of the University of Cape Town and St. George’s University of London. Patients gave written informed consent for blood and CSF samples to be used for research purposes.+ cell count was 27 cells/μL (IQR 14–51). All patients were treated with amphotericin B 1mg/kg plus flucytosine 100mg/kg daily for fourteen days. Twenty nine patients received adjunctive IFNγ 100μg subcutaneously on days 1 and 3, and thirty patients received IFNγ 100μg subcutaneously thrice weekly for the first two weeks of therapy. Mortality at 2-weeks was 16% (14). All patients were ART naïve at study enrollment, and ART was initiated at a median of 23 days post initiation of antifungal therapy. IRIS occurred in 14% of those who started ART (9/65), occurring a median of 60 days post ART initiation , 44% (40) were male, and the median CD4itiation .Baseline CSF cytokine concentrations are shown in PCA indicated that co-correlated pro-inflammatory cytokines and chemokines represented by PC1 scores, and those represented by PC2 scores function as independent variables. To assess whether proinflammatory cytokines or chemokines were related to other clinically important variables in cryptococcal meningitis we explored the relationship of PC1 and PC2 with peripheral blood CD4, CSF lymphocyte counts, markers of CSF macrophage (or resident CNS macrophage like cell) activation, CSF arginase activity, baseline CSF fungal burden, rate of clearance of infection, 2-week mortality and IRIS . PC1 scoFungal burden was negatively correlated with PC1 and PC2 indicating that patients with high fungal burdens tended to have pauci-inflammatory CSF cytokine profiles with high MCP-1 and MIP-1α chemokine expression, while lower fungal burdens were associated with more robust CSF inflammatory responses and lower chemokine levels. Importantly PC1 scores showed a positive correlation with more rapid clearance of cryptococci from the CSF (an association that remained significant after adjustment for treatment group), consistent with the hypothesis that robust pro-inflammatory responses contribute to fungal clearance. Also in keeping with this observation, PC1 scores were lower in patients who had died within 2 weeks compared to those who survived . This as+-cell count, and the previously described risk factors for mortality, baseline fungal burden and altered mental status, as described above (p = 0.004), and in a linear regression model adjusting for treatment group and the known risk factors for IRIS, baseline fungal burden and CSF white cell count (p = 0.004).Amongst patients who survived, PC2 but not PC1 scores, differentiated patients who developed IRIS following the initiation of ART . PC2 sco10 CSF lymphocyte count , and associated with IRIS  and logA sensitivity analysis examining associations between PC scores and outcomes restricted to the patients in the trial control arm who did not receive adjuvant interferon-γ immunotherapy was performed. In this smaller sample, the association between PC1 and rate of clearance of infection from the CSF and mortality were no longer significant . The associations between PC2 and IRIS remained significant in both crude (p = 0.01) and adjusted analysis (p = 0.03).Taken together, our data suggest that multiparameter measurements of CSF cytokines and chemokines in patients presenting with cryptococcal meningitis may predict the risk of early mortality or development of IRIS following ART. To test whether our data could be used to predict these clinical outcomes, we employed machine-learning algorithms and leave one out cross-validation to classify each patient after training on the data from all the other patients in the study. To take advantage of high dimensional pattern recognition we used support vector machine learning; and to assess whether a minimum number of variables could classify patients correctly, decision tree analyses was used . Support+ T-cells [The phenotype of the baseline CSF immune response in patients with HIV-associated CM was shown to be associated with both microbiological and clinical outcomes, and predictive of subsequent IRIS. For the first time we have demonstrated that more robust pro-inflammatory CNS cytokine responses, characterized by higher levels of CSF IL-6 and IFNγ are associated with evidence of increased markers of innate effector cell activation, and, in keeping with previous reports , more ef T-cells , and hig T-cells  are assoCryptococcus. IL-17 concentrations were closely correlated with IFNγ levels, and associated with rate of clearance of infection and survival. Of note, IL-17 production was not observed in response to cryptococcal mannoprotein stimulation of CD4+ T-cells from a subset of patients included in this study[+ T-cells. Other cytokines associated with Th17-type T-cell responses, including IL-21, IL-22 and IL-23 were not present in detectable quantities in the majority of patients studied, further supporting this supposition. γδ+ T-cells, CD8+ T-cells, NK cells, and neutrophils are all capable of producing the cytokine [In keeping with animal models , 23, 40,his study, suggestcytokine , althougCryptococcal infection is associated with alternative activation of effector cells [Cryptococcus for some time, and had had active CNS disease for several weeks prior to presentation [We found no evidence for a detrimental Th2-type response in CM patients, nor evidence for arginase activity, which in mouse models of or cells . Levels or cells . Reasonsor cells , and argor cells , is not or cells . It alsoentation . Many ofentation , 48, andIn terms of cryptococcal IRIS, previous studies have identified poor baseline inflammatory responses, rapid immune reconstitution from this low baseline, and a high organism or antigen burdens as key risk factors . Low per+ T-cell response to Cryptococcus is associated with disease severity and outcome in HIV-associated CM [Cryptococcus-specific memory CD4+ T-cells are capable of promoting a cellular infiltrate into the CNS in response to local production of chemokines such as MCP-1 and MIP1α by microglial cells following exposure to Cryptococci. We hypothesize that these infiltrating cells then stimulate expression of pro-inflammatory cytokines by both resident glial cells and trafficked cells of myeloid and lymphoid lineages, leading to activation of microglial cells and macrophages, with resultant restriction of intracellular growth of Cryptococci, resulting in lower fungal burdens at presentation, faster clearance on infection on treatment, and improved survival. With the evolution of this protective, predominantly Th1-type inflammatory cytokine response, compensatory parallel increases in both Th2-type cytokines such as IL-4 and regulatory cytokines such as IL-10 occur. In the absence of any effective Cryptococcus-specific CD4 response, microglial cells and CNS macrophages are unable to effectively restrict cryptococcal growth, leading to high organism burdens, poor clearance of infection during treatment, and high mortality. These patients with severe immune suppression at baseline, indicated by low peripheral CD4 counts and CSF lymphocyte counts, also have markedly up regulated CNS MCP-1 and MIP1α chemokine expression, which, following immune restoration with ART, leads to an influx of inflammatory cells, excessive local inflammation, and IRIS.In a previous study we have demonstrated that the phenotype of the peripheral CD4iated CM . IFNγ an+ cell count and CSF lymphocyte count. This may suggest that the source of these CSF cytokines is infiltrating T-cells and monocytes (which may be mistaken for lymphocytes on CSF cell counts). Alternatively such incoming cells may facilitate or stimulate cytokine production by resident CNS immune cells. Interestingly a consistent and significant inverse relationship was seen between the levels of the chemo-attractant chemokines MCP-1, MIP-1α and peripheral CD4+ cell count and CSF lymphocyte count. This finding is consistent with existing data suggesting that chemokine production by glial cells and monocytes/macrophages is not significantly compromised in patients with late stage HIV-infection [+ T-cell help. Secondly, when exploring the correlations between baseline CSF immune parameters and fungal burdens it is not possible to definitively attribute causality. Whilst it seems plausible that the association between more vigorous CSF inflammatory responses and lower organism burdens means that variation in the patient’s ability to mount an effective inflammatory response plays a key role in determining disease severity, it is possible that very high organism burdens paradoxically lead to down regulation of the host inflammatory responses. Finally, these data were derived from a clinical trial comparing differing three treatment regimens leading to different rates of fungal clearance . The cytokine and chemokine levels used in these analyses were all determined prior to the administration of any antifungal therapy, meaning all baseline comparisons are unaffected by treatment group. And in all analyses relating to rate of clearance of infection, IRIS, and mortality outcomes, adjustment for treatment group was made. In each of these cases the associations seen in unadjusted analysis remained robust, or were strengthened following adjustment, supporting the validity of our findings. The sensitivity analysis restricted to the study control group (i.e. those who did not receive adjuvant interferon-γ immunotherapy) was underpowered as a result of the small sample size. However even in this small sample, although the mortality associations were no longer significant, the association between PC2 and IRIS remained statistically robust.Despite the prospective nature of our study, with comprehensive clinical and immunological data, there are a number of limitations. Firstly, the cellular source of the CNS cytokine production is not possible to elucidate from these data. Unlike in previous smaller studies , 51, levnfection , and tha+ T-cell counts and CSF lymphocyte counts at disease presentation had high CNS chemokine expression, and were at high risk of subsequent IRIS following ART initiation.In conclusion, the presence of a CSF inflammatory response consisting of Th1, Th2 and Th17 type cytokines has been shown to correlate with markers of innate effector cell activation, more rapid clearance of infection and survival in patients with HIV-associated cryptococcal meningitis. We did not find evidence of a dichotomous Th1/Th2 response. Rather Th1, Th2 and Th17 type cytokine concentrations correlate closely with each other, and the presence of this combined inflammatory response is beneficial, while its absence is detrimental. Patients with low peripheral CD4S1 Figth centile, are shown on days 1, 3, 7, and 14. No significant differences in the change in cytokine levels between days 1 and 3 were seen in the interferon-γ treated patients compared to controls. Between day 1 and day 7 there were larger reductions in IL-6, IL-17, RANTES and VEGF concentrations in IFNγ treated patients than controls, mirroring the more rapid decline in fungal burden .Median chemokine and cytokine levels, with error bars to 75(TIFF)Click here for additional data file.S2 FigThe associations between PC1 and baseline CSF IL-6, IFNγ, and IL-8 concentrations; and PC2 and baseline CSF MCP-1, MIP-1α, and GM-CSF concentrations.(TIFF)Click here for additional data file.S1 Data File6/L), baseline fungal burden , rate of clearance of infection , day 1 CSF opening pressure (cm H2O), mortality at 2 weeks, antiretroviral initiation status, immune reconstitution inflammatory syndrome (IRIS), and day 1, 3, 7, and 14 CSF levels of interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), interleukin (IL)-2, IL-4, IL-6, IL-8 (chemokine (C-X-C motif) ligand 8), IL-10, IL-12p70, IL-17, IL-21, IL-22, IL-23, monocyte chemotactic protein-1  ligand 2), macrophage inflammatory protein-1α  ligand 3), RANTES (chemokine (C-C motif) ligand 5), granulocyte-macrophage colony-stimulating factor , vascular endothelial growth factor (VEGF), soluble CD14 (sCD14) and neopterin concentrations (log10picograms/mL), and enzymatic activity of arginase.Variables included are adjuvant treatment category (interferon gamma administered), CD4 cell count (cells/μL), abnormal mental-status (defined as a Glasgow Coma Score <15 or seizures), CSF lymphocyte count (x 10(DTA)Click here for additional data file.


Due to their low CNS penetrance, there are concerns about the capacity of non-conventional PI-based ART  to preserve neurocognitive performance (NP).We evaluated the NP change of aviremic participants of the SALT clinical trial  switchin2=0.16) on NP evolution was similar to TT (reference), even after adjusting  by significant confounders  (r2=0.25). A total of 158 patients were included . They haNP stability was observed after 48 weeks of follow up in the majority of patients whether DT or TT was used to maintain HIV-suppression. Incidence rates of NP impairment or NP impairment recovery were also similar between DT and TT.


The difference was almost completely the result of the spinal CSF volume reduction , while the cranial CSF volume was not considerably altered . This report indicates that LP and VP shunts in IIH do not considerably change the cranial CSF volume, while the reduction of CSF volume after LP shunt placement affects almost exclusively the spinal part of the CSF system. Our results suggest that an analysis of both the cranial and the spinal part of the CSF space is necessary for therapeutic procedures planning and for an early recognition of numerous side effects that often arise after shunts placement in IIH patients.Lumboperitoneal (LP) and ventriculoperitoneal (VP) shunts are a frequent treatment modality for idiopathic intracranial hypertension (IIH). Although these shunts have been used for a long time, it is still not clear how they change the total craniospinal CSF volume and what portions of cranial and spinal CSF are affected. This report for the first time presents the results of a volumetric analysis of the total cranial and spinal CSF space in a patient with IIH. We performed an automated segmentation of the cranial and a manual segmentation of the spinal CSF space first with an LP shunt installed and again after the LP shunt was replaced by a VP shunt. When the LP shunt was in place, the total CSF volume was smaller than when the VP shunt was in place (222.4 cm Idiopathic intracranial hypertension (IIH) is a condition characterized by elevated intracranial pressure without evidence of structural intracranial abnormalities ,2. Altho2O. On the basis of all the findings and clinical symptoms the patient was diagnosed with IIH (A previously healthy child was hospitalized at the age of 6 years due to headache, nausea, vomiting, and loss of vision. A brain MR scan did not show any structural abnormalities, and all standard laboratory tests were in a physiological range. Lumbar CSF pressure in a lateral decubitus position was 32 cm Hwith IIH . No signWe performed a volumetric analysis using automated segmentation of the cranial CSF volume on high-resolution T1 slices analyzed by CIVET 1.1.11 software  (Montrea3, which is considerably less than normal (3, which falls within the normal range (3, but the spinal CSF volume increased to 104.9 cm3 (3 smaller when the LP shunt was in place than when the VP shunt was in place. This difference was a consequence of a reduced spinal CSF volume, while the cranial CSF volume remained the same. Although the intracranial subarachnoid space was somewhat reduced after LP shunt placement (Volumetric analysis of the CSF space showed that LP shunting induced CSF overdrainage predominantly in the spinal part. The spinal CSF volume was 49.3 cmn normal ,11. Howeal range . Interes04.9 cm3 . Thus, tlacement , and thelacement , in bothThe presented results suggest that LP shunts reduce the spinal CSF volume, while VP shunts keep the cranial and spinal CSF volume in the physiological range. As after the replacement of the LP with a VP shunt, our patient’s condition improved, it seems that normal cranial and spinal CSF volumes are the preconditions for a good clinical outcome in IIH.The observed CSF volume changes in our patient can be partially explained by different biophysical characteristics of the cranial and spinal intradural space -16 and bOur results indicate that VP and LP shunts differently affect the cranial and spinal CSF volume in patients with IIH. This is clearly demonstrated by the overdrainage induced by the LP shunt placement, when only the spinal portion of the CSF was reduced while the cranial portion remained unaffected. Our results emphasize the importance of a total cranial and spinal CSF space evaluation before neurosurgical procedures or during postoperative follow-up, which has been rarely performed so far. Such an evaluation could enable the selection of appropriate therapeutic procedures and early recognition of numerous side effects that occur after the placement of different types of shunts.


Muscular dystrophies (MD) are heterogeneous group of diseases characterized by progressive muscle dysfunction. There is a large body of evidence indicating that angiogenesis is impaired in muscles of MD patients. Therefore, induction of dystrophic muscle revascularization should become a novel approach aimed at diminishing the extent of myocyte damage. Recently, we and others demonstrated that administration of granulocyte colony-stimulating factor (G-CSF) resulted in clinical improvement of patients with neuromuscular disorders. To date, however, the exact mechanisms underlying these beneficial effects of G-CSF have not been fully understood. Here we used flow cytometry to quantitate numbers of CD34+ cells, endothelial progenitor cells, and different monocyte subsets in peripheral blood of pediatric MD patients treated with repetitive courses of G-CSF administration. We showed that repetitive cycles of G-CSF administration induced efficient mobilization of above-mentioned cells including cells with proangiogenic potential. These findings contribute to better understanding the beneficial clinical effects of G-CSF in pediatric MD patients. Muscular dystrophies (MD) are a heterogeneous group of muscle diseases characterized by progressive muscle weakness and wasting , 2. DespTo date, experimental therapies mainly focused on Vascular Endothelial Growth Factor- (VEGF-) related strategies. It is well established that VEGF function as a potent promotor of angiogenesis and promyogenic factor. In dystrophin deficient muscles VEGF was shown to promote myofiber regeneration and protect cells from apoptosis . MoreoveNotably, there is a growing body of evidence that monocytes/macrophages are also important players in muscle regeneration. It should be noted that two distinct and functionally different subpopulations of macrophages are present in regenerating muscle tissue, namely, MI  and MII  macrophages. MI macrophages are referred to as proinflammatory cells and are involved in immune activation, phagocytosis, and muscle cell lysis. In contrast, MII macrophages are usually considered to exert anti-inflammatory properties as they have been shown to regulate inflammatory cell function and participate in vascularization process. This subpopulation is able to support muscle cell regeneration, by inducing satellite cell proliferation and tissue revascularization . HoweverRecently, the members of our group demonstrated that G-CSF administration brought beneficial clinical effects in pediatric patients with MD . G-CSF iHere we wished to assess the effects of repeated cycles of G-CSF administration on mobilization of bone marrow derived stem/progenitor cells  and different monocyte subsets in pediatric patients with MD. In parallel, we set out to analyze the effects of G-CSF administration on angiopoietins that similarly to EPC are involved in angiogenesis  or a marker that is associated with changes of monocyte/macrophage phenotype, namely, soluble CD163 (sCD163).μg/kg of body weight/day for five consecutive days (course 1). Such treatment course was repeated after 1 month (course 2) and after 2 months (course 3).A total of eleven muscular dystrophy patients were enrolled in this study. Detailed clinical characteristics of all patients are summarized in μL (for monocytes) and 600 μL (for EPCs) of whole blood were stained with monoclonal antibodies and incubated for 30 min at room temperature, in the dark. Thereafter, erythrocytes were lysed by adding 2 mL of FACS lysing solution (BD), followed by 15 min incubation in the dark. Cells were washed twice with cold PBS  and fixed with CellFix (BD Biosciences). Fluorescence-minus-one (FMO) controls were used for setting compensation and to assure correct gating. Specimen acquisition was performed using FACSCalibur flow cytometer (BD Biosciences). Obtained data were analysed using FlowJo ver. 7.6.5 software (Tree Star).Fresh EDTA-anticoagulated whole blood samples were stained with a panel  of mouseAngiopoietin-1, Angiopoietin-2, and sCD163 levels in EDTA-plasma samples from patients with MD were quantified by means of commercially available enzyme-linked immunosorbent assays (ELISA). To determine sCD163 plasma levels all samples were initially diluted 1000-fold with reagent diluent (1% BSA (Sigma-Aldrich) in PBS). Next, the specimens were assayed using sCD163 DuoSet ELISA kit (R&D Systems), according to the manufacturer's instruction. In order to determine Ang-1 and Ang-2 levels samples were directly assayed using Ang-1 DuoSet ELISA kit and Ang-2 DuoSet ELISA kit (both from R&D Systems). Finally, the protein levels in the specimens were calculated from a reference curve generated by using reference standards. The samples were analyzed with automated light absorbance reader (LEDETEC 96 system). Results were calculated by MicroWin 2000 software.p < 0.05. The results are presented as median (interquartile range).Statistical analysis was carried out using GraphPad Prism 6 (GraphPad software). Wilcoxon test was used to compare changes in monocytes and EPCs numbers and plasma protein levels in single treatment course. Kruskal-Wallis test with post hoc Dunn's multiple comparison test was used to determine differences between all treatment courses. Spearman correlation coefficient was used to determine correlations between plasma protein levels and cell subsets. The differences were considered statistically significant at p > 0.05).First, we analyzed the effect of G-CSF treatment on hematopoietic stem/progenitor cells mobilization in children with MD. We observed substantial increase in CD34+ cell numbers after course 1 (from 908 (309–1839) to 2327 (1896–3965), p > 0.05). In parallel, we assessed the levels of two major angiopoietins, Ang-1 and Ang-2, during treatment with G-CSF and found that none of them was affected by this therapy  following repetitive courses of G-CSF treatment. We found significant increase in EPC numbers after course 1 (from 34 (19–75) to 84 (73–206),  therapy .p > 0.05).Next we set out to investigate changes in absolute numbers of different monocyte subsets. We found that G-CSF administration induced mobilization of CD14++CD16−, CD14++CD16+, and CD14+CD16++ monocytes in all studied individuals . Moreovep < 0.05, p > 0.05, p > 0.05).Next, we assessed the effects of G-CSF treatment on sCD163 levels. We observed substantial increase in sCD163 levels in all studied individuals undergoing initial treatment . InteresFinally, we investigated whether plasma Ang-1, Ang-2, and sCD163 levels were correlated to numbers of CD34+ cells, EPCs, and monocytes subsets in peripheral blood. We did not find any significant correlations among above-mentioned parameters.p = 0.015 for course 1; p = 0.007 for course 2; p = 0.031 for course 3; data not shown) and EPCs, in all studied individuals. Interestingly from clinical point of view, the growth rate of analyzed cell populations did not differ between courses of treatment . Similar to our study, de Kruijf et al. reported in mice model that multiple cycles of recombinant human G-CSF administration (up to 12 cycles) did not lead to bone marrow HSC pool depletion [G-CSF-induced mobilization of hematopoietic stem/progenitor cells is usually delayed, with peak levels achieved within 5–7 days. In fact, in present study we observed a substantial increase of CD34+ cells, including hematopoietic stem cells (HSCs) . IntereIn summary, to our knowledge, this is the first report showing that repetitive G-CSF treatment can induce efficient mobilization of cells with proangiogenic potential, namely, EPC and putative proangiogenic monocytes. These findings could help better understand the beneficial clinical effects of repetitive G-CSF administration in MD pediatric patients. Nevertheless, the clinical safety of such treatment in this group of patients needs to be carefully addressed in further follow-up studies.


Febrile neutropenia (FN) is a common complication among patients with chemotherapy-induced myelotoxicity and is associated with a number of negative outcomes including prolonged hospitalization, increased medical costs, increased risk of mortality, dose reductions, and delays. Granulocyte-colony-stimulating factor (G-CSF), granulocyte–macrophage-colony stimulating factor (GM-CSF), and pegylated G-CSF are effective at reducing risk and duration of neutropenia-related events. However, despite guidelines, the use of G-CSF and pegylated G-CSF in the United States has not been consistent and pattern of care studies have focused primarily on G-CSF. A number of studies found that G-CSF is underutilized in patients undergoing chemotherapy treatments associated with a high risk of FN, while being over utilized in patients with a low-risk FN. Wide variations in overuse, underuse, and misuse of G-CSF are associated with a number of physician and patient factors. Improved awareness of the guidelines, feedback to providers regarding proper usage, and understanding of chemotherapy regimens associated with very low risks as well as high risks (>20%) of FN is some of the approaches that could lead to improving care. Neutropenia is a common complication among patients with chemotherapy-induced myelotoxicity. Severe neutropenia (SN) and febrile neutropenia (FN: neutropenia with fever) are associated with prolonged hospitalization, serious infections and the use of broad-spectrum antibiotics, increased medical costs, decreased quality of life, and increased mortality , 2. Gran9/L accompanied by fever. In the United States, neutropenia-related hospitalization is estimated to occur in 34.2 cases per 1000 chemotherapy-treated patients, translating to approximately 60,000 cases every year [Neutropenia is marked by abnormally low levels of white blood cells due to chemotherapy or other factors such as marrow infiltration from cancer, which ultimately predisposes patients to potentially life-threatening infections . As noteery year , 12. Repery year . In addiery year . Neutropery year –17.G-CSFs are biological growth factors that support the proliferation, differentiation, and activation of granulocytes. The United States and European guidelines support the routine use of G-CSF as primary prophylaxis  in patients receiving chemotherapy regimens where the risk of FN is >20% , 18–20. As a secondary prophylaxis , G-CSF reduces: time for neutrophil recovery, the incidence of FN, hospitalization, and the administration of broad-spectrum antibiotics . While sIt should be noted that despite its effectiveness, G-CSF use is not without some adverse events. The most frequently patient-reported adverse event is mild-to-moderate bone pain . In addiAs mentioned earlier, United States and European guidelines –20 suggeA recent examination of over 25,000 cancer patients admitted to the hospital for FN found no difference between the percentage of low-risk patients (62.1%) and high-risk patients (65.9%) receiving a G-CSF to treat  their existing FN . AnotherIn a population-based, observational, multiregional cohort study of lung and colorectal cancer patients by Potosky and colleagues, only 17% of patients undergoing chemotherapy regimens at high risk (≥20%) for FN received prophylactic G-CSF . Thirty-Given the effectiveness of G-CSF when used properly, it is important to identify the factors that lead to the inconsistency between guideline recommendations and actual clinical use of G-CSF. The current review has identified a number of potential factors that can be either classified as factors associated with the physician or the patient. Electronic databases  were searched to identify studies published up to January 2014 using combinations of the following search terms “G-CSF,” “G-CSF,” “guidelines,” “predictors,” “patient (physician) characteristics,” “underutilization,” and “overutilization.”We found that the use of G-CSF can vary greatly not only between different cancer practices but between physicians within the same practice . For exaOne of the most salient factors for the use of G-CSF is the setting in which the patient receives treatment. In two early surveys of ASCO members, doctors in HMOs or academic settings were less likely to use G-CSF as primary or secondary prophylaxis than physicians who practiced in fee-for-service settings , 32, 33.Taken together, these studies suggest that physicians in an academic setting use G-CSF less than physicians in a fee-for-service setting. However, when taking into consideration how G-CSF is used, the differentiation is more nuanced. Prophylactic use of G-CSF may be underutilized by physicians in an academic setting and more often used by physicians in a fee-for-service setting. However, using G-CSF to treat FN occurs less frequently in academic settings compared to a fee-for-service setting. One possibility for this difference may be due to issues of reimbursement, which is less of a concern for physicians in fee-for-service than in an academic setting. In fact, Bennett and colleagues  found thFurther evidence for the role of compensation was found by Potosky and colleagues, who reported that patients in HMO plans, where physician compensation is less tied to the drug and services administered, were less likely to receive a prophylactic G-CSF than patients in a non-HMO . The autIn a 1999 study, physicians with higher hematology training, and with a higher number of patients with hematologic malignancies (more exposure to this population), were more likely to use G-CSF as primary prophylaxis . PatientThe guidelines for prophylactic use of G-CSF take into consideration a number of patient factors. Factors leading to a higher risk of FN and more likely use of prophylactic G-CSF includes being female, being over the age of 65, having advanced disease, or presenting with serious comorbidities , 19. HowMembers of racial and ethnic minorities do not always have the same access to or receive the same level of health care quality as nonminority patients . There iAlthough a severe comorbidity score is associated with an increased likelihood of G-CSF use either prophylactically or reactively in patients undergoing intermediate- or low-risk chemotherapy regimens , a studyDu and colleagues found that the use of G-CSF varied widely based on geographic region . For exaFew studies have assessed patients' attitudes or knowledge of neutropenia and G-CSF use. One recent discrete choice survey of almost 300 cancer patients found that these patients chose G-CSF options with the lowest out-of-pocket expenses, the fewest injections, and lowest risk of disruption to chemotherapy schedule and infection requiring hospitalization most frequently . In partAnother factor that may impact the primary prophylaxis of G-CSF is the cancer type of the patient. In a retrospective database study of patients that received G-CSF as a primary prophylaxis, breast cancer was the most common tumor type, followed by non-Hodgkin's lymphoma and lung cancer . ResultsRamsey and colleagues  examinedIn another study, patients undergoing an intermediate-risk cancer regimen were more likely to have received G-CSF either as primary prophylaxis, as secondary prophylaxis or to treat FN than patients undergoing a low- or high-risk regimen . SimilarUse of G-CSF has also been found to vary depending on the stage of the disease. For instance, in breast cancer patients, only 11% of patients with stage I received a G-CSF compared with 24% of patients with stage III and 17% of patients with stage IV . This stDespite the guidelines, many patients are receiving G-CSF when risks of FN are low and many are not receiving G-CSF when risks are high (>20%). In practice, many regimens have <20% risk and should not receive primary prophylaxis. Furthermore, many clinicians are using G-CSFs reactively to treat FN which is counter to current guidelines. As identified in this review, the reasons for the inconsistent prescribing patterns G-CSF are multifactorial and include differences among physician experience and training, practice setting  reimbursement, and the geographical location of care. As well as factors inherent to the patient or disease including race, geographic location of care, comorbidity, disease severity, chemotherapy regimen, and patients' beliefs and attitudes toward G-CSF.Physician training and experience are associated with their adherence to guidelines. Specifically, Cabana and colleagues found that a lack of awareness and familiarity of guidelines were two of the most common barriers to physicians proper use of clinical guidelines . Both ofThis review also highlighted the importance of the hospital setting in which clinicians are working which may be a proxy for issues involving reimbursement. Specifically, prophylactic GCSF may be underutilized by physicians in an academic setting and more often used by physicians in nonacademic hospitals. As first suggested highlighted by Bennett and colleagues , this diWith respect to the patient factors, there is some initial evidence of racial disparity in G-CSF and additional research is needed to determine the breadth of race differences and whether they are confounded by cancer type. Furthermore, it is clear that cancer-type, severity of the disease, and chemotherapy regimen are each a factor that is associated with the differential use of G-CSF prophylaxis. Additional research is needed to more clearly understand these effects separately and in conjunction with one another given their confounding impact. Finally, additional research is needed to more closely examine patients' attitudes toward and knowledge of neutropenia and G-CSF. Insuring that patients have a better understanding of neutropenia's impact and the potential benefits (and risks) of G-CSF could lead to more appropriate use of G-CSF.


Aim: To compare the acceptability and efficacy of orally administered commercially available midazolam syrup and injection midazolam mixed in honey for performing venepuncture.Materials and methods: This double blind randomized controlled trial enrolled 40 anxious and healthy 2 to 6 years olds. All subjects received either syrup midazolam or injection midazolam mixed in honey (0.5 mg/kg) per orally, prior to venepuncture as per their group assignment. Primary outcome measures in this trial was acceptability of midazolam. Secondary outcome measures included sedation depth, success of venepuncture, observer and parental satisfaction and parental perception of child's pain.Results: Although the acceptability of syrup midazolam (95%) was higher than injection midazolam (80%), there was no significant difference among two groups with respect to any primary or secondary outcome (p > 0.05).Conclusion: Syrup midazolam can serve as a suitable alternative to injection midazolam; thus, eliminating the procedural steps of mixing injection midazolam with any vehicle.How to cite this article: Srivastava B, Mittal N, Mittal P. Acceptability and Efficacy of Commercial Oral Preparation of Midazolam for brief Painful Procedure: A Randomized Double Blind Clinical Trial. Int J Clin Pediatr Dent 2014;7(3):153-156. Owing to anxiety as well as lack of cooperation by young child due to various behavioral issues; venepuncture, at most of the times becomes a patience testing procedure in pediatric practice.The pediatric population seems to have undergone a qualitative transformation; being less cooperative than earlier. The evolving contemporary parenting which is more protective with inability to control children's behavior is probably accountable for this.34 The scenario becomes worse when there is an urgency to treat as in case of a painful abscessed tooth or trauma. Under such circumstances, the practitioner has to resort to pharmacotherapeutic means of behavior management.Initially, the most conservative traditional behavioral management approaches such as tell-show-do, modeling, contingency management, etc. are attempted; which, however, remain unsuccessful in good number of cases. Restraint and HOME are commonly employed measures to gain control over tantrum throwing children; but, these obviously have psychological trauma associated with them, with greater number of parents and dentists recognizing it.8 because of availability of multiple routes, short half life (1.5-2 hours), high potency (twice as that of diazepam), rapid onset of action (10-30 minutes depending upon the route), reliable dose dependent anxiolytic effects, and low grade anterograde amnesia.A wide variety of sedative agents through various routes have been tried for procedural dental sedation. Midazolam has been extremely popularInjection midazolam has been widely used for oral administration after mixing with any pleasantly flavored masking agent, i.e. fruit juices, sugar syrups or honey. Recently premixed oral preparation of midazolam has become commercially available. The commercial preparation has advantage of eliminating procedural steps of mixing the drug with masking agent. If, in case, homogenous mixing of injection midazolam with masking agent is not ensured, there are chances of rejection of drug solution due to residual bitter taste of drug and hence, loss of compliance for next increment as in case of young apprehensive child. This may result in lesser dose of drug being administered than required; thus, compromising the efficacy. Further, mixing with the nonstandardized juices/solutions may result in unpredictable bioavailability and thus, non-uniform efficacy.There is rarity of data regarding acceptability and efficacy of commercial oral preparation of midazolam. Also, to the best of our knowledge, no study has been conducted on Indian children who may vary in their coping abilities, pain threshold, level of anxiety, etc. Keeping this in mind, the present study was planned to report on efficacy and acceptability of commercial oral midazolam preparation as a premedicament before venepuncture.The present prospective randomized control trial was conducted in the Department of Pedodontics and Preventive Dentistry at Santosh Dental College and Hospital, Ghaziabad, Uttar Pradesh, India. The study was approved by University ethical committee review board.9 a score of ≤4 was a prerequisite for inclusion in this study. Only children belonging to physical status American Society of Anesthesia (ASA) I were included. Children with known history of allergy to benzodiaz-epines, narrow angle glaucoma, mental retardation or learning disabilities, impaired renal and hepatic functions, obstructed nasal passages, raised intracranial or intraocular pressure, allergy to soya milk or egg, etc. were excluded from this trial.A total of 40 anxious children in the age range of 2 to 6 years, requiring dental treatment under IV sedation were included in the present study. Baseline anxiety was assessed by first author (N) as per Ven ham's a n xiet y rating scale;Children with history of fever or those with a significant cough with/without sputum production were either excluded or if possible, postponed for about 2 to 4 weeks after complete resolution of symptoms. Also, the compliance to follow fasting guidelines and failure to obtain informed consent from parents/guardian resulted in exclusion from trial.The selected children were assigned to the two groups using the block randomization method with five blocks of variable number. Three blocks were prepared with eight patients in each block; one block was made with ten patients and one block had six patients. The decision to allot the child to either of two groups was based upon randomly choosing a sealed envelope containing details of sedative agent to be administered. The sealed envelopes were prepared beforehand by an investigator (BS) not further involved in outcome assessment in this study.10 Subjects in group A were premedicated with commercially available premixed midazolam syrup with predominant orange favor for oral use  in a dose of 0.5 mg/kg 20 minutes prior to venous cannulation. Subjects in group B were premedicated with injection midazolam  mixed in honey in a dose of 0.5 mg/kg 20 minutes prior to venous cannulation. The drug was administered via oral transmucosal route using a calibrated needleless syringe in small aliquots so as to prolong contact with oral mucosa. After drug administration, child was taken to a quiet room accompanied by one parent. Twenty minutes after premedica-tion, venous access was obtained and response of child was noted down. Following cannulation, the sedation for accomplishment of miscellaneous dental procedures was maintained with 1 to 1.5 mg/kg IV bolus of propofol mixed with 2% of 1 ml lignocaine followed by 25 to 75 μg/kg/min of propofol infusion.All subjects followed NPO guide lines as per ASA guide-lines.The drugs were administered by using opaque needleless syringes so as to keep the outcome assessor unaware of test drug.2; any complications and their management.The data for each patient was entered on preprinted pro-formas which included demographic characteristics , total dose of midazolam, baseline anxiety, sedation depth, success of venepuncture, observer and parental satisfaction and parental perception of child's pain, vital signs, i.e. heart rate, NIBP, respiratory rate, SpO7 as follows: 1 = accepts readily, 2 = dislikes  but accepts, 3 = accepts with great difficulty, 4 = could not be premedi-cated because of extreme resistance.Primary outcome measure in this trial was acceptability of midazolam. The test solution was administered in 4 to 5 divided doses and acceptability was rated according to ease of administration of next increment using the four point acceptability scale11 success of venepuncture, observer and parental satisfaction and parental perception of child's pain.Secondary outcome measures included sedation depth measured using Houpt's sedation rating scale,Success of venepuncture was measured as 1 = successfully completed, 2 = aborted.12Observer as well as parental satisfaction was recorded on a five point Likert scale as follows: 1 = not satisfied, 2 = somewhat satisfied, 3 = satisfied, 4 = very satisfied, and 5 = excellent. Parental perception of child's pain was noted on a 0 to 10 mm Verbal analog scale.®, Inc, Redmond, Wash) and then imported to SPSS (version 16.0) for analyses. Kolmogorov-Smirnov test was used to test the normality of data. Chi-square test was used for intergroup comparison for variables such as acceptability of midazolam, success of vene-puncture, observer and parental satisfaction. For quantitative data following non-normal distribution, i.e. Houpt's sedation scores, parental satisfaction scores, Mann-Whitney U test was applied for intergroup comparison. While for variables such as age and weight, following normal distribution as shown by results of Kolmogorov-Smirnov test; independent t-test was used. A two-tailed (α = 2) probability (p) value less than 0.05 (p < 0.05) was considered to be statistically significant.All data were first entered on Excel spreadsheet (Microsoft OfficeBaseline demographic characteristics were similar in both groups . Both ofWe observed an acceptability of 95% in group A and 80% in group B (p > 0.05). The venepuncture could not be performed in two patients in group B. However, similar sedation quality was observed in both groups with respect to sleep (p = 0.511), crying (p = 0.769), movement (p = 859) and overall behavior (p = 0.725).Parental as well as observer satisfaction was higher in group A, however, the difference between two groups was not statistically significant (p = 0.287). Similarly the VA S scores were lesser in group A, the difference between two groups was again not statistically significant (p = 0.483).To the best of our knowledge, this is the first trial to report on acceptability of commercial oral preparation of midazolam in Indian children. Although the acceptability of midazolam via oral route has been tested previously; in those studies, injection midazolam for parenteral use (which has a bitter taste) was administered orally in combination with various sweet tasting masking agents. No data exists on acceptability and efficacy of commercial oral preparation of midazolam.In our study, one child accepted the drug with great difficulty in group B while we did not face such problem in group A. Anyhow; we did not find any statistically signi-ficant difference in acceptability and efficacy among two groups. None of the children totally refused to accept drug solution in either of two groups, thus showing its acceptability by uncooperative children in preschool age.8 reported similar acceptability among mida-zolam in strawberry syrup and placebo strawberry syrup. This could have been the reason that despite the pleasant taste of commercial preparation, we did not report any superior acceptability over injection midazolam in honey. For the similar reasons, we reported no significant difference in efficacy among two study groups.Similar acceptability scores, however, cannot be simply attributed to taste of drug solution solely, as the disliking for a particular drug can be part of general uncooperative and apprehensive nature of children. Previously also, Kapur et al13 however; reported superior efficacy of parenteral midazolam mixed in grape favored Syraptala® syrup over commercially favored cherry favored midazolam syrup . In their discussion, authors mentioned that the commercial preparation had a potential to precipitate into crystals of insoluble complex of midazolam and saccharine. The presence of this precipitate might have caused a lack of uniformity in the product and may result in the administration of a super- or subpotent dose, resulting in lesser efficacy. The syrup used in our study did not pose any such problems. But, similar acceptability was reported for midazolam syrup and midazolam-syraptala mixture.An American study,6 used raspberry syrup and reported an acceptability of 87%. While using strawberry syrup, an acceptability of 80 to 86% has been reported.78 Better acceptability with commercially available midazolam syrup than previously conducted studies where the bitter taste of midazolam was masked by using sweet tasting masking agents was found. This is an encouraging finding and suggestive of possible successful use of midazolam syrup as an alternative to injection midazolam.Parnis et alSpitting of the drug solution by children is very common and this can be attributed to drug's taste as well as behavioral profile of child. This can result in lesser than required drug dose being administered and hence, insufficient level of sedation. Latter may result in difficult or failed procedure and hence, defeating the purpose of sedation. Two children in group B accepted the drug with great difficulty and these two children had to be administered rescue sedation intramuscular ketamine to accomplish venepuncture. Initial unsuccessful attempts with midazolam solution might have led to increased parental apprehension and dissatisfaction among both observer as well as parents. This could have led to poorer observer and parental satisfaction scores as well as higher VAS score among parents.Both syrup midazolam and injection midazolam in honey are equally efficacious and equally acceptable by young and anxious children. Hence, syrup midazolam can serve as a suitable alternative to injection midazolam; thus, eliminating the procedural steps of mixing injection midazolam with any vehicle.


Infections are still the most common complications of cerebral shunt procedures. Even though fungal etiologies are considered to be rare, they are associated with significant morbidity and mortality. Due to their uncommonness, diagnostic procedures and optimal therapy are poorly defined. We report a case of Candida tropicalis infection of ventriculo-peritoneal cerebrospinal fluid (CSF) shunt in a 49-year-old immune competent male treated with voriconazole (VOR).Microbiological and CSF markers  of fungal infection, biofilm production capacity, sensitivity of serial isolates of the pathogen, and the concentration of the antifungal drug have been monitored and related to the clinical course of this infection.Despite appropriate treatment with VOR, in terms of adequate achieved CSF drug concentrations and initial effective therapeutic response, loss of VOR susceptibility of the C tropicalis and treatment failure were observed.Biofilm production of the C. tropicalis isolate might have had a significant role in treatment failure. Of interest, clinical and microbiological unfavorable outcome was anticipated by persistence of BDG in CSF. Rising titers of this marker were associated with relapse of fungal infection. Until now, few cases of Candida tropicalis infections of CNS have been reported in the medical literature.,3 Therefore, risk factors, prognosis, diagnostic procedures, and optimal therapy are poorly defined. Despite appropriate therapy, the referred mortality of patients affected by C tropicalis meningitis approached 30%.,2 We describe a case of an infection of the cerebrospinal fluid (CSF) shunt device in a middle-aged patient sustained by C tropicalis. Peculiarities of this report of such a rare infection are that measurement of -β-d-Glucan (BDG) in the CSF, biofilm production capacity and modifications of the azole susceptibility of serial isolates of the pathogen, as well as antifungal drugs CSF concentration have been monitored and related to the clinical outcome.2pontocerebellar acoustic neuroma with placement of a ventriculo-peritoneal (VP) shunt and received an successful 10-day Fluconazole (FLU) treatment course for a catheter-related C tropicalis urinary tract infection. CSF analysis, performed at the admission, revealed glucose concentration of 34.2 mg/dL, protein level of 332 mg/dL, and leukocytes cell count of 471 cells/μL (75% polymorphonucleocytes). No signs of persistent Candida urinary tract infection were found in serial urine analysis and cultures performed during this second hospitalization. Fungal meningitis was diagnosed on the basis of C tropicalis isolation from the CSF culture. The same pathogen was also cultured from the removed VP shunt, while blood cultures were negative. Intravenous voriconazole (VOR) was started  and an external ventricular drainage (EVD) was inserted. The clinical course and the main laboratory findings, expressed in function of time, are shown in Fig. A 49-year-old immune-competent male was admitted to the Neurosurgical Intensive Care for fever (up to 38°C) associated with ideomotor slowdown, severe confusional state, and meningism. Two months prior hospitalization, the patient underwent surgical removal of a C tropicalis was still cultured from CSF and consequently EVD was changed. On the day 31 of VOR treatment, CSF cell count and glucose concentration achieved normal values and CSF culture yielded no microbial growth. With apparent C tropicalis microbial eradication from CSF, a clinical improvement of patient's condition in terms of Glascow Coma Score  (3 mg/kg/daily) along with FLU  and EVD was changed again. On the day 66 of antifungal therapy, the patient died for a concurrent bacterial sepsis, showing no further significant clinical improvement of his meningitis after the replacement of VOR by LAMB along with FLU. CSF cultures remained positive for C tropicalis. Given that interventions and all investigations described in this case report were performed as part of the standard health care whose informed consent was obtained, no ethical approval was either sought or obliged.33.1susceptibility of C tropicalis in CSF samples was tested by means of the minimal inhibitory concentrations (MICs) measurement , Italy), according to CLSI M27A3 guidelines. The susceptibility of the C tropicalis isolates was monitored and the changes recorded over the time (Table 2 dilution factor) of the VOR MICs was observed in serial CSF C tropicalis isolates.The antifungal 3.2C tropicalis strains was measured through XTT-reduction dehydrogenase assay and expressed as the ratio of the mean sample absorbance performed in triplicate experiments, over the 50% mean absorbance of a reference C. albicans strain , in terms of biofilm index (BFI).,6 Clinical isolates with BFI ≥0.5 were considered biofilm-producing strains. The aim of this experiment was to determine eventual time-related changes in biofilm-forming ability of strains of C tropicalis isolated from the CSF samples. All the isolates of C tropicalis were biofilm producers. The BFI fell from 2.1 to 1.1, with a 52% reduction at day 40. The recurrence of the isolation of C tropicalis on the day 48 was accompanied by a rise in the BFI .Biofilm production by 3.3Limulus amebocyte lysate pathway, using a commercially available diagnostic kit , according to the manufacturer instructions. Glucan-free dilution tubes and pipette tips were used, and all assays were performed in a biosafety cabinet that had not been used to manipulate fungal cultures. Individuals performing the assay were blinded to all diagnostic and clinical information. This analysis was performed posthumously on CSF samples stored in the biobank. Our results showed a progressive decrease in the levels of CSF BDG (from 5230 to 1233 pg/mL), even though the values were constantly high and the negativity of the marker was never reached. Of note was a new gradual rise in the BDG in the CSF samples some days before the new isolation of C tropicalis  over the concentration of 0.1 to 50 mg/L. Validation data for accuracy and precision were coefficient of variation between 2.4% and 7.3%; intra-day accuracy was in the range of 95% to 106.5 %.VOR plasma concentration was determined by high-performance liquid chromatography (HPLC), using Perkin Elmer chromatograph  with wavelength ultraviolet (UV) detector set at 255 nm. An aliquot (0.5 mL) was pipetted into a 2 mL polypropylene tube  and acetonitrile (1.0 mL) added. The mixture was vortex mixed briefly and after standing for 10 minutes at room temperature the mixture was centrifuged at 1200C tropicalis.A median CSF VOR concentration of 2 mg/L (range 0.27–3.82 mg/L) was registered, while the mean blood level of VOR was 2.48 mg/L (range 1.91–3.05 mg/L). The CSF/plasma VOR concentration ratio was 0.8. The CSF VOR concentrations observed were always higher than the coeval MIC of 4VOR should be used as a first-line treatment for CNS Candida infections. The rationale for the use of this drug in this setting is linked to its ability to reach high concentrations in the CSF.–11 Lutsar et al reported that in patients treated with VOR for CNS fungal infections, the median CSF/plasma ratio was 0.46 (range 0.22–1.0). Wiederhold et al, testing 173 CSF samples, confirmed a similar result (CSF/plasma ratio 0.52) and described that the median quantifiable CSF level of VOR was 2.47 mg/L (range 0–15.3 mg/L) with CSF concentrations more heavily distributed toward the lower end of the range. However, the CSF concentration and the ratio may vary depending on the serum drug concentrations and is influenced by several variables such as liver disease, age, genetic polymorphism of the cytochrome CYP2C19, and comedications. These variables may lead to large inter- and intra-patient differences in VOR pharmacokinetic, perhaps sometimes associated with a decreased efficacy. Therefore, therapeutic drug monitoring (TDM) is required to improve the efficacy and the safety of VOR therapy in patients with invasive mycoses.The Italian Consensus for invasive candidiasis management (ITALIC) has suggested that C tropicalis isolates, ranging from 0.06 to 0.25 mg/L, were always below the resistance breakpoint that is >0.5 mg/l, according to the CLSI interpretation. However, a progressive increase of the MIC values was observed during the course of VOR treatment and a value of 0.25 mg/L was observed on day 48 of VOR therapy. To date, according to new CLSI VOR clinical breakpoints for C tropicalis (available only after the occurrence of the described case), MICs values for VOR of 0.25 to 0.5 mg/L should be interpreted as intermediate sensitivity. Of interest, a recent Italian study reported that 99% of the isolates of C tropicalis tested were highly susceptible to VOR  with only 1 out of 102 isolates showing dose-dependent susceptibility. These data were confirmed by the results of a large multicenter study conducted in Europe and North and South America where only 2.2% and 3.5% of 3127 C tropicalis isolates showed MICs for VOR of 0.25 and ≥0.5 mg/L, respectively. As a matter of fact, in our case, we cannot exclude that the above-mentioned loss in susceptibility to VOR might have affected the efficacy of therapy.VOR was used in our case and the drug achieved plasma and CSF levels that should have provided a clinical efficacy (CSF/plasma concentration ratio of 0.8). Furthermore, the MICs of VOR treatment failure, LAMB as well as FLU therapy was attempted on the basis of previous apparently favorable in vivo and in vitro observations of this antifungal agents combination.,16 However, the efficacy of this salvage therapy could not be fully evaluated, as the patient finally died for bacterial superinfection.Following C tropicalis to form biofilm on devices is considered a potential virulence factor and might have represented another limitation for therapy with VOR (or LAMB) in our case.,18 Such a phenomenon might be strongly implicated in the final VOR treatment failure despite susceptibility of fungal isolates and prompt removal and substitution of EVDs.Further insights might be gained by the observation of biofilm production by all consecutive fungal isolates. Indeed, the ability of BFI has been observed in serial isolates during antifungal therapy. To this end, recent studies showed that VOR fails to eradicate Candida spp. in mature biofilm, but seems to display in vitro efficacy in the reduction of biofilm formation.–21 Of course, knowledge of the clinical significance of these observations requires for further investigations.Of interest, as shown in Fig.  Therefore, due to these possible bias, serum and/or BDG seem to be a suitable marker in this setting.The therapeutic efficacy of VOR was initially assumed by the progressive restoration of the normal physical-chemical and microbiological CSF parameters. These findings were also indirectly confirmed by the gradual decline of CSF BDG observed until a few days before the recurrence of positive CSF culture under VOR therapy. Erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and BDG might be used for monitoring efficacy of antifungal therapy. Nevertheless, ESR and CRP are nonspecific inflammatory markers and can be affected by concomitant conditions such as concurrent bacterial infections that compromise their usefulness in monitoring the fungal disease in critically ill patients, as it occurred in our patients.CSF BDG as a marker for the detection of fungal infections in the CNS and for clinical therapy monitoring.–28 In our case, during VOR treatment, the retrospective analysis in serial samples of CSF demonstrated persistence of BDG, even when cultures yielded no microbial growth, albeit with a progressive reduction of titers. On the basis of these observations, persistence of BDG in the CSF might suggest the need for maintaining the antifungal treatment despite negative cultures. On the contrary, clearance of this marker might suggest the optimal timing to reposition a VP shunt. Not less important, a sudden increase of CSF BDG values during antifungal therapy, as that observed in our case before the C tropicalis recurrence, might represent a warning sign for the treatment failure.Recent studies, performed in animal models and on human beings, proposed  According with the results of the fungal meningitis rabbit model proposed by Petraitiene et al, plasmatic levels of β-glucan lower than CSF levels hint a compartmentalization of this polymeric carbohydrate, with higher concentrations within the CNS not readily crossing the blood–brain barrier. Taken together, these data could indicate that serum BDG values may not be helpful in determining invasion of the CNS by a fungal pathogen.On the contrary, serum BDG levels do not seem to be concordant with CSF BDG levels: in fact in our case, BDG values were significantly higher in CSF than in blood. As previously suggested, this gap could suggest isolated CNS infection without significant spillover into venous blood or that BDG may be more rapidly cleared in blood than in CSF.Candida infections, as proposed by ITALIC, is not shared by the most recent guidelines for the “Management of Candidiasis,” updated by the Infectious Diseases Society of America (IDSA) in 2016. In these recommendations, VOR is not cited as a possible choice in the treatment of CSN Candida infections, although it is recognized that it can achieve significant CSF concentration, potentially active against susceptible isolates, and it has been shown to be efficacious in small case series.–10,12 Wide variability in serum levels, need of the TDM, common drug–drug interactions with potential toxicity might have discouraged the use of VOR in these Guidelines.Finally, despite the data supporting the efficacy of VOR in CNS fungal infections, use of this azole as first-line therapy in CSF 5C tropicalis meningitis wherein appropriate treatment with VOR, in terms of adequate achieved CSF drug concentrations, was associated with loss of VOR susceptibility of the offending pathogen and treatment failure. Of interest, clinical and microbiological unfavorable outcome was anticipated by persistence of BDG in CSF. Eventually, rising titers of this marker were associated with clinical and microbiological relapse of fungal infection. Biofilm production of the C tropicalis isolate might have had a significant role in treatment failure. The importance of all these data should be confirmed by further studies.We described a rare case of


Liver transplantation has received increased attention in the medical field since the 1980s following the introduction of new immunosuppressants and improved surgical techniques. Currently, transplantation is the treatment of choice for patients with end-stage liver disease, and it has been expanded for other indications. Liver transplantation outcomes depend on donor factors, operating conditions, and the disease stage of the recipient. A retrospective cohort was studied to identify mortality and graft failure rates and their associated factors. All adult liver transplants performed in the state of São Paulo, Brazil, between 2006 and 2012 were studied.+ ≥ 141.01 mg/dl [RR = 1.70 (95% CI: 1.47–1.97)], RNI ≥ 2.71 [RR = 1.64 (95% CI: 1.41–1.90)], body surface ≥ 1.98 [RR = 0.81 (95% CI: 0.68–0.97)] and donor age ≥ 54 years [RR = 1.28 (95% CI: 1.11–1.48)], male gender [RR = 1.19(95% CI: 1.03–1.37)], dobutamine use [RR = 0.54 (95% CI: 0.36–0.82)] and intubation ≥ 6 days [RR = 1.16 (95% CI: 1.10–1.34)] affected the negative outcome rate.A hierarchical Poisson multiple regression model was used to analyze factors related to mortality and graft failure in liver transplants. A total of 2,666 patients, 18 years or older,  were investigated. Outcome variables included mortality and graft failure rates, which were grouped into a single binary variable called negative outcome rate. Additionally, donor clinical, laboratory, intensive care, and organ characteristics and recipient clinical data were analyzed. The mortality rate was 16.2 per 100 person-years (py) (95% CI: 15.1–17.3), and the graft failure rate was 1.8 per 100 py (95% CI: 1.5–2.2). Thus, the negative outcome rate was 18.0 per 100 py (95% CI: 16.9–19.2). The best risk model demonstrated that recipient creatinine ≥ 2.11 mg/dl [RR = 1.80 (95% CI: 1.56–2.08)], total bilirubin ≥ 2.11 mg/dl [RR = 1.48 (95% CI: 1.27–1.72)], NaThe current study confirms that both donor and recipient characteristics must be considered in post-transplant outcomes and prognostic scores. Our data demonstrated that recipient characteristics have a greater impact on post-transplant outcomes than donor characteristics. This new concept makes liver transplant teams to rethink about the limits in a MELD allocation system, with many teams competing with each other. The results suggest that although we have some concerns about the donors features, the recipient factors were heaviest predictors for bad outcomes. Liver transplantation has gained increased attention in the medical field since the 1980s due to the introduction of new immunosuppressants and improved surgical techniques. These factors have also improved outcomes, and a huge expansion in transplants and institutions performing this procedure has occurred. Currently, liver transplantation is the treatment of choice for patients with end-stage liver disease, and this treatment has been expanded to other indications as well.et al is useful for calculating the liver graft risk based on donor variables )*0.725)*(mass [kg])0.425), gender and cause of death ;Donor clinical data: age, body mass index—BMI—(mass [kg]/height [m]+ (sodium) (mg/dl), serum GOT  (mg/dl), serum GPT (glutamic-pyruvic transaminase) (mg/dl), alkaline-phosphatase (mg/dl), glutamyl-transferase (mg/dl), total bilirubin (mg/dl), and creatinine (mg/dl);Donor laboratory data: serum NaDonor intensive care data: intensive care days; intubation days; use of dopamine, dobutamine, and noradrenaline;Organ characteristics: cold ischemia time (hours) and warm ischemia time (hours);2) and body surface ((0.007184*(height [cm])*0.725)*(mass [kg])0.425);Recipient clinical data: age (year), body mass index (mass [kg]/height [m]+ (mg/dl);Recipient laboratory data: creatinine (mg/dl), total bilirubin (mg/dl), serum international normalized ratio (INR), serum NaOther features: recipient MELD Score (0.957*ln[serum creatinine]+0.378*ln[serum bilirubin]+1.120*ln[INR]+0.643)*10 , outcome (death and graft failure) and outcome causes .Individual donor and recipient variables available in the database were grouped as follows:The origin of the organ, according to the organ collection center (OCC), was the geographical context variable available in the database. The São Paulo Transplant Center (SPTC) coordinates 10 OCCs, including 4 in São Paulo City  and 6 in countryside states  .Outcome measures included the mortality rate and graft failure rate, grouped into a single binary variable (yes/no) called the negative outcome (NO) rate. Continuous quantitative independent variables were binarized by percentile rank, with the third quartile serving as the cutoff reference.The study was reviewed and approved by the Research Ethics Committee of the Clinics Hospital, São Paulo University Medical School (Protocol Number #9949–13), in accordance with Brazilian and international regulations for research with human subjects. The institutional review board (IRB) waived the individual requirement for written informed consent, and they requested written consent from the director of SPTC because this was a retrospective study. The board also requested confidentiality of the individuals’ data, which was ensured at all stages of the project. The following information was de-identified: name, address, zip code, registration, donor's name and donor registry. None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given.A hierarchical multiple regression model was considered to study factors related to mortality and graft failure in liver transplants. This analysis is generally used to explain the relationship between variables in models with a set of empirical propositions already indicating the relationship strength and direction between predictors and outcomes. The order of predictor entry in the regression equation was defined by the researcher based on a pre-established conceptual framework .Building a conceptual framework requires knowledge of the biological and temporal determination affecting outcomes . Using tThe position of a specific set of variables regarding the outcome was based on the arithmetic mean of the scoring system, named the expert score (ES). Organ characteristics data were the most proximal terms (4 to 5 points). Medial terms (3 to 4.99 points) included donor clinical data, donor laboratory data, donor intensive care data and recipient clinical data. The distal terms (1 to 2.99 points) included recipient laboratory data .Data are shown as absolute frequency, means with standard deviations (SD), proportion with confidence intervals 95% CI), and medians with interquartile ranges (IQRs) unless otherwise stated 5% CI, an. Crude iTo estimate survival time until death, graft failures and negative outcomes were used based on the Kaplan-Meier method ,13. To sTo improve the different models, predictor variables were tested for collinearity with the variance inflation factor (VIF) as well as for the presence of influential values. Model accuracy was evaluated using a cross-validation system ,15.http://www.r-project.org/) for statistical treatments. All the significance levels were set to p<0.05.The database was exported to the Statistical Package for the Social Sciences (SPSS) 22 for Windows  and R-GUI version 3.0.2 , with a mean of 2.07 (SD 1.87) per person. The median follow-up duration was 1.75 years . The transplantation midpoint time was 2009 (SD 1.55). Thus, most transplants were concentrated between 2008 and 2010, as shown in We recorded 897 (33.6%) deaths and 100 (3.8%) cases of graft failure . Fig 3C + higher than 141 mg/dl and body surface lower than 1.98  and AIC  values, was IV; this model explained a greater amount of variance compared with an empty model , model I , model II  and model V . However, it was not significantly better than model III . After adjustment in the Poisson multiple regression model, most analyzed variables maintained or improved (donor body surface and dobutamine use) their statistical significance and effect. Only donor total bilirubin was lower than 0.81 mg/dl and exhibited no significant effect after the adjustment .The MELD score was implemented as an allocation system for liver transplants in the USA in February 2002 . This moThe post-transplant survival  in this Although liver transplant waiting list mortality is closely correlated with MELD score, and these advanced cirrhotic patients urgently require transplantation, extended criteria grafts in very ill patients can lead to worse post-transplant outcomes. However, survival benefit studies show that these patients benefit more from this procedure, even with some risk of a mismatch –25. ThisRecipient creatinine, bilirubin and prothrombin time are biochemical markers of liver and kidney function and were shown to be much more relevant than any other donor characteristics. These markers are part of the MELD calculation, a score that is associated with short-term survival in patients with terminal hepatic insufficiency . A high Another recipient factor associated with better outcomes in our study was the body surface of the donor above 1.98. This finding is likely related to better nutrition in these patient groups. In a situation with a high percentage of critically ill patients, malnutrition is responsible for raising morbidity and mortality indices .These results demonstrated that scores based only on donor data, such as the DRI (donor risk index)  and ECD Other studies have evaluated recipient data  or both Regarding donor gender, some authors have reported an increased risk of graft loss as high as 20% when female donor organs have been transplanted into male recipients. However, others did not find such differences . Other sDonor maintenance in many cases is not ideal in Brazil. Some hospitals do not have enough ICU beds for every patient and experience frequent staff shortages. Besides the mentioned problem, our data demonstrated that objective factors related to the donor do not have a big influence on patients outcomes. Donor liver enzymes, days in the ICU, BMI and vasoactive drugs had no negative impact on outcomes, which is in contrast with previous studies ,31,36,37The finding that a lower rate of NO was associated with dobutamine use in the maintenance of the donor was interesting. In our best study model (model IV), the probability of negative outcome was 46% lower in cases with donors using dobutamine. It is important to highlight the effect of this factor in association with other modeled variables [crude RR = 0.63 (95% CI: 0.43–0.92) vs. adjusted RR = 0.54 (95% CI: 0.36–0.82)]. Dobutamine is a catecholamine derivative with specificity for beta-1 adrenergic receptors and is commonly used as a cardiotonic agent after cardiac surgery and during dobutamine stress echocardiography . NoradrePrior to the database analysis, we performed a survey with transplant experts to evaluate the factors most significantly associated with negative outcomes following liver transplants. This analysis resulted in the theoretical framework analyzed herein . The resThese experts are familiar with the specialized literature and attend international meetings regularly; however, despite their experience in the field, their knowledge of global-scale data can obscure their recognition of the realities in their own countries. The contrast between the survey and data presented herein reinforces the need for region-specific versus internationally standardized scores.The MLG multiple model was used to assess the impact of factors on the NO rate and no survival analysis, as used in previous studies ,39,40. IThe large retrospective cohort dataset was a strength in this study . AdditioWe found results lower than expected regarding patients and graft survival, but after analyzing it we could understand that they are mailing due to recipient factors. As we could see in other countries the MELD system allocation, with many teams competing with each other for not enough organs and pushing the limits for the sicker patients is harmful and bring down the outcomes. However, instead to look for another allocation system as other authors, the present study allowed a better dealing with the MELD system. We are now, in our region, trying to avoid the futile transplant. We are paying more attention on very sick patients, with high MELD score, especially in those with many organs support or after recovering from septic treatments. These measures are improving the results.We demonstrated the greater importance of recipient disease stage vs. donor factors in predicting post-transplant results. This study leaded to a better dealing with the MELD system and it is improving the results. Future studies will be conducted with this dataset to develop a national liver transplant algorithm. We intend to use the variables that have been shown to impact on the probability of negative outcome raised in this study and intelligent computing tools to develop a model applied to the Brazilian reality.The current study confirms that both donor and recipient characteristics must be taken into consideration with respect to post-transplant outcomes and prognostic scores. Our data demonstrated that recipient health status was more important than donor status in predicting transplant outcomes. These results bring a better comprehensive of the harmful of pushing the limits of transplanting very sick patients. Moreover, it also allows us to better deal with MELD allocation system in an organs shortage reality associated with a very competitive scenario.


Tick-borne encephalitis (TBE) is a serious acute central nervous system infection that can result in death or long-term neurological dysfunctions. We hypothesize that changes in sphingosine-1-phosphate (S1P) concentration occur during TBE development.S1P and interleukin-6 (IL-6) concentrations in blood plasma and cerebrospinal fluid (CSF) were measured using HPLC and ELISA, respectively. The effects of S1P on cytoskeletal structure and IL-6 production were assessed using rat astrocyte primary cultures with and without addition of plasma gelsolin and the S1P receptor antagonist fingolimod phosphate (FTY720P).In vitro, treatment of confluent rat astrocyte monolayers with a high concentration of S1P (5 μM) results in cytoskeletal actin remodeling that can be prevented by the addition of recombinant plasma gelsolin, FTY720P, or their combination. Additionally, gelsolin and FTY720P significantly decreased S1P-induced release of IL-6.We report that acute inflammation due to TBE virus infection is associated with elevated levels of S1P and IL-6 in the CSF of infected patients. This elevated concentration is observed even at the earliest neurologic stage of disease, and may be controlled by glucocorticosteroid anti-inflammatory treatment, administered to patients unresponsive to antipyretic drugs and who suffer from a fever above 39°C. TBE is associated with increased concentration of S1P and IL-6 in CSF, and this increase might promote development of inflammation. The consequences of increased extracellular S1P can be modulated by gelsolin and FTY720P. Therefore, blocking the inflammatory response at sites of infection by agents modulating S1P pathways might aid in developing new strategies for TBE treatment. Sphingosine-1-phosphate (S1P) is a product of sphingomyelin (SM) metabolism. It is present in most eukaryotic organisms. S1P regulates cell function both intracellularly and by binding to extracellular receptors . In the 2-regulated protein, can also act as a universal carrier or scavenger of S1P, and its function may include interference with S1P actions [S1P is abundant in plasma where it is bound to high-density lipoproteins and albumin . Recentl actions . Such an actions . In the  actions . An infl actions . TBE, a  actions . Even th actions ,18. We h actions , and itsS1P (S9666) was purchased from Sigma Aldrich . (S)- fingolimod phosphate FTY720P; B-0721) was purchased from Echelon Biosciences Inc. . Recombinant human plasma gelsolin (rhGSN) was obtained from Biogen-Idec Inc . Stock solutions of S1P were prepared in 0.3 M NaOH, since pH strongly affects the aggregation behavior of S1P [P; B-0721Human blood and CSF specimen collection was performed in the Department of Neurology and Department of Infectious Diseases and Neuroinfections at the Medical University of Białystok (Poland). Blood was collected in heparinized syringes and centrifuged at 1,500 g for 5 minutes at 4°C. The protocol for this study was approved by the Ethics Committee for Research on Humans, Medical University of Białystok . At the time of patient recruitment, written consent was obtained from all subjects. All individuals were undergoing lumbar puncture for diagnostic purposes, and most of the TBE patients received a second lumbar puncture 10 to 12 days later to monitor the course of the disease. Following lumbar punctures, CSF cells were counted, and the rest of the material , along with plasma samples, were frozen and kept at −80°C.Clinical and laboratory characteristics of the patient groups are given in Table Borrelia burgdorferi in serum and CSF of LNB patients. MS patients included in the study were in the process of MS diagnosis, and their EDSS  scores were between 0.5 and 4.0 (1.7 ± 0.9). Finally, they were diagnosed according to McDonald’s criteria [LNB was diagnosed according to the European Federation of Neurological Societies criteria . In all criteria  as relapet al. [17-S1P, Avanti Polar Lipids, Inc., Alabaster, USA) were added to 250 μl of plasma or CSF, and the samples were ultrasonicated in ice-cold water for one minute. Lipids were then extracted by the addition of chloroform, 1 M NaCl, and 3 N NaOH. The alkaline aqueous phase containing S1P was transferred to a fresh tube. The residual S1P in the chloroform phase was re-extracted twice with methanol/1 M NaCl  solution, and then all the aqueous fractions were combined. The amount of S1P was determined indirectly after dephosphorylation to sphingosine, with the use of alkaline phosphatase . To improve the extraction yield of released sphingosine, chloroform was carefully placed at the bottom of the reaction tubes. The chloroform fraction containing the dephosphorylated sphingoid base was washed three times with alkaline water (pH adjusted to 10.0 with ammonium hydroxide) and then evaporated under a nitrogen stream. The dried lipid residues were re-dissolved in ethanol, converted to their o-phthalaldehyde derivatives, and analyzed using an HPLC system  equipped with a fluorescence detector and a C18 reversed-phase column . The isocratic eluent composition of acetonitrile/water  and a flow rate of 1 ml/min were used. Column temperature was maintained at 33°C by use of a column oven .S1P concentration was measured by the method described in Min et al. . Briefly2. Neuronal cells were removed through a series of trypsinizations, and the remaining astrocytes were maintained at 37°C and 5% CO2 in DMEM + GlutaMax  supplemented with 5% fetal bovine serum. The purity of astrocyte cultures was 80% or more, as determined by immunofluorescence staining for glial fibrillary acidic protein [Mixed cortical cultures were obtained from prenatal rats and maintained for 14 days, or until confluence was reached, in Neurobasal™ media supplemented with 0.4 M GlutaMax and B27 (50× dilution)  at 37°C and 5% CO protein ,25. In apost-hoc Bonferroni analysis test. Differences between means were evaluated using the Student’s t-test, with P <0.05 being taken as the level of significance.Data are reported as a mean ± SE (standard error) or SD (standard deviation). Data analysis was performed using one-way analysis of variance (ANOVA) tests with a P <0.001) than in control subjects (383 ± 23.3 nM)  compared to the control group (0.72 ± 0.16 nM). These results are in close agreement with our previous study in which we showed an intrathecal increase of S1P in early stage MS [P <0.02) and CSF (P <0.05) of TBE patients. More precisely, before treatment S1P levels were 433.2 ± 19.2 nM and 2.29 ± 0.22 nM in plasma and CSF, respectively, and after treatment they were 370 ± 24.9 nM and 0.95 ± 0.21 nM in plasma and CSF, respectively. Average S1P concentration in plasma of TBE patients not treated with glucocorticosteroids remained stable (439.4 ± 21.0 nM versus 446 ± 35.8 nM). However, in the CSF of TBE subjects not treated with glucocorticosteroids, S1P concentration continued to increase during the course of the disease and was significantly higher (P <0.05) in samples obtained during the second lumbar puncture performed 10 to 12 days after the first, compared to the samples taken during the first lumbar puncture, which was performed at the time of diagnosis (1.79 ± 0.39 nM versus 1.48 ± 0.23 nM). This difference suggests an essential role of S1P in progression of these inflammatory processes. The initial S1P concentration in the CSF of subjects in poor general condition treated with glucocorticosteroids was higher compared to the initial S1P concentration in non-treated patients who were in better general condition. We did not observe significant changes in basic CSF parameters after glucocorticosteroid treatment  and LNB (538.0 ± 30.5 nM) patients was significantly higher (stage MS . They alstage MS . Compari1.8 nM anThere is currently no specific treatment for TBE infection, and treatment with glucocorticosteroids is still used in the most severe cases in countries with a high prevalence of the disease ,28. In tAstrocytes are the most abundant cells in the brain  and chanAstrocytes release a variety of cytokines in response to CNS infections and injuries ,38. IL-6in vivo experiments with FTY720P would be required to demonstrate a causal role of S1P in the pathogenesis of TBE.Our results indicate that S1P, but not GSN or FTY720-P, induced production of IL-6, and treatment with GSN or FTY720-P inhibited IL-6 release by rat astrocytes stimulated with S1P Figure . These rAcute CNS inflammation due to TBE virus infection is associated with an elevated S1P concentration in the blood and CSF of infected patients. In a cell culture system, the functions of astrocytes in an inflammatory state, induced by a high concentration of S1P, can be restored to their normal state by the administration of rhGSN, FTY720P, or their combination. This observation could be of clinical significance and might be useful for developing new treatments for pathological CNS conditions associated with rising concentrations of S1P in the blood and CSF, such as are produced by TBE.


Cerebrospinal fluid (CSF) is a major part of the central nervous system (CNS) extracellular fluid, and fine regulation of its composition is vital to the brain’s health. Although CSF dynamics has been studied for an entire century, many of its aspects are still insufficiently understood. Today there are two hypotheses ,2 on CSFAccording to the traditional hypothesis, CSF is formed inside the brain ventricles, mostly by secretion from the choroid plexuses, and it circulates along the ventricles and subarachnoid space to be absorbed across the arachnoid villi into the dural venous sinuses, and/or viacranial and spinal nerves paraneural sheaths into the lymphatic system. Since substance exchange occurs between the CNS extracellular interstitial fluid (ISF) and CSF, it is assumed that CSF serves as a sink for the removal of various metabolites out of the CNS by its unidirectional pulsatile flow and absorption -5. This The emerging concept of CSF physiology described as microcirculatory/microvessel hypothesis suggests that CNS microvessels are instrumental in fluid filtration and reabsorption inside the brain and spinal cord parenchyma, as well as inside the CSF system ,8-10. ItCroatian Medical Journal (CMJ) is dedicated to the CSF dynamics, and we hope that it will provide the readers with new data and views that can help them in their research projects. Due to the significance of the new CSF dynamics concept, this issue’s editorial is supplemented by a reprint of the article by Bulat and Klarica published in the Periodicum Biologorum in 2005, which first presented the new hypothesis on fluids physiology  in a 6-month-old infant, which cannot be controlled using standard operative drainage techniques . There is also the case of the oldest living patient with hydranencephaly, which raises some questions regarding the CSF turnover and homeostasis in a person with no brain parenchyma inside the supratentorial space . Also, this issue includes a case of a severe aqueductal stenosis lasting for 5 years without any detectable CSF movements, which is not accompanied with hydrocephalus development . In addition, Bechter and Shmitz describe time dynamics of contrast distribution from lumbar subarachnoid space into the psoas muscle tissue in one patient, and discuss the importance of their findings for pain research.Due to the importance of his reserch for disputing the traditional CSF physiology hypothesis, we dedicate a part of this editorial to the work of Prof. emeritus Marin Bulat (1936-2012), who started the CSF physiology research in Croatia .Marin Bulat began his scientific career at the Ruđer Bošković Institute under the mentorship of Prof. Zlatko Supek. In his master's (1964) and PhD thesis (1966), which determined the course of his scientific work, he explored the role of serotonin and its metabolites inside the CSF system and the brain. His early papers describe the serotonin passage from CSF into the brain by means of diffusion process (without any restrictions) depending on the concentration gradient ,13. Sero3H - benzylpenicillin, phenolsulphonphtalein) or inulin inside the CSF system and CNS, it was observed that they were distributed in all directions and that the distribution intensity depended on the rate of their elimination into the CNS capillaries  caused by to-and fro pulsations will always, after their application into the lateral ventricles (LV), create an illusion of a unidirectional bulk CSF circulation . On the contrary, after application of these substances into other parts of the CSF system, they are distributed in all directions  . BesidesIn his fight against the conventional notions, Prof. Bulat found inspiration and strenght in the thought of Claude Bernard, one of the most distinguished physiologists and medical scientists of the nineteenth century: ”When we meet a fact which contradicts a prevailing theory, we must accept the fact and abandon the theory, even when the theory is supported by great names and generally accepted.” He never gave up, and he managed to show his students and colleagues the way to persevere in science. He used to say that science is like a candle that burns low, and that this flame should be preserved and carried on with extreme attention, scientific integrity, and hard persistent work. He passed on this difficult task to all of us who are now, together with our foreign colleagues, trying to save that flame of science and to pass it on to new generations.Instead of conclusion let's thank Prof. emeritus Marin Bulat with another famous sentence by Claude Bernard: “A man of science rises ever, in seeking truth; and if he never finds it in its wholeness, he discovers nevertheless very significant fragments; and these fragments of universal truth are precisely what constitutes science.”